BIOLOGICAL CHEMISTRY.1. INTRODUCTION.IN the present Report fields of active research in biological chemistry havebeen covered which have not been reported upon before in Annual Reports.The metabolism of aromatic compounds by bacteria is now a growing andimportant field and has applications in relation to the destruction of herbi-cides by soil bacteria and the disposal of aromatic wastes in sewage. Thehighly toxic organophosphorus compounds were originally investigated aspotential chemical-warfare agents, but have now found use in agricultureas insecticides, Many of them are powerful inhibitors of esterases, and astudy of their biochemical behaviour has given an insight into the mechanismof enzyme action and, in particular, has enabled us to characterize theactive centres of esterases.The field of steroid chemistry and metabolismis now expanding at a tremendous rate, and a report on some aspect of thesteroids is almost an annual feature of some section or other of the Reports.It is not surprising, therefore, that a review on some facet of steroid bio-chemistry should be included in these Reports. Perhaps the most fascinat-ing advance in the metabolism of these compounds is the identification ofthe origin in vivo of all the carbon atoms of the cholesterol molecule. In-cluded in these Reports is also a review on the enzymes which split the estersof sulphuric acid. Until recently the sulphatases were a relatively neglectedgroup of enzymes, but in the last five years or so, interest in them has revived,and at present there is a considerable output of work in this field.A numberof sulphatases of very different specificity, have been found. Furthermorean important advance has been made during the year in elucidating thebiological synthesis of sulphuric acid esters, with the characterization of theso-called “ active sulphate.”R. T. W.2. BIOCHEMISTRY OF THE OXIDATIVE METABOLISM OF AROMATICDuring the past decade, fairly precise knowledge has been obtained ofthe way in which certain micro-organisms utilise aromatic compounds inmetabolism. Zobell dealt with the bacteriological aspects of this pheno-menon.and by Stanier ; more recently, Stanier has contributed a valuable chapteron its enzymology. The subject has not been previously reviewed in theseReports; it appears appropriate now to do so.COMPOUNDS BY MICRO-ORGANISMS.The biochemistry of the process was last reviewed by HappoldC.E. Zobell, Bact. Rev., 1946, 10, 1 ; Adv. Enzymol., 1950, 10, 443.a F. C. Happold, Biochem. Soc. Symp., No. 5, 1950, 85.R. Y. Stanier, “ Symposium sur le metabolisme microbien : l l e Congrhs inter-* R. Y. Stanier, ‘‘ Methods in Enzymology,” ed. by S. P. Colowick and N. 0. Kaplan,national de Biochimie,” 1952, p. 64.Academic Press, New York, 1955, Vol. 2, 273280 BIOLOGICAL CHEMISTRY.Ring-cleavage Metabolism of Aromatic Compounds.-( a) Micro-ovgaizisms.Several types of micro-organisms 5* classified into the following six families,Coccaceae, Mycobacteriaceae, Bacteriaceae, Pseudomonadaceae, Spirillaceae,and Bacillaceae, are known, which grow aerobically in a simple mineral-saltmedium, with an aromatic compound as sole source of organic carbon.During growth of the micro-organism the benzene ring undergoes fission, thecompounds formed being utilised in the diverse processes associated with cellmetabolism.Such organisms have been isolated from soil, sewage, andmammalian faxes, and are widely distributed in nature. Indeed, the bio-logical function of these micro-organisms by virtue of this property may beregarded as an essential step in the " carbon " cycle. Their use as industrialscavengers is common, since percolation of phenolic waste products throughsewage beds provides a cheap method of detoxicating aromatic compoundspotentially harmful to aquatic life.That this property is not confined to the lower bacteria (eubacteria), hasbeen amply demonstrated.Thus, amongst the fungi, A s$ergiZZzis ~ p p . ; ~PeTLiciZZizim ~ p p . , ~ * Oospora spp.,lO and Neurospora spp.ll are capable ofsome of these reactions, as well as certain soil l2 and wood-rotting fungi.13(b) Plants. The specific problem of aromatic-ring catabolism has notbeen extensively studied in plants, owing, no doubt, to the experimental diffi-culties involved. A variety of plants form glycosides from certain foreignphenols,l* e.g., o-chlorophenol is converted into o-chlorophenyl P-gentio-bioside ; glycoside formation may be a detoxication mechanism immobilisingtoxic phenolic by-products of metabolism, because the plant has no excretorysystem. The occurrence of a bewildering variety of aromatic compoundsin the plant world, however, shows the presence of a very efficient mechanismfor the synthesis of the benzene nucleus, and enzymes which hydroxylatephenolic substances (e.g., polyphenol oxidases) are abundant in plant tissues.In mammals, unnatural aromatic substances are usuallydetoxicated by hydroxylation and conjugation, before excretion.That thecapacity for ring fission has not been lost entirely is shown by the old observ-ation by Jaffe,15 since confirmed by several workei-s,l6 that the administrationof benzene to rabbits and dogs, gives a small amount of tram-trans-muconicacid in the urine. With the naturally occurring aromatic amino-acids, thereexist in liver tissue fairly well characterised enzyme systems l7 whichdisrupt the benzene ring of tyrosine, after manipulation of the side chain.(c) Mammals.P.H. H. Gray and H. G. Thornton, Zentr. Bakt., 2 Abt., 1028, 73, 74.F. Bernheim, J. Bact., 1941, 41, 387; J , Biol. Chenz., 1942, 143, 383.A. J. Kluyver and J. C. M. van Zijp, Antonie van Leeuwenhoek. J . Microbiol. Serol.,8 D. J. D. Hockenhull, A. D. Walker, G. D. Wilkin, and F. G. Winder, Bioclzem. J , ,1961, 17, 315.1952, 50, 605.M. Isono, J . Agvic. Chem. SOC. (Japan), 1953, 27, 255; 1954, 28, 196.lo S. Landa and J. Eliasek, Chern. Listy, 1956, 50, 1834.11 S. R. Gross, K. D. Gafford, and E. L. Tatum, J . Biol. Chena., 1956, 219, 781.12 M. E. K. Henderson and V. C. Farmer, J.Gen. MicvoEioZ., 1955, 12, 37.l3 G. Fahreus, Kgl. Lantbvukgs-Hogskol. Ann., 1949, 16, 618.14 L. P. Miller, Science, 1940, 92, 42.l5 M. Jaffe, 2. phjsiol. Chem., 1909, 62, 58.16 D. V. Parke and R. T. \Villiams, Biochmz. J., 1952, 51, 339.1' W. E. Knox, ref. 4, p. 207EVANS : OXIDATIVE METABOLISM OF AROMATIC COMPOUNDS. 281A noteworthy fact is the close similarity in the biochemical pathway of ring-cleavage metabolism of the naturally-occurring aromatic amino-acids in allforms of life studied.Criteria Used to Establish the Participation of a Presumed Intermediate ina Metabolic Bathway.-As Kluyver l8 pointed out many years ago, no singleapproach to the analysis of the activities of the living cell can ever justifythe belief that our conceptual schemes bear a one-to-one correspondence tothe real course of events.This is particularly true of the topic underreview ; briefly, the evidence and criteria used are as follows :Most bacterial oxidationsof aromatic compounds are complete, the carbon skeleton of the substratebeing converted exclusively into carbon dioxide and cell material. How-ever, as Evans Is first showed, intermediates sometimes accumulatetransiently in cultures, and may be isolated chemically from the culturefluid. A variant of this traditional method of approach is the study of cell-free enzyme preparations made from organisms after growth on the originalsubstrate, on any likely intermediate in vitro. The interpretation of results,from the point of view of origin of the isolated products, always presentsdifficulty, but the probability of their being real intermediates is vastlyincreased if marked compounds are used.It may be fairly claimed that thisapproach has nearly always given the initial clue.Observationswhich have led to current ideas on the factors which control the enzymaticconstitution of a microbial cell go back to the work of Wortmann.20 It wasKarstromJ21 however, who designated as “ adaptive ” those enzymes whichare produced as a specific response to the presence of the homologous sub-strate in the culture medium. He differentiated them from the “con-stitutive ” enzymes which are always formed by the cells of a given species,irrespective of the composition of the medium.Stanier 22 in 1947 found that the ring-fission enzymes, elaborated by hisPseudomonas strains of aromatic-ring splitters, were strictly adaptive innature ; these enzymes were demonstratively present in the bacterial cellsonly when growth occurred in the presence of the aromatic substrate.Heconceived the idea of using this phenomenon as the basis for a refined typeof kinetic analysis to determine the nature of the intermediates that lie onan adaptively-controlled metabolic pathway. This valuable technique wascalled “ simultaneous ” or “ successive ” adaptation, but for various reasons,Cohn et al.23 now propose the more accurate terminology of “ sequential(a) Chemical isolation from growing czdtwes.(b) The use of the techniqz4e of “ sequential induction.”18 A.J. Kluyver, ‘‘ The chemical activities of micro-organisms,” Univ. London1 9 W. C. Evans, Biochem. J., 1947, 41, 373.2O J. Wortmann, 2. physiol. Chem., 1882, 6, 287.2 1 H. Karstrom, Ergebn. Enzymforsch., 1937-38, 7, 350. See also R. J. Dubos,Bact. Rev., 1940, 4, 1 ; J. Monod, Growth, 1947, 11, 323; “Third Symp. SOC. Gen. Micro-biol.,” ed. by E. F. Gale and R. Davies, Camb. Univ. Press, 1953.22 R. Y . Stanier, J . Bact., 1947, 54, 339. See also R. J. Fitzgerald, F. Bernheim,and D. B. Fitzgerald, J . Biol. Chem., 1948, 175, 195; G. S. Eadie, F. Bernheim, andR. J. Fitzgerald, ibid., 1948, 176, 857.23 M. Cohn, J. Monod, M. R. Pollock, S. Spiegelman, and R. Y . Stanier, Nature,1963, 172, 1096.Press, London, 1931282 BIOLOGICAL CHEMISTRY.induction.” The principle 24 can be illustrated by considering a hypotheticalmetabolic pathway, each step of which is catalysed by a specific, inducedenzyme :A+B---+C+D+E+etc.EA EB EC ED EECells potentially capable of carrying out these reactions will be devoid of therelevant enzymes if they have not been exposed by the conditions of growthto compound A (the inducer).When placed in contact with A, such cellsrespond by producing the enzyme E A , catalysing the step A- B. Theformation of B will in turn provide the necessary activation for the formationof Eg, and so on. Thus, cells fully induced to dissimilate A will also beconditioned to metabolise B, C , D, etc. If one adapts the cells to an inter-mediate in the chain (say C ) , they will then also be adapted to the laterintermediates, D, E, etc., but not necessarily to the earlier ones.Consider,however, the case of a compound X, which may appear on chemical groundsto be a possible intermediate in the dissimilation of A, and which is likewisepotentially attackable by a specific, inducible enzyme Ex. In view of theknown high specificity of the inductive response, it is extremely improbablethat cells specifically adapted by exposure to A will also be adapted to X,if X i s not a member of the reaction chain. Thus, by adapting an organismto a given primary substrate, and then analysing its adaptive patterns withrespect to postulated intermediates, evidence can be obtained as to whichof these compounds are actually operative. It is, of course, necessary tomake a parallel test with “ unadapted ” cells, grown in the absence of allthe compounds under test, in order to make certain that the relevant enzyrnesare not always present in the cells (Le., constitutive).In practice theapplication of this technique is extremely simple ; washed cell suspensionsgrown on the aromatic substrate whose metabolic pathway is under in-vestigation, are incubated separately, the various postulated intermediatesnow being used as substrates, the rate of oxygen uptake being measuredmanometrically. The absence of a lag period indicates compliance withthis criterion of sequential induction ; the presence of a lag period, howeversmall, is taken to mean that the induced enzymes were not originally presentin the cells, the delay representing the time required for their synthesis.The validity of the biochemical inference drawn from sequential-inductionexperiments rests on some assumptions, which may not always be correct,e.g.: (i) Rigid specificity of the inductive response to a particular chemicalstructure ; if this is not so, then false positive conclusions are possible.22* 25-27(ii) Free permeability of the cell to all compounds tested; should this con-dition not hold then false negative results may beIt is now known that these assumptions do not always obtain, and theuse of this method alone cannot provide unequivocal evidence for the2924 R. Y. Stanier, Ann. Rev. MicrobioE., 1961, 5, 36.25 S. Spiegelman, M. Sussman, and B. Taylor, Fed. Proc., 1950, 9, 120.28 R.Y . Stanier, B. P. Sleeper, M. Tsuchida, and D. L. Nacdonald, J. Bact., 1950,27 S. R. Gross and E. L. Tatum, Science, 1955, 122, 1141.2* R. Y. Stanier, Bad. Rev., 1950, 14, 179.59, 137.S. Dagley, M. E. Fewster, and F. C. Happold, J. Gen. MicrobioE., 1963, 8, 1EVANS : OXIDATIVE METABOLISM OF AROMATIC COMPOUNDS. 283existence of a metabolic pathway, but should be supported wherever possibleby other evidence. In spite of these limitations, it is the Reporter’s viewthat Stanier’s technique contributed very materially, by providing inde-pendent evidence, towards the establishment of the evidence of biochemicalpathways of aromatic-ring cleavage first put forward mainly as a result ofisolation.(c) Preparation of the specif;c enzymes catalysing each stt@ in the proposedmetabolic pathway.The purification of the specific enzymes catalysingeach step in the reaction chain is a desirable objective, not always realisedbecause of lability or the particulate nature of some of them. Their use, ina study in vitro of each postulated reaction, usually provides information asto mechanism, co-factor requirements, and identity of intermediates whichdo not accumulate in cultures.Isolation and Selection of Strains.-The isolation of specific microbes withthe required properties (viz., ability to grow freely in pure culture containinga simple salt mixture as basal medium, together with the aromatic compoundas sole substrate) is usually accomplished by the selective culture method,first applied by Winogradsky30 and developed by Beijerin~k.~~ The soilor sewage bed is continuously perfused 32 with the simple medium for a periodof days, the use of toxic amounts of the substrate being guarded against;for aromatic ring splitters, O * O l - O * l ~ & wjv is a suitable strength.It ishelpful to estimate the concentration of the aromatic substrate in the primarymixed culture medium. This may stay constant for days, and nothingseems to be happening ; suddenly its disappearance becomes manifest, andthe whole may be gone in a few hours. When active metabolism of thesubstrate is in progress, subcultures are made into simple media, thuscausing preferential growth of a certain type of germ, ultimately leading toa predominance of the conditionally fittest.From such enrichment cultures,after plating out on simple media (stiffened with either agar or silica gel), thedesired organism is isolated as a pure strain by the traditional methods ofmicrobiology.The initial work on the oxidative metabolism of phenol used Vibrio 0/1,isolated by Happold and Key 33 from sewage effluent containing spent gas-works liquors. Subsequently, Pseudomonad-type organisms isolated fromsoil were also employed in expanding the work to include other aromaticcompounds, since the capacity of Vibrio 011 is limited. Careful selection ofstrain, and appropriate adaptation of the bacterial cells, is an essentialpreliminary to enzymic studies of these metabolic pathways.Chance-isolated and artificially produced mutants of various micro-organisms have become highly successful tools in the elucidation of bio-synthetical pathways, e.g., Davis’s use of several polyauxotrophs of30 S.Winogradsky, “ Microbiologie du sol,” Oeuvres complktes, Masson, Paris, 1949.31 M. W. Beijerinck, “ Verzamelde Geschriften,” Delft, 1921-1940, Vol. 1-6.32 Several perfusion apparatuses have been devised ; Professor F. C. Happold usedminiature sewage-beds in the laboratory in the 1930’s. H. Lees and J. H. Quastel,Chem. and Ind., 1944, 238; L. J. Audus, Nature, 1946, 158, 419; F. M. Collins andC. M. Sims, Nature, 1956, 178, 1073.33 I;. C. Happold and A. Key, J . Hygiene, 1932, 32, 573.34 B. D. Davis, in ref. 3, p. 32; idem, “ Amino Acid Metabolism,” ed. by W. D.McElroy and B.Glass, The Johns Hopkins Press, Baltimore, 1955, p. 799284 BIOLOGICAL CHEMISTRY.Escherichia coli in aromatic biosyntheses. Recently Gross, Gaff ord, andTatum 11 have used a mutant strain of Newospora cyassa, Y7655a, producedby treatment of Neurospora macroconidia with N-methylbis-2-chloroethyl-mine, in a very elegant study of the ring-cleavage metabolism of proto-catechuic acid.AROMATIC SUBSTRATELow energy release reactlolls J,HYDROXY LATEDINTERMED-IATESInduced enzymes [ AROMATICCARRIER 3 FINAL HSYSTEMS ACCEPTORSALIPHATIC c INTERMEDIATESr e l e a s eII materrIe a c t ionsalOxidative metabolism of aromatic compounds.The oxidative metabolism of aromatic compounds, dealt with in thisReport, implies the ability of these micro-organisms to produce enzymeswhich manipulate the benzene ring in such a way that the products formed,at some stage, enter the main terminal respiratory cycle of the cell, expressedschematically above.There exists an anmobic type of microbial metabolismof aromatic compounds, the so-called methane fermentation investigatedinitially by S ~ h n g e n , ~ ~ and later by Buswell and his collaborator^,^^ aboutwhich hardly anything is known; the dissimilation of benzoic acid by thisprocess conforms to the overall equation :4C,H&02H + 18H20 -w 15CH, + 13C0,Oxidative Metabolism of Phenol by Micro-organisms.-Biological oxidationof phenolic compounds by the aerobic oxidases, with quinone formation andsubsequent polyrnerisation leading to coloured complex products, is wellknown since the work of Raper37 and his collaborators.Beijerinck hadisolated Vibrio tyrosiyzatica, which contained a tyrosinase system, from thecanal waters of Delft, and Happol<38 had shown that all bacteria whichgave the " direct oxidase reaction'' of Gordon and McLeod, possessed acatechol oxidase system. This background knowledge indicated that somemicro-organisms possessed enzymes which hydroxylated the aromatic ring.35 N. L. Sohngen, " Het onstaan en verdwijnen van waterstof en methaan onderden invloed van liet organische leven," Thesis, Delft, 1906.36 G. E. Symons and A. M. Buswell, J. Avner. Chewz. Soc., 1953, 55, 2028.37 H. S. Raper, Physiol. Rev., 1928, 8, 245.3* F. C. Happold, Biochem.J., 1930, 24, 1737EVANS : OXIDATIVE METABOLISM OF ARQMATIC COMPOUNDS. 286In 1939, Evans and Happolda9 studied the breakdown of phenol by aVzbrio (called O/l-oxidase l), previously isolated by Happold and Key.This organism had the amazing capacity of growing profusely, with aeration,in a simple mineral-salt medium containing phenol (up to 0.1% w/v) as soleorganic source of energy. During growth, the phenol was metabolised, andno pigment was produced; the biochemistry of this process was unknown atthis time. The transient accumulation of compounds giving (a) reactionscharacteristic of o-dihydric phenols, (b) Rothera and Gerhardt positivereactions indicative of a P-oxo-acid, was established. Catechol was readilyisolated from cultures, but the nature, and isolation of the fi-oxo-acidproved more elusive, although it was known not to be identical with aceto-acetic acid.The isolation was accomplished by Milby40 in 1948, and theoxo-acid identified as fi-oxoadipic acid. Washed cells of Vibrio 011 utilised10 atoms of oxygen per molecule of phenol, approximately 9 atoms permolecule of catechol, with the production of roughly 4 molecules of carbondioxide, in both cases. Evans l9 therefore suggested that the oxidativemetabolism of phenol proceeded via catechol, followed by ring cleavage, to ap-oxo-acid. As far back as 1928, Tausson41 believed that the benzenenucleus was split with the formation of muconic acid, which was thenrapidly oxidised to carbon dioxide, by phenanthrene-oxidising bacteria.Evans had considered this possibility, but the inability of Vibrio O / l togrow or metabolise trans-trans-muconic acid, excluded it from being anintermediate.In 1950, Hayaishi and Hashimoto 42 reported the preparationand purification of an enzyme from Pseudomoizas spp. which acted on catecholyielding an acid, tentatively identified as cis-cis-muconic acid. A crudeenzyme preparation which catalysed the reaction :catechol + 0, + H,O -+ p-oxoadipic acidwas found by Stanier and Hayaishi 43 to be incapable of giving the oxo-acidwhen the muconic acid isolated by the Japanese workers42 was used assubstrate-a fact which excluded it from being a true intermediate in thereaction. At this stage, the investigations by Elvidge, Linstead, Sims, andOrkina on the stereochemistry of the muconic acids threw light on themicrobiological problem.These workers isolated a new cis-trans-muconicacid from the alkali-catalysed isomerisation of a related unsaturated lactone.This new acid resembled very closely the cis-cis-muconic acid which can beprepared by the chemical oxidation of phenol.45 When the cis-cis-acid ismerely boiled with water, it is inverted into the new cis-traPzs-isomeride.This means that many samples of cis-cis-acid used by previous workersmust have been inverted by the usual processes of purification. The roleof these muconic acids in the enzymic ring-cleavage conversion of catecholinto p-oxoadipic acid was settled in 1951 by Evans and Smith; 46 the pure30 W. C. Evans and F. C .Happold, J . SOL. Chem. Ind., 1939, 58, 55.41 W. 0. Tausson, Planta, 1928, 5, 239.42 0. Hayaishi and 2. Hashimoto, Med. J. Osaka Uiziv., 1950, 2, 33.43 R. Y. Stanier and 0. Hayaishi, Science, 1951, 114, 326.44 J. A. Elvidge, R. P. Linstead, P. Sims, and B. A. Orkin, J., 1950, 2235.45 J. Boeseken and R. Engelberts, Proc. Acad. Sci., Amsterdam, 1931, 34, 1292.4 6 W. C. Evans and B. S. W. Smith, Biochem. J . , 1951, 49, x.B. A. Kilby, Biochcm. J., 1948, 43, v; 1951, 49, 671286 BIOLOGICAL CHEMISTRY,cis-cis-isomer (1) is metabolised by intact cells, and is converted non-oxidatively into p-oxoadipic acid (3) by a crude catechol-oxidising cell-freeenzyme preparation, whereas the cis-trans- and the trans-trans-isomer arebiologically inactive. Evans et aL4’ also found that a lactone, y-carboxy:methyl-Aa-butenolide (2), related to cis-cis-muconic acid, was converted bythe same enzyme preparation, into p-oxoadipic acid.Kilby 40 has shownthat intact cells further metabolise this oxo-acid by a C(4)-CtD split, tosuccinate and acetate, which are then capable of entering the terminalcycle of respiration. The scheme 1 below suggested by Evans, Smith,Linstead, and Elvidge 4T is supported by (a) isolation evidence, (b) sequentialinduction, and (c) a study of the individual enzymes concerned.SCHEME 1. Oxidative metabolic pafhway of phewol by micro-ovganisnzs.Nature of the Enzymes Involved in the Individual Steps of Scheme 1.-Some of the enzymes involved in this oxidative pathway4 can now beprepared, cell-free, starting froin fully adapted cells, and by employing theusual disintegration methods, followed by fractionation, at low temperatures.The bacterial enzymes which hydroxylate aromatic substrates are very labile,and active cell-free preparations have not been described. However, this isbelieved to have been accomplished recently by Dagley and Patel 48 in thecase of the enzyme which hydroxylates P-hydroxybenzoic acid to proto-catechuic acid.In view of the interest of biochemists in aromatic hydroxyl-ations generally, advances in this field are to be expected (Udenfriend andhis collaborators 49 have prepared microsomal preparations from liver whichhydroxylate several aromatic compounds in the presence of reduced tri-phosphopyridine nucleotide and oxygen).The Japanese workers 42* 50 purified pyrocatechase from acetone-driedcells of adapted Pseudomonas spp.and showed its dependence on ferrousions. It appears also that mercapto-compounds, e.g., glutathione, alsostimulate activity. The lactonising and lactone-splitting enzymes have beenseparated very elegantly by Sistrom and Stanier,S1 thus completing the step-wise enzymic degradation of cis-cis-muconic acid through y-carboxymethyl-4 7 W. C. Evans, B. S. W. Smith, R. P. Linstead, and J. A. Elvidge, Nature, 1951,48 S . Dagley and M. D. Patel, 1956 (personal communication).49 C. Mitoma, H. S. Posner, H. C . Reitz, and S. Udenfriend, Arch. Biochem. Bio-50 0. Hayaishi, M. Katagiri, and S. Rothberg, J . Amer. Chem. SOC., 1955, 7’7, 2914.61 W.R. Sistrom and R. Y . Stanier, J . Biol. Chem., 1954, 210, 821; Nature, 1954,168, 772.phys.. 1956, 61, 431.174, 513EVANS : 0XID.YrIVE METABOLISM OF -1ROMATIC COhfP0UNI)S. 287h"-butenolide to p-oxoadipic acid. The lactonising enzyme requires eitherMg++ or Mn++ ions for activity, whereas the lactone-splitting enzyme doesnot. The lactonising enzyme catalyses a remarkable pair of reactions :IIHAC'COTc i s - c i s -5 %y 2 -H-C-HI0 rE IICH(+)9 5 %c0,-IH-C-HThe iwo reversible reactions of m u c o ~ i c acid atid y-carboxynzetl~yl-A'-bute~iolide rafalvsedby i'he lactonising enzyme, with the equilib~ium values for pH 8.(a) is the reversible interconversion of cis-cis-muconic acid and the (+)-lactone,at pH 8.0 the equilibrium mixture containing 95% lactone, and in (b) thecis-trans-isomer is converted into an equilibrium mixture with the (-)-lactone(20% of lactone at pH S), although more slowly than the natural substrate.Since the double bond in the lactone ring has a cis-configuration, it is evidentthat the lactonising enzyme must act on the trans-bond of cis-trans-muconicacid. The enzyme cannot interconvert the two geometric isomers or thetwo enantiomorphs.The basis of this pair of reactions is an ability of theenzyme to distinguish between two chemically identical atoms or bonds.Thus in the forward reaction only one of the two component bonds of the@-double bond is split, and in the back reaction only one of the two&hydrogen atoms is removed.The ability of an enzyme to distinguish be-tween chemically identical groups has been demonstrated before 5 2 9 % anda three-point attachment of substrate to enzyme has been proposed as themechanism.Oxidative Metabolic Pathways of the Benzoic Acids by Micro-organisms.-The metabolism of benzoic acid and its derivatives demonstrates the limiteddiversity of pathways employed by these bacteria in manipulating thearomatic ring before cleavage.Benzoic 55-57 and salicylic 593 6o acid give rise to catechol (4) by whatappears to be a single oxidative decarboxylation step. Evidence for thisrests on sequential induction, and isolation of catechol from cultures ofthese substrates. Sleeper 58 subsequently used benzoic acid labelled in thecarboxyl and the C(l) position with 14C, in this bacterial oxidation, and52 V.R. Potter and C. Heidelberger, Nature, 1949, 164, 180.53 H. F. Fischer, E. E. Conn, B. Vennesland, and F. H. Westheimer, J . Biol. Chent.,54 A. G. Ogston, Nature, 1948, 162, 963.5 5 W. H. Parr, R. A. Evans, and W. C. Evans, Biochern. J., 1949, 45, xxis.5 6 R. A. Evans, W. H. Parr, and W. C. Evans, Nature, 1949, 164, 674.5 7 B. P. Sleeper and R. Y . Stanier, J . Uact., 1950, 59, 117.5 8 B. P. Sleeper, ibid., 1951, 62, 657.58 N. Walker and W. C. Evans, Biochem. J . , 1952, 52, xxiii.60 B. S. Roof, T. J. Lannon, and J. C. Turner, Proc. SOC. Exp. Biol. Med., 1953, 84.1953, 202, 687.38288 BIOLOGICAL CHEMISTRY.showed that, (1) catechol derived from [~arboxy-~~C]benzoic acid is inactive,while that derived from [l-14C]benzoic acid has the same specific activityas the parent compound; (2) all the radioactivity of the catechol derivedfrom [l-14C]benzoic acid is in carbon atoms 1 and 2 ; (3) from the completeoxidation of benzoic acid the radioactivity of the carboxyl group is foundalmost exclusively in respiratory carbon dioxide; that of carbon atom 1 islargely found in carbon dioxide, but a significant percentage appears in thebacterial cells and supernatant liquid.Walker and Evans 59 showed that m-hydroxybenzoic acid gives gentisicacid (5) before ring cleavage, by isolation and adaptation evidence ; this wasconfirmed by Roof, Lannon, and Turner.Go Ring cleavage of gentisic acidoccurs through a new pathway and the formation of 4 : 6-diox0hept-~ A-ene-dioic acid (6) as the immediate product has been suggested, but not proved.Q" \6" 0." (4)as schemrz I.C02HCO2H oc0cH'2 asscheme I .I FH2 + o c o 2 2 (3!02H- unknown pathway.H2C, CO2HHO / Co2H (7)OHSCHEME 2.p-Hydroxybenzoic acid gives protocatechuic acid,lS which then under-goes ring cleavage with the formation of p-oxoadipic acid (7).55 UsingPseudomonas fluorescens adapted to protocatechuic acid, Stanier andIngraham 61 prepared a cell-free enzyme, termed " protocatechuic acidoxidase," able to cause cleavage of this substrate with the formation of anextremely labile intermediate, isolated as a crystalline trisodium salt, andidentified as a p-carboxymuconic acid (8) 62 (probably the cis-&isomer).During purification of the oxidase, they obtained, as a by-product, a " de-carboxylase " enzyme preparation, which converted cis-cis-p-carboxy-muconic acid into p-oxoadipic acid.The latter reaction is undoubtedlymore complex, and remains obscure.Oxidative metabolic pathways of the benzoic acids by micro-organisms.131 R. Y . Stanier and J. L. Ingraham, J . BioZ. Chem., 1964, 210, 799.62 D. L. MacDonald, R. Y . Stanier, and J. L. Ingraham, ibid., p. 809EVANS : OXIDATIVE METABOLISM OF AROMATIC COMPOUNDS. 289Recently, Gross, Gafford, and Tatum l1 have shown that a mould,Neurospora crassa, can convert protocatechuic acid into p-oxoadipic acid, bythe aid of enzymes inductively produced. They made a detailed comparativestudy of this reaction, using [2 : 6-14C]protocatechuic acid, and purifiedenzymes prepared from Neurospora and Pseudomonas, with unexpectedresults.From the mould they prepared two enzymes, the first convertingprotocatechuic acid into cis-cis-P-carboxymuconic acid (similar to proto-catechuic oxidase of Pseudomonas) ; the second enzyme converted thecarboxymuconic acid into p-carboxymuconolactone [ (-)-P-ca.rboxyy-carboxymethyl-AQ-butenolide] (9). A delactonising enzyme, or enzymes,then converts p-carboxymuconolactone into b-oxoadipic acid with loss ofcarbon dioxide. The prepared p-carboxymuconolactone was not attackedby extracts of Pseudomonas jaorescens, which were fully capable of convert-ing protocatechuic acid into p-oxoadipic acid ; this lactone, then, cannotbe an intermediate in the latter reaction. Isotopic evidence also showedconclusively that the conversion of p-carboxymuconic acid into p-oxoadipicacid is different in the two enzyme systems. Dagley and Pate14* haveisolated a new substance (m.p. 235"), the constitution of which remains-tobe determined, as a product of reaction of Pseudomonas enzyme on proto-catechuic acid. It contains no phenol or enol groups; analysis gives(C,H,O,),, and alkaline titration indicates two carboxyl groups per moleculeif 12 = 1. The additional carboxyl group on @-carboxymuconic acid intro-duces new possibilities for lactone formation, and it may well be that thePseudomonas enzyme forms a different lactone from p-carboxymucono-lactone, as an intermediate stage on the way to p-oxoadipic acid.Evans 63 made a preliminary study of the breakdown of phthalic acidby a soil Pseudomonad; none of the monohydroxyphthalic acids was de-tected, neither did they satisfy the criteria of sequential induction, but4 : 5-dihydroxyphthalic acid was identified chromatographically in growingcultures, and was adapted.Its subsequent pathway has not been in-vestigated.Oxidative Metabolic Pathways of Phenylalanine, Tyrosine, and theirDerivatives by Micro-organisms-Suda and Takeda 64 found that Pseudo-monas spp. already adapted to tyrosine were also adapted to oxidise homo-gentisic acid ; a cell-free enzyme preparation, homogentisicase, whichcatalysed the rupture of the ring was prepared. Like pyrocatechase, thisenzyme required ferrous ions as a co-factor, implying the existence inbacteria of a pathway for tyrosine metabolism similar to that existing in themammal.Jones, Smith, and Evans 65 isolated homogentisic acid from atyrosine culture of Vibrio 011. Meanwhile, Kluyver and van Zijp 7 hadshown the production of homogentisic acid from phenylacetic acid byAspergillus Niger. Hockenhull et at. ,a working with Penicillium chryso-genum, showed that the acetic acid side chain was partially degraded tobenzaldehyde before ring cleavage. Isono identified o-hydroxyphenyl-acetic acid, from phenylacetate cultures of a similar mould species, and63 W. C . Evans, Biochem. J., 1966,61, x.64 M. Suda and Y. Takeda, Med. J . Osaka Univ., 1950, 2, 37.66 J. D. Jones, B. S. W.Smith, and W. C . Evans, Biochem. J., 1952, 51, xi.REP.-VOL. LIII 290 BIOLOGICAL CHEMISTRY.subsequently produced a mutant strain of Penicillium chrysogenum causingthe accumulation of homogentisic acid.Dagley, Fewster, and Happold 29 carefully examined the adaptivepatterns of Vibrio O / l grown on phenylalanine and tyrosine ; their resultssupport the pathway shown in scheme 3, as far as homogentisic acid. Thefate of this intermediate in the microbial pathway is only presumed to be thesame as that of those catalysed by liver enzymes, recently elucidated byseveral workers,66 i.e., the formation of 4 : 6-dioxo-oct-2-enedioic acid, itsisomerisation and subsequent hydrolysis to fumaric and acetoacetic acid,have not yet been demonstrated by using microbial enzymes.The pathways of metabolism of mandelic acid and P-hydroxymandelicacid were established by Stanier 67 and by Gunter,68 respectively. Gun-salus, Stanier, and Gunsalus 69 succeeded in separating four soluble enzymesconcerned with the conversion of mandelic into benzoic acid, from Pseudo-monasjuorescens, and these reactions can now be formulated as follows :racemase dehydrogenaseD( -)-Mandelic acid 4-t L( -/-)-Mandelic acid - - 2Hcarboxylase dehydrogenase 1Benzoylformic acid -- Benzaldehyde TYNt ____l_t TPNHBenzoic t i acid DPNf - DPNHdehydrogenase 2CO,Abbreviations used are: TPN+, DPN+, TPNH, and DPNH for oxidised andSmith, Jones, and Evans 70 showed that a soil Pseudomonad oxidisedp-cresol to p-hydroxybenzoic acid. Dagley and Patel 71 have investigatedthis pathway in more detail, and confirmed the reaction sequence shown inscheme 3.Oxidative Metabolic Pathway of Tryptophan by Micro-organisms.-Theproduction of indole from tryptophan, through elimination of the alanineside-chain, is carried out by E.coli grown under certain cultural condition^.^^Bacteria of the Pseudomonas group, however, are capable of the completeoxidation of this amino-acid. The adaptive patterns of such organisms wereindependently examined by Suda, Hayaishi, and Oda,73 and Stanier andTsuchidaS7* The former found that Pseudomonas spp. adapted to trypto-phan were fully adapted to oxidise kynurenine, anthranilic acid, andcatechol. Stanier and Hayaishi 75 found marked differences in dissimilatorypatterns between various Pseudomonas strains, with respect to tryptophan ;most of them oxidise it through anthranilic acid and catechol (referred toas the " aromatic pathway "), whilst a few employ a different route, throughreduced triphospho- and diphospho-pyridine nucleotides.6 6 W.E. Knox, ref. 4, p. 292.6 7 R. Y . Stanier, J. Bact., 1948, 55, 477.68 S. E. Gunter, ibid., 1953, 66, 341.69 C. F. Gunsalus, R. Y. Stanier, and I. C. Gunsalus, ibid., 1963, 66, 548.70 B. S. W. Smith, J. D. Jones, and W. C. Evans, Biockem. J., 1952, 50, xxviii.71 S. Dagley and M. D. Patel, ibid., 1955, 60, xxxv.72 F. C. Happold, Adv. Enzymol., 1950, 10, 52.73 M. Suda, 0. Hayaishi, and Y. Oda, Med. J. Osaka Univ., 1950, 2, 21.74 R. Y. Stanier and M.Tsuchida, J . Bact., 1949, 58, 45; 1951, 62, 355.75 R. Y. Stanier and 0. Hayaishi, Science, 1961, 114, 326; J . Biol. Chem., 1962,195, 735EVANS : OXIDATIVE METABOLISM OF AROMATIC COMPOUNDS. 291kynurenic acid (designated the ‘‘ quinoline pathway ”). Yet a few rarestrains carried out various blocked oxidations of tryptophan with accumul-ation of indole, anthranilic acid, or kynurenic acid.CH,. CH-CO, H 0 )(1H2 4OHfCH 2 * CO. CO 1H 0 OHCO2HICH-OH 0- OHC02 HI 6- OH 6 OH7 OH 6“OH \scheme 2 .f lSCHEME 3. Oxidative metabolic pathway of phenylalanine, tyrosine, and derivativesby Micro-organisms .SCHEME 4. Oxidative metabolic pathwa-y of tryptophan by micro-oqanisms.Cell-free extracts capable of oxidising L-tryptophan to $-oxoadipic acidwere obtained by Hayaishi and Stanier; 76 this complex extract containeda tryptophan osidase-peroxidase system, kynureninase, and anthranilic acidoxidase, in addition to the ring-cleavage enzymes.Detailed study of theenzymatic step-reactions which result in the conversion of tryptophan intoanthranilic acid indicates a close similarity to the analogous reactions in themammal; 77 these are depicted in scheme 4.713 0. Hayaishi and R. Y . Stanier, J . Bact., 1951, 62, 691.$7 W. E. Knox, ref. 4, p. 242292 BIOLOGICAL CHEMISTRY.Oxidative Metabolic Pathways of Miscellaneous Aromatic Compounds byMicro-organisms.-An increasing number of synthetic aromatic compoundsare used as herbicides, insecticides, food preservatives, etc., and it is becomingimportant to know something about their metabolism, from a fundamentalpoint of view, since many of them are physiologically active, and because ofthe potential hazards to life should they accumulate in toxic amounts.Acomplex milieu like soil often causes the disappearance of a great varietyof organic chemicals by biological means. Provided such versatile organismscan be grown in simplified media, with a reasonably rapid utilisation of thesubstrates, an attractive biochemical approach to the study of their meta-bolism presents itself. These media will now be summarised.(a) The nitrophenols. An industrial biological purification plant detoxic-ating nitrophenols, afforded Pseudomonad-type organisms capable of growingin a mineral-salt medium containing 0.02% of o- and p-nitrophenol.Evansand Simpson 78 showed that the nitro-group was substituted by hydroxyl inthe aromatic ring, which was further metabolised by ring-fission. Theeliminated nitro-group appeared in the medium as the nitrite ion ; this noveltype of reaction is catalysed by a very labile enzyme. Jensen and Gun-derson 79 isolated soil bacteria capable of decomposing organic nitro-compounds (e.g., dinitro-o-cresol), and confirmed elimination of the nitro-group as nitrite.Benzenesulphonic acid is also attacked by soil Pseudomonads withreplacement of the sulphonic acid group by hydroxyl.80(b) The chlorophenoxyacetic acid herbicides. Several workers 81-88 havedescribed the disappearance of the chlorophenoxyacetic acids when dilutesolutions (0.01 yo wlv) were percolated through soil.Almost as many speciesof organisms were isolated from these enrichment cultures. Audus s2(Bacterium globiforme group), Jensen and Petersen 84 (Flavobacteriumaquatile), Stapp and Spicher 85 (Flavobacterium peregrinum n. sp.), Rogoffand Reid B6 (Corynebacterium), and Steenson and Walker 87 (Achromobactersp.) have all studied the biology of this process. Evans and his collabora-tors,88? 90 in a study of the metabolic pathway of these herbicides, encounteredthe difficulty, mentioned by previous workers, of the poor growth and slowutilisation of the 2 : 4-dichlorophenoxyacetic acid (2 : 4-D) by these organisms,in liquid media containing mineral salts. With P-chlorophenoxyacetic acid(CPAA) this difficulty is not serious, with the Gram-negative motile rod-likeorganism isolated from soil under conifer litter.Evans and Smith 8878 W. C. Evans and J. R. Simpson, Biochem. J., 1953, 55, xxiv.7O H. L. Jensen and K. Gunderson, Nature, 1954, 175, 341; Acta Agric. Scand.,80 J. R. Simpson, MSc. Thesis, Univ. of Wales, 1954.*I P. S. Nutman, H. G. Thornton, and J. H. Quastel, Nature, 1945, 155, 498.88 L. J. Audus, Nature, 1950, 166, 356.83 A. S. Newman and J. R. Thomas, Proc. Soil Sci. Soc. Amer., 1950, 14,84 H. L. Jensen and H. I. Petersen, Acta Agric. Scand., 1962, 2, 216.135 C. Stapp and G. Spicher, Zentr. Bakt., 2 Abt., 1954, 108, 225.86 M. Rogoff and J. J. Reid, J. Bact., 1956, 71. 303.87 T. I. Steenson and N. Walker, Plant and Soil, 1956, 8, 17; J .Gen. Microbial.,8 8 W. C. Evans and B. S. W. Smith, Biochem. J., 1964, 67, xxx.1956, 0, 100.160.1957, 16, 14EVANS : OXIDATIVE METABOLISM OF AROMATIC COMPOUNDS. 293isolated 4-chloro-2-hydroxyphenoxyacetic acid,89 and 4-chlorocatechol,from CPAA cultures, and these presumed intermediates also obeyed thecriteria of sequential induction; recently, Evans and Moss have isolateda p-chloromuconic acid 91 (probably the cis-tram-isomer) as a product ofreaction of washed cells on 4-chlorocatechol, and there is evidence frominfrared spectroscopy of the formation of a related lactone (possibly p-chloro-muconolactone) .92 Biological specificity of the enzymes is again in evidencehere, since the particular isomer of p-chloromuconic acid actually isolatedwas not acted upon by the cells, inversion having presumably occurredduring the process.A point of interest, in view of the behaviour of the cellenzymes towards other substituents in the benzene nucleus, is that withCPAA, the halogen is not eliminated before ring-fission. Chloride-ion pro-duction occurs after the p-chloromuconic acid stage. Progress in the elucid-ation of the microbial metabolic pathway of 2 : 4-D has been slow ; cultureswere shown 93 chromatographically to contain traces of 2 : 4-dichlorophenol,3 : 5-dich1orocatecho1, and a hydroxyaromatic acid with a different RF from2 : 4-dichloro-6-hydroxyphenoxy-acetic acid." Scheme 5 shows intemedi-ates identified to date.HCC I CI CIand furtherO.CH,.CO,H OH i" metabolismunidentifiedr ing- cl e avageC l o - _ _ - + C l o / _3 C'I$OH / _tICI CL CII productsSCHEME 5.Oxidative metabolic pathway of the chlorophenoxyacetic acids bymicro-organisms.(c) Naphthalene and certain halogenated derivatives. Naphthalene and itsderivatives have had limited use as soil insecticides, and TattersfieldQ5studied their persistence in various soils. Since then, numerous authorshave isolated organisms which grow in a medium containing naphthaleneand mineral salts; Strawinski and Stoneg6 in 1943 isolated salicylic acidfrom such cultures. Walker and Wiltshire 97u isolated (+)-trans-1 : 2-di-hydro-1 : 2-dihydroxynaphthalene and salicylic acid from naphthalenecultures of an aerobic motile Gram-negative rod (untyped) , and showed thatthese presumed intermediates also obeyed the criteria of sequential induction.a9 J.P. Brown and E. B. McCall, J., 1955, 3681.90 W. C. Evans and P. Moss, Biochem. J., 1957, 65, SP.9 1 J . Boeseken and C. F. Metz, Rac. Trav. chim., 1935, 54, 345.92 0. Neunhoeffer, Ber., 1935, 68, 1774.ga B. S. W. Smith, Ph.D. Thesis, Univ. of Wales, 1954.94 G. VV. K. Cavil1 and D. L. Ford, J., 1964, 665.95 F. Tattersfield, Ann. Apfil. Biol., 1928, 15, 67.O 6 R. J. Strawinski and R. W. Stone, J . Bact., 1943, 45, 16.97 N. Walker and G. H. Wiltshire, (a) J . Gen. Microbial., 1953, 8, 273; ( b ) ibid.,1955, 12, 478294 BIOLOGICAL CHEMISTRY.The formation of the (+)-tram-diol by micro-organisms has its analogy inthe naphthalene metabolism of mammals, where both (+)- and (-)-isomersare formed.98 They 97b also studied the breakdown of l-chloronaphthaleneby a suitable organism, identifying 8-chloro-1 : 2-dihydro-1 : 2-dihydroxy-naphthalene and 3-chlorosalicylic acid as intermediates.H OHcarechol pathwayCO2H (schemes 1 3nd 2)further metabolized(patthway unknown)SCHEME 6.Oxidative metabolic pathway 01 naphthalene by micro-organisms.The stages between naphthalenediol and salicylic acid remain to be dis-covered ; results with the halogenated hydrocarbon have, however, estab-lished that Cc4) in 8-chloro-1 : 2-dihydro-1 : 2-dihydroxynaphthalene be-comes the carboxyl carbon atom of 3-chlorosalicylic acid. Scheme 6 depictsour knowledge of naphthalene metabolism.W. C. E.3.ORGANOPHOSPHORUS COMPOUNDS AND ESTERASES.Abbreviations used throughout this Report are given below.Before 1939 many organophosphorus compounds were prepared bySchrader in Germany, and the earliest account of their toxicity was pub-lished in 1932.2 These substances are amongst the most toxic known andlittle information was available about their biochemical properties untilafter the war, because of their potential use as chemical-warfare agents.They are used widely in agriculture as insecticides, but they have alsoaroused the interests of chemists and biochemists. Many are powerfulinhibitors of esterases, reacting stoicheiometrically by the straight-forwardkinetics of a bimolecular reaction. The bimolecular rate constants may beas high as 6.3 x lo7 (1. mole-l min.-l a t 25" at pH 7.4) for the reaction ofisopropyl methylphosphonoff ~ o r i d a t e .~ This Report will consider tE emechanism of inhibition by these compounds and of reactivation of theinhibited enzyme, attempts to characterise chemically the active centre ofesterases, and the enzymic hydrolysis of anti-esterase organophosphoruscompounds.Some organophosphorus compounds, e.g., 00-diethyl O-9-nitrophenylphosphorothionate and octamethylpyrophosphoramide, are only weakinhibitors of esterases, but are toxic owing to their conversion by mammals98 Cf. L. Young, Biochenz. SOC. Symp., No. 5, 1950, p. 27; E. Boyland, ibid., p. 40.1 The following abbreviations have been used throughout : Diisopropyl phosphono-fluoridate, DFP ; Tetraethyl pyrophosphate, TEPP ; Diethyl p-nitrophenyl phosphate,E600.* W. Lange and G. Kruger, Ber., 1932, 65, 1698.3 B. J. Jandorf, J . -4grir. Food Chem., 1956, 4, 853ALDRIDGE ORG.4NOPHOSPHORUS COMPOUNDS ANL) ESTEHASES. 295and insects into substances which inhibit cholinesterase. This aspect willnot be considered here and has recently been reviewed.4TABLE 1. Enzymes inhibited by organophosphorus compozcnds.EnzymeCh ymotrypsinTrypsinCholinesterase (true andLiver esterase and milk lipaseAce t yle s terase (orange andpseudo)wheat B-esterase)Cholesterol esteraseOrganophosphorus compoiindDiisopropyl phosphorofluoridateDiphenyl phosphorochloridateDiethyl phosphorofluoridothionateTetraisopropyl pyrophosphateTetrapropyl dithionopyrophosphateTetraethyl pyrophosphateDiethyl p-nitrophenyl phosphateDiisopropyl phosphorofluoridateTetraethyl pyrophosphateDiethyl p-nitrophenyl phosphateOS-diethyl O-p-nitrophenyl phosphorothiolateDiisopropyl phosphorofluoridateDiisopropyl phosphorofluoridate and analogues11 Organophosphorus compoundsDiisopropyl phosphorofluoridateTetraethyl pyrophosphateDiisopropyl phosphorofluoridateTetraethyl pyrophosphateDiisopropyl phosphorofluoridateDiethyl p-nitrophenyl phosphateDiethyl p-nitrophenyl phosphateDiisopropyl p-nitrophenyl phosphateDiisopropyl phosphorofluoridateRef.56,#JP7897101 ,1112131415iii,,.17,,,,Mechanism of Anti-esterase Action.-Organophosphorus compounds aregeneral inhibitors of enzymes which possess carboxylic esterase activity.However, not all esterases are inhibited by organophosphorus compounds,e.g., A-esterase which hydrolyses P-nitrophenyl esters of carboxylic acids.16118It was apparent that theinhibition of esterases was in some ways different fromthat of inhibitors previously studied.It was shown by Jansen, Fellowes-Nutting, Jang, and Balls for chymotrypsin and by Boursnell and Webb 19 forpseudocholinesterase that the phosphorus from the inhibitor was extremelytightly bound and was not removed by treatment with trichloroacetic acid.The inhibited chymotrypsin could be repeatedly recrystallised without losingany phosphorus. When crystalline chymotrypsin is inhibited by DFP,74 J. E. Casida, J . Agric.Food Chem., 1956, 4, 772.6 E. F. Jansen, M. D. Fellowes-Nutting, R. Jang, and A. K. Balls, J . Bid. Chent.,6 E. F. Jansen, A. L. Curl, and A. K. Balls, ibid., 1951, 190, 557.7 E. F. Jansen. M. D. Fellowes-Nutting, R. Jang, and A. K. Balls, ibid., 1950,8 B. S. Hartley and B. A. Kilby, Nature, 1950, 166, 784.B E. F. Jansen and A. K. Balls, J . Biol. Chem., 1952, 194, 721.1949, 179, 189.185, 209.10 B. A. Kilby and G. Youatt, Biochim. Biophys. Acta, 1952, 8, 112.11 A. Mazur and 0. Bodansky, J . BioE. Chem., 1946, 163, 261.12 J . F. Mackworth and E. C. Webb, Biochem. J., 1948, 42, 91.1s W. N. Aldridge, ibid., 1953, 53, 62.14 E. C. Webb, ibid., 1948, 42, 96.18 E. F. Jansen, M. D. Fellowes-Nutting, and A. K. Balls, J . B i d . Chem., 1948,16 W.N. Aldridge, Biochem. J., 1953, fi3, 110.17 D. K. Myers, A. Schotte, H. Boer, H. Borsje-Bakker, ibid., 1955, 61, 621.1) R. Goutier, Biochirn. Biophys. Actu, 19S6, 19. 624.19 J. C. Boursnell and E. C. Webb, Nature, 1949, 164, 875.175, 975296 BIOLOGICAL CHEMISTRY.by TEPP,2O or by one molecule of acid is liberated for each moleculeof enzyme inhibited. This has also been shown to be true for crystallinetrypsin inhibited by E600.1° One molecule of phosphorus is bound to onemolecule 0: enzyme 6* ‘ 9 99 21*22 and in every case the rate of reaction ofinhibitor with enzyme shows the characteristics of a bimolecular re-action.l5~ 22-26 Pure crystalline cholinesterases are not available and soindirect methods have had to be used to gain information on their mechanism.After inhibition of cholinesterases by DFP containing 32P the phosphorus isfirmly bound to the enzyme.19*28-30 From the effect of temperature uponthe rate of reaction, the apparent energies of activation are 10-1 1 kcal./molefor true cholinesterase and E600,27 and 14-15 kcal./mole for pseudo-cholinesterase and NN’N”N”’-tetraisopropylpyrophosphorotetramide.26The evidence so far agrees with phosphorylation of the enzyme and not withsimple absorptive or ionic binding as the mechanism of inhibition for allcarboxylic esterases by organophosphorus compounds.A mechanism has been suggested by Nachmansohn and Wilson31 forthe hydrolysis of acetylcholine by cholinesterase which may be representedas follows :Ac - [ F i - - T C ] + (2) ChOH + + AcOH b c A F’ - -t L h ----Ch + HsO= enzyme, Ac-CH = acetylcholine, ChOH = choline, AcOH = acetic acid.In this process the formation of the acetylated enzyme (reactions 1 and 2)is the rate-limiting step, while its hydrolysis (reaction 3) is comparativelyfast.The inhibition of cholinesterase by organophosphorus compoundswould fit into such a scheme if the phosphorylated enzyme were ~table,3~-34the rate-limiting step being its hydrolysis. It appears that the inhibitorscombine with the enzyme active centre, for the inhibition is easily preventedby the presence of substrate.23* 35-37 Wilson 32 showed that the activity oftrue cholinesterase inhibited by TEPP slowly returned, and Aldridge 2720 A. K. Balls and E. F. Jansen, Adv.Enzynzol., 1952, 13, 321.11 E. F. Jansen, M. D. Fellowes-Nutting, and A. K. Balls, J . Biol. Chem., 1949,179, 201.na B. J. Jandorf, H. 0. Michel, N. K. Schaffer, R. Egan, and W. H. Summerson,Discuss. Faraday Soc., 1955, 20, 134.2s W. N. Aldridge, Biochem. J., 1950, 46, 451.94 Idem, ibid., 1954, 57, 692.86 W. N. Aldridge and A. N . Davison, ibid., 1962, 51, 62.aa A. N . Davison, ibid., 1956;, 60, 339.27 W . N . Aldridge, ibid., 1963, 54, 442.28 H. 0. Michel and S. Krop, J . Biol. Chew., 1961, 190, 119.29 H. 0. Michel, Fed. Proc., 1952, 11, 269.30 E. F. Jansen, R. Jang, and A. K. Balls, J . BioZ. Chem., 1952, 196, 247.*1 D. Nachmansohn and I. B. Wilson, A&. EnzymoE., 1961, 12, 259.32 I. B. Wilson, J . Biol. Chern., 1951, 190, 11.8s Idem, ibid., 1951, 199, 113.34 W.N . Aldridge, Chem. and Ind., 1954, 473.36 J. A. Cohen, M. G. P. J. Wamnga, and B. R. Bovens, Biochim. Biopbys. Ada.3% F. Hobbiger, Brit. J. Phaumacol., 1964, 9, 169.87 A. S. V . Burgen, ibid., 1949, 4, 219.1961, 6, 469ALDRIDGE ORGANOPHOSPHORUS COMPOUNDS AND ESTERASES. 297demonstrated that true cholinesterase inhibited by dimethyl fmitrophenylphosphate produced an inhibited enzyme which was spontaneously reactiv-ated following first-order kinetics, and having a half-life at 37" and pH 7.6of approximately 1.5 hr. The energy of activation of this process was1 6 1 5 kcal./mole. The rate of return of enzyme activity is identical afterinhibition by dimethyl P-nitrophenyl phosphate , dimethyl phosphoro-fluoridate, tetramethyl pyrophosphate, and 00-dimethyl S-$-nitrophenylphosphorothi0late,3~ indicating that in each case a dimethyl phosphorylatedenzyme is produced. Diethyl phosphorylated rat pseudo-cholinesterase isalso unstable s9 and a similar examination has been made.38It is clear therefore that the enzyme is dialkyl phosphorylated, and thisis linked to the enzyme by a covalent bond which may be hydrolysed.Itremains to examine how far the behaviours of esterases with organophosphorusinhibitors and with substrates are similar. With a series of diethyl phenylphosphates where the lability to hydrolysis has been modified by sub-stituents in the aromatic ring, the inhibitory power was directly related totheir rate of hydrolysis under standard conditions.25* 40 This is consistentwith hydrolysis of the phosphate ester being an essential part of the in-hibitory process, though it must be emphasised that such a correlation mayonly be demonstrated when a series of closely related analogues are examined.The scheme given above for the hydrolysis of acetylcholine provides for twosites in the enzyme active centre-an anionic site to bind the quaternarynitrogen atom, and an esteratic site to which the carbonyl oxygen isatta~hed.~l On these grounds it would be expected that variation of thealkoxy-groups attached to the phosphorus would influence the inhibitorypower. In fact, changes of the alkoxy-groups of organophosphorus com-pounds from 00-dimethyl to 00-diisopropyl alter their inhibitory power indifferent directions against true and pseudo-cholinesterase.13 Similarly,lengthening the acyl group of choline esters changes their rates of hydrolysisby true and pseudo-cholinesterase and there is a good parallelism between thesubstrate and inhibitor specificities of these enzymes.13 The esteratic siteis clearly important in determining inhibitor potency.Several compoundshave been prepared containing a quaternary nitrogen atom in the labilegroup. The phosphostigmines m(dialkoxyphosphiny1oxy) -NNN-trimethyl-anilinium methyl sulphate are highly active inhibitors of cholinesterase 4 1 ~ 4 2and the quaternary salts are more active than the tertiary compounds.42~*Methylation of 00-diethyl S-ethylthioethyl phosphorothiolate to produce thesulphonium derivative increased its inhibitory power one hundred-fo1d.u3-(Diethoxyphosphinyloxy)-l-methylquinolinium methyl sulphate is anextremely powerful inhibitor of true cholinesterase (50% inhibition at 37"for 20 min.by 1.5 x 10-lo~) and the quaternary is more active than the36 W. N. Aldridge and A. N. Davison, Biochem. J., 1963, 55, 763.38 A. N. Davison, ibid., 1953, 54, 5S3.40 W. N. Aldridge and A. N. Davison, ibid., 1962, 52, 663.41 A. S . V. Burgen and F. Hobbiger, Brit. J . Pharmacol., 1951, 6, 593.42 K. J. M. Andrews, F. R. Atherton, F. Bergel, and A. L. Momson, J., 1962, 780.43 F. Hobbiger. Chem. and Ifid.. 1954, 1574.44 J. R. Fukuto, R. L. Metcalf, R. B. Marsh. and M. Maxon, J . Amev. Chem. Soc.,1055, 77, 3670298 BIOLOGICAL CHEMISTRY.tertiary comp~und.~~~ 45 Although changing the tertiary atom to aquaternary atom will also influence the stability of the compound, alterationsof the magnitude just described indicate that the anionic site can be utilisedin the inhibitory process.Both factors, lability to hydrolysis and ( ( fit ”upon the enzyme surface, are important as they undoubtedly are with sub-strates. Thus acetylthiocholine is hydrolysed by true cholinesterase at ahigher rate than a~etylcholine.~~ The structures of these compounds are sosimilar that it is unlikely that the ( ( fit ” upon the enzyme active centre isdifferent, but acetylthiocholine is more unstable to hydroly~is.~~ Althoughthese two factors play a part, an inhibitor, di-n-propyl 2 : 2-dichlorovinylphosphate, has been recently e~amined,~ whose activity is difficult to explain.This substance is very stable to hydrolysis, does not react with catechol orpicolinohydroxamic acid (see section on reactivation), and does not possess astructure which would be regarded as in any way resembling that of acetyl-choline.Reaction (I), the formation of the hypothetical reversible complexbetween inhibitor and enzyme, has not been demonstrated.Such a re-versible complex between DFP and cholinesterase was originally claimed tohave been dem~nstrated,~’ but this has now been Thisfailure to detect the reversible complex is not surprising, for enzyme-substrate complexes have been detected onlyIt should be noted that phosphorus is not an essential constituent of amolecule which will produce an inhibition similar to that described above.Myers and Kemp 51 have shown that a variety of organic acid fluorides willinhibit cholinesterase, e.g., NN-dimethylcarbamoyl fluoride, chloromethane-sulphonyl fluoride, and toluene-9-sulphonyl fluoride.Earlier, kineticstudies on the inhibition of cholinesterase by [2-(NN-dimethylcarbamoyl-oxy)-5-phenylbenzyl] trimethylammonium bromide, which had always beenconsidered a typical reversible inhibitor, had indicated that the inhibitionpassed through a stage analogous to the phosphorylated enzyme, Le., thedimethylcarbamoyl-enzyme which is readily hydrolysed to the originalenzyme.52 Myers has now shown that NN-dimethylcarbamoyl fluorideproduces a similarly unstable inhibited enzyme, whereas NN-diethyl-carbamoyl fluoride produces a stable inhibited enzyme and behaves similarlyto organophosphorus inhibitor^.^^ The isolation of chymotrypsin acetate,an intermediate in the hydrolysis of p-nitrophenyl acetate by chymotrypsin,is also of interest (see (‘ degradation of inhibited enzymes ”).Reactivation of the Phosphorylated Enzyme.-For many years the inhibi-tion of esterases by organophosphorus compounds was considered to beirreversible.However, Wilson showed that inhibition of electric-eel5045 K. J. M. Andrews, F. R. Atherton, F. Bergel, and A. L. Morrison, J., 1954, 1638.46 G. B. Koelle, J . Pharmacol., 1950, 100, 158.47 D. Nachmansohn, M. A. Rothenberg, and E. A. Feld, Arch. Biochem., 1946, 14,49 D. Keilin and T.Mann, R o c . Roy. Soc., 1937, B, 122, 119.50 B. Chance, Acta Chem. Scand., 1947, 1, 236.51 D. K. Myers and A. Kemp, Nature, 1954, 173, 33.61 D. K. Myers, Biochem. J . , 1952, 62, 46.53 Idem, ibid.. 1956, 62, 556.197.T. B. Wilson and M. Cohen, Biochim. Biophys. Acta, 1953, 11, 147ALDRIDGE : ORGANOPHOSPHORUS COMPOUNDS AND ESTERASES. 899cholinesterase by TEPP produced an inhibited enzyme which was slowlyreactivated on st0rage.~2 Later it was found that the dimethyl phosphoryl-ated true cholinesterase z7 and the diethyl phosphorylated pseudo-cholin-esterase 39 were much more unstable. Hestrin had previously shown thatcholinesterase will catalyse the formation of acetohydroxamic acid fromacetylcholine and hydroxylamine,54 and Wilson has demonstrated that therate of the spontaneous reactivation of the diethyl phosphorylated enzymecould be increased by hydroxylamine and also by choline.32 TEPP anddiethyl phosphorofluoridate both produced an inhibited enzyme which wasreactivated at the same rate by hydroxylamine, but the enzyme inhibitedby DFP was much more stable.% Diethyl phosphorylated chymotrypsinmay also be partially reactivated by hydr~xylamine,~~* 56 This demon-stration, that in addition to its slow reactivation by hydrolysis, the inhibitedenzyme could be reactivated much faster by hydroxylamine, led to a searchfor more powerful nucleophilic reactivators.A large variety of hydroxamicacids and oximes have now been examined.221 57-69 Pyridine-2-aldoximemethiodide,'* 67 picolinohydroxamic acid,63 bishydroxyiminoacetone andmonohydroxyiminoacetone 579 6O are particularly effective.This reactiv-ation by water,27 by hydr~xylamine,~~~ 56 or by bishydroxyiminoacetone e,~is temperature-dependent and has a high energy of activation. Reactivationby hydroxylamine may be prevented by the addition of positively chargedions such as tetramethyl- or tetraethyl-ammonium,% Trimethylamine willslow down reactivation by nicotinohydroxamic acid methiodide and pyridine-2-aldoxime methiodide,61* 62 and acetylcholine that by pyridine-2-aldoximemethiodide. 67 Such results are entirely consistent with attachment of thereactivator at the anionic site of cholinesterase.68 Although a large numberof oximes which are not positively charged are effective,57 the statementthat the tertiary pyridine-2-aldoxime is a million times less effective thanthe quaternary methiodide for the reactivation of diethyl phosphorylatedcholinesterase (electric eel) 84 is difficult to explain except on the basis of theparticipation of the anionic site.This does not preclude the possibility thatreactivators may act without attachment to the anionic site. Similarsituations exist in the hydrolysis of such esters as triacetin by cholinesterase 70and in the high inhibitory power of DFP for cholinesterase. Reactivation byS. Hestrin, J . Biol. Chem., 1949, 180, 879.~55 L. W. Cunningham and H. Neurath, Biochim. Biophys. Acta, 1953, 11, 310.56 L. W. Cunningham, J . Biol. Chem., 1954, 207, 443.I 7 A. F. Childs, D. R.Davies, A. L. Green, and J. P. Rutland, Brit. J . Pharmacol.,s8 I. B. Wilson, J . Amer. Chew. SOC., 1955, 77, 2383.59 I. B. Wilson and E. K. Meislich, ibid., 1963, 75, 4628.6o D. R. Davies and A. L. Green, Biochem. J., 1956, 63, 529.61 F. Hobbiger, Brit. J . Pharmucol., 1955, 10, 356.6s I. B. Wilson and S. Ginsburg, Arch. Biochem. Biophys., 1955, 54, 569.64 Idem, Biochim. Biophys. Acta, 1955, 18, 168.66 H. Kewitz and I. B. Wilson, Arch. Biochem. Biophys., 1956, 60, 261.67 D. R. Davies and A. L. Green, Discuss. Furuduy Soc., 1955, 20, 269.6a I. B. Wilson, ibid., p. 119.68 B. J. Jandorf, E. A. Crowell, and A. P. Levin, Fed. Proc., 1955, 14, 231.7O D. H. Adams, Biochim. Biophvs. Acta, 1949, 8, 1 .1955, 10, 462.Idem, ibid., 1956, 11, 295.I. B.Wilson, S. Ginsburg, and E. K. Meislich, J . Amer. Chew. Soc., 1955, 77, 4286300 BIOLOGICAL CHEMISTRY.ammonium molybdate is not prevented by trimethylamine,62 and is prob-ably related to the catalysis of the hydrolysis of '' energy-rich " phosphatecompounds 71-73 and also of inhibitors such as DFP.74While reactivation of diethyl phosphorylated cholinesterase has beenrelatively easy to demonstrate, the enzymes inhibited by DFP were muchmore difficult to reactivate 66 It has now been establishedthat this is due to an alteration in the inhibited enzyme and not to experi-mental artifacts. The ease of reactivation of diisopropyl phosphorylatedcholinesterase is related to the time of incubation of the inhibited enzymebefore reactivation.22* so* 61* 62* 68 In most experiments the enzyme has beenincubated in the presence of the inhibitors, but the change is not due tosecondary reactions of the inhibitor with the enzyrne.e2 Experimentalartifacts are eliminated since the change can occur in vivo.Hobbiger62has shown that a human myasthenic after repeated doses of TEPP hadalmost all his blood cholinesterase activity inhibited and in a non-reactivat-able form. This is also true after prolonged administration of dimethylphosphorus inhibitors to rats.7s The conversion of the reactivatable intothe non-reactivatable inhibited enzyme occurs most rapidly withtrue cholinesterase inhibited by 00-dimethyl, 00-diisopropyl, 0-iso-propyl methyl, and is much slower after 00-diethyl phosphorus com-pounds.22* 80, 68v 69* 76 This difference in ease of conversion is unexplained ;it is unexpected both on chemical grounds and when account is taken of theknown substrate specificity of the enzymes.More information is requiredon the rate of transformation for a series of inhibitors and several enzymesof different substrate specificities. It has been demonstrated that diiso-propyl phosphorylated pseudo-cholinesterase (human) is transformed morerapidly than the corresponding inhibited true cholinesterase (human) .61$62In general, the transformation is temperature-dependent 22s 5 8 s 60 and forthe methyl 0-isopropyl phosphorylated cholinesterase (true) it is markedlycatdysed by acid.60Reactivation studies have been almost all upon inhibited cholinesterases.Diethyl phosphorylated chymotrypsin can be partially reactivated byhydroxylamine 55, 513 and diisopropyl phosphorylated chymotrypsin slowlyby nicotinohydroxamic acid methiodidegg There has so far been no con-clusive demonstration of a progressive transformation of a reactivatable intoa non-reactivatable inhibited enzyme with enzymes other than the cholin-esterases.The difference between these two forms of cholinesterase is atthe moment absolute-one form may be reactivated, whereas the othercannot even with the most effective reactivators or by ammonium molybdate.Recently, several papers have been published showing that these re-activators react readily with the inhibitors themselves. Hydroxylaminereacts with a variety of inhibitor^,'^ and detailed studies have been made of7 1 F.Lipmann, Adv. Enzymol., 1941, 1, 112.7' F. Lipmann and L. C. Tuttle, J . BioZ. Chem., 1944,158, 671.T. Winnick and E. M. Scott, Avck. Biochem., 1947, 12, 201.74 T. Wagner-Jauregg. B. E. Hackley, T. A. Lies, 0. 0. Owens, and R. Proper, J .75 M. Vandekar, Biochervt. J., 1957, 65, 1 ~ .76 B. J. Jandorf, J . Amer. Chem. SOC., 1966, 78, 3686.Amer. Chem. SOC., 195s. 77, 922ALDRIDGE : ORGANOPHOSPHORUS COMPOUNDS AND ESTERASES. 301the reaction of hydroxamic acids and oximes with organophosphorus com-pounds. 7-79Degradation of the Inhibited Enzymes,-The straight-forward chemicalreaction of organophosphorus compounds with esterases and the theory thatthey react as substrates for the enzyme, but that the dialkylphosphorusmoiety remains attached to or near to the active centre, have led to attemptsto degrade the enzyme to obtain information of the chemical structure andarchitecture of the active centre.The use of inhibitors containing 32P madeit easier to trace the fragment to which the phosphorus was attached. Forthis work it is essential to have the enzyme of reasonable purity so that forthis aspect of the problem cholinesterase has been little used.When recrystallised diisopropyl phosphorylated chymotrypsin is hydro-lysed by acid, phosphoserine can be isolated from the hydrolysates.80s *lPhosphoserine can also be isolated from diisopropyl phosphorylated pseudo-cholinesterase, liver ali-esterase, trypsin, red-cell ali-esterase, and red-cellcholinesterase.82 It was realised that the radioactive phosphorus may havemigrated during the drastic acid hydrolysis.s0* 81 That this was a distinctpossibility was shown when it was found that a diisopropyl phosphoryl groupon the amino-group of serine will easily migrate to the hydroxy-group,a andthat serine phosphate is a very stable substance. Cohen and his co-workershave used much less drastic methods of degradation. After degradation ofdiisopropyl phosphorylated chymotrypsin with proteolytic enzymes, apeptide has been isolated containing one molecule each of proline, leucine,aspartic acid, and serine, and 2-3 molecules of glycine per diisopropylphosphoryl group.Alkaline hydrolysis of the peptide yielded diisopropylphosphate, whereas acid hydrolysis gave serine phosphate.This work hasbeen extended to diisopropyl phosphorylated trypsin and liver esterase andsimilar peptides were obtainedSa6 A similar result has been obtained withtrypsin inhibited by DFP, with %-labelled isopropyl groups.S6 Althoughthe diisopropyl phosphoryl group has been shown to be attached to serine,many workers believe that the group has migrated since the initial in-hibitory process. Chemically the hydroxyl group of serine does not reactwith DFP 87 and the only amino-acids reacting at all readily with DFP arehistidine 8a and tyrosine.8e Photochemical oxidation of chymotrypsin inthe presence of methylene-blue leads to the loss of one molecule of histidine77 R. Swidler and G. M. Steinberg, J .Amer. Chem. SOC., 1956, 78, 3594.18 B. E. Hackley, P. Plapinger, M. Stolberg, and T. Wagner-Jauregg, ibid., 1966,7O A. L. Green and B. Saville, J., 1966, 3887.80 N. K. Schaffer, S. C. May, and W. H. Summerson, J . Bid. Chem., 1963, m, 69.81 Idem, ibid., 1954, 206, 201.82 J. A. Cohen, R. A. Oosterbaan, M. G. P. J. Warringa, and H. S. Jansz, Discuss.83 P. Plapinger and T. Wagner- Jauregg, J . Amer. Chem. SOC., 1953, 75, 6767.82 R. A. Oosterbaan, P. Kunst. and J. A. Cohen, Biochim. Biofikys. Acta, 1965,16,299.85 R. A. Oosterbaan. H. S. Jansz, and J. A. Cohen, ibid., 1966, 20, 402.86 G. H. Dixon, S. Go, and H. Neurath, ibid., 1956, 19, 193.87 T. Wagner-Jauregg, J. J. O’Neill, and W. H. Summerson, J . Amev. Chem. SOC.,88 T. Wagner- Jauregg and B.E. Hackley, ibid., 1953, 75, 2126.89 R. F. Ashbolt and H. N. Rydon, ibid., 1952, 74, 1865.So L. Weil, S. James, and A. R. Bucbert, Arch. Biochem. Biophys., 1963, (8, 266.77, 3651.Faraday SOC., 1965, 20, 114.1951, 73, 5202302 BIOLOGICAL CHEMISTRY.(out of a total of 2) and approximately 3 of 7 the tryptophan residues.22This photo-oxidised chymotrypsin failed to react with DFP. Also diiso-propyl phosphorylated chymotrypsin was more difficult to oxidise in thisway. From the variation of cholinesterase activity with pH, it has beensuggested that the acylated or phosphorylated site in the enzyme may be thebasic nitrogen atom of the glyoxaline ring in h i ~ t i d i n e , ~ ~ and, by using similarcriteria, histidine has been implicated in the catalytic activity of pseudo-cholinesterase, chymotrypsin, trypsin, and liver e ~ t e r a s e .~ ~ - ~ ~ Hartley andKilby 96 observed that the hydrolysis of 9-nitrophenyl acetate by chymo-trypsin was a two-phase process and was inhibited by organophosphoruscompounds. This has now been clearly demonstrated to be a primaryformation of acetylchymotrypsin followed by a slow hydrolysis of thisintermediate ; the acetylchymotrypsin has been 98 No evidencefor the formation of acetylglyoxaline was obtained by measuring the differ-ence in the ultraviolet spectra of acetylchymotrypsin and chymotryp~in.~~Gutfreund has also concluded from kinetic studies that the evidence pointsto an acetylation of the hydroxyl group of serine followed by its transferto histidine.lW Histidine catalyses the hydrolysis of DFP, and it has beenshown that diisopropyl phosphorylglyoxaline is highly unstable.88 All thisevidence explains the attraction of the idea that histidine is involved in thehydrolysis of substrates by esterases.The suggestion of a transfer of the phosphorus moiety from one group toanother on the enzyme surface has been correlated with the change ofinhibited cholinesterase from a reactivatable to an irreversible stage.61However, such a change has only been conclusively demonstrated for theinhibited cholinesterase.It is, of course, possible that this change occursextremely rapidly with chymotrypsin and trypsin. Only recently havepeptides containing histidine been isolated. Hydrolysis with 12~-hydro-chloric acid at 37" for 3 days of methyl O-isopropyl phosphorylated chymo-trypsin has yielded a peptide containing glycine, aspartic acid, methyl-phosphonylserine, glutamic acid, alanine, and valine.A papain digest ofthe same enzyme gave a peptide containing, in addition to these amino-acids,histidine, proline, leucine, cystine, and threonine.lo1, lo2 It appears from thepeptides so far isolated, that the histidine is some distance from serine. Iftransfer takes place, then it is reasonable to suppose that it must be becausethe peptide chains are so folded that the two amino-acids are in juxta-position. Dixon and Neurath lo3 have attempted to prevent such a migra-91 I. B. Wilson and F. Bergmann, J . Biol. Chem., 1950, 186, 683.92 K. J.Laidler, Trans. Faraday SOC., 1955, 51, 560.93 H. Gutfreund, ibid.. p. 441.94 F. Bergmann, R. Segal, A. Shimoni, and M. Wurzel, Biochem. J., 1966, 63, 684.95 B. R. Hammond and H. Gutfreund, ibid., 1966, 61, 187.96 B. S. Hartley and B. A. Kilby, ibid., 1952, 50, 672.9 7 A. K. Balls and F. L. Aldrich, Proc. Nat. Acad. Sci., U.S.A., 1955, 41, 190.913 A. K. Balls and H. N. Wood, J . Biol. Chem., 1956, 219, 245.99 G. H. Dixon, W. J. Dreyer, and H. Neurath, J . Amer. Chem. SOC., 1956,78, 4810.100 H. Gutfreund and J. M. Sturtevant, Biochem. J., 1956, 83, 656.101 N. K. Schaffer, S. Harshman, R. R. Engle, and R. W. Drisko, Fed. Proc., 1955,102 N. K. Schaffer, R. R. Engle, L. Simet, R. W. Drisko, and S . Harshman, ibid.,108 G. 13. Dixon and H. Neurath, Biochim.Biophys. Acta, 1966, 20, 672.14, 275.1956, 15, 347ALDRIDGE ORGANOPHOSPHORUS COMPOUNDS AND ESTERASES. 303tion in trypsin by treatment with urea immediately after inhibition. Nodifference in labelling by 32P from DFP was found between an inhibitedenzyme so treated and a control. No reaction between trypsin and DFPoccurred in the presence of 8~-urea. Evidence has been given that E600will react with trypsin in 8 M - ~ r e a , ~ ~ * l O ~ but other workers have failed toconfirm these observations.lo6The work described in this section may be summarised : From a varietyof inhibited enzymes, serine phosphate has been isolated. On the basis thatthe phosphate moiety was attached to the serine before degradation of theprotein and, since the enzyme is inhibited, this serine is very near the activecentre, it appears that the amino-acid composition around the active centreof a variety of esterases is very similar.On chemical grounds it is notgenerally considered that the phosphorus is primarily attached to the serine,but attempts to show a two-stage process for enzymes other than cholin-esterases have so far been unsuccessful.Hydrolysis of Organophosphorus Compounds by Enzymes.-Mazur lo7 firstdemonstrated that DFP was hydrolysed by enzymes present inmammals. Since then, the enzymic hydrolysis of a variety of organo-phosphorus compounds has been establi~hed-E600,~~~ lo8 ethyl NN-di-methylphosphoroamidocyanidate,lN* l10 TEPP,25s ll1 isopropyl methyl-phosphonofluoridate,ll28 113 and 3-(diethoxyphosphinyloxy)-l-methylquinol-inium methyl s ~ l p h a t e .~ ~A source of confusion in the examination of these enzymes has beentheir overlapping specificity. ll1 For instance, the enzyme which hydrolysesDFP present in rabbit plasma is not the same enzyme as that present in hogkidney.ll* Water-soluble enzymes of rat and hog liver are activated bymanganese and cobalt, and hydrolyse DFP and its di-rt-butyl analogue atthe same rate. The water-insoluble enzymes of these livers hydrolyse di-n-butyl phosphorofluoridate at a greater rate than DFP and are activated bycalcium and inhibited by manganese, cobalt, and magnesium.l15Of particular interest is the work of Mounter and his co-workers on theenzyme of hog kidney which hydrolyses DFP.This is activated by man-ganese and also by glyoxaline, histidine, and other metal-chelating agentssuch as 2 : 2'-dipyridyl.ll6-ll8 It was concluded 118 that only definitemolecular structures involving glyoxaline or pyridine derivatives activate thehydrolysis of DFP in the presence of manganese. These compounds mayeither prevent or inhibit the activation of the hydrolysis by cobalt; in thelo4 T. Viswanatha and I. E. Liener, J . Biol. Chem., 1955, 215, 777.l o 6 Idem, Nature, 1955, 176, 1120.lo6 J. I. Harris and B. S. Hartley, Biochim. Biophys. Acta, 1956, 21, 201.A. Mazur, J . Biol. Chem., 1946, 164, 271.108 W. N. Aldridge, Biochem. J., 1953, 53, 117.loQ K. B. Augustinsson, Biochim. Biophys. A d a , 1954, 13, 303.K. B. Augustinsson and G.Heimburger, A d a Chem. Scand., 1954, 8, 553.l11 Idem, ibid., p. 1533.I l 2 F. C. G. Hoskin, Canad. J . Biochem. Physiol., 1956, 34, 75.llS P. A. Adie, F. C. G. Hoskin, and G. S. Trick, ibid., p. 80.llP L. A. Mounter, J . Biol. Chem., 1954, 209, 813.116 L. A. Mounter, C . S. Floyd, and A. Chanutin, ibid., 1953, 204, 221.117 L. A. Mounter and A. Chanutin, ibid., 1953, 204, 837.ll@ Idem, ibid., 1954, 210, 219.Idem, ibid., 1955, 215, 705304 BIOLOGICAL CHEMISTRY.presence of cobalt, proline and hydroxyproline are effective activators.118These observations may be compared with the pure chemical studies on thenon-enzymic catalysis of the hydrolysis of DFP by metal chelate com-pounds. 74 Copper sulphate increases the rate of hydrolysis of DFP, whereasother metals such as iron (ferrous), palladium, chromium, nickel, and cobaltare inactive or only slightly active.On the other hand, copper complexesof amino-acids, glyoxaline, ethylenediamine, o-phenanthroline, and 2 : 2‘-dipyridyl are highly active. The half-life of DFP at pH 7.6 at 38”alone or in the presence of glyoxaline or copper sulphate or both is 2 days,5 hr., 5 hr., and 20 min., respectively. It is of interest that copper was byfar the most active metal and chelate compounds of nickel and cobalt weremuch less effective, and iron and manganese were inactive. For the enzymichydrolysis of DFP, manganese is the most active metal.ll6 With bidentatedonor groups, such as 2 : 2’-dipyridyl and ethylenediamine, the 1 : 1 chelatecompounds are more effective than the 1 : 2 compounds.74 Copper ions willalso catalyse the hydrolysis of thiophosphoric esters such as 00-diethylO-P-nitrophenyl phosphorothionate.119It was natural to attempt to find a more physiological substrate for theenzymes which hydrolyse such foreign substrates as DFP and E600. Froma variety of criteria, it was concluded that the enzyme in rabbit plasma whichwill hydrolyse E600 will also hydrolyse 9-nitrophenyl acetate, propionate,and butyrate,lo8 and this has recently been confirmed after electrophoreticseparation of the enzyme.18 The enzyme apparently cannot distinguishbetween carboxyl or phosphate esters. Recently, it has been stated thatthe enzyme in hog kidney which hydrolyses DFP, also hydrolyses acet-amido-acids, such as N-acetyl-valine, -leucine, -methionine, and -alanine. 120With these two enzymes, it seems that enzymes with esterase activity whichare not inhibited by organophosphorus inhibitors also hydrolyse them.Originally it was thought that the enzyme of rat pancreas was an exception;it is not inhibited by a variety of inhibitors such as TEPP, DFP, NN’N”N“’-tetraisopropylpyrophosphoramide, and NN-diisopropylphosphorodiamidicfluoride,24- 121 but is inhibited by E6U0.24 It was originally considered thatthis inhibition was reversible,24 but this has recently been disp~ted.1~9 121Conclusions.-Organophosphorus compounds do not inhibit enzymesother than carboxylic esterases.The inhibitory process may be representedas follows :H = enzyme, R-X = inhibitor with X the labile group, Re = reactivatable,ll@ J.A. A. Ketelaar, H. R. Gersmann, and M. M. Beck, Nature, 1956, 117, 392.1 2 O L. A. Mounter, Fed. Proc., 1966, 15, 317.lal P. Desnuelle, M. J. Constantin, and L. Sarda, BUZZ. SOC. CAim. b i d , 1956, 38, 625CALLOW : METABOLISM OF STEROIDS. 306The evidence is overwhelming, at least for chymotrypsin, trypsin, andtrue and pseudo-cholinesterase, that the organophosphorus inhibitors arehydrolysed (reaction 2) by the enzyme, but that the intermediate, thephosphorylated enzyme, is stable, whereas with natural substrates it isunstable. These esterases therefore do not distinguish between carboxylicand phosphate esters; this is also true for an enzyme which hydrolysesE600 and 9-nitrophenyl acetate, and another hydrolysing DFP and acet-amido-acids.Although it is likely that this reaction scheme applies to thesetwo enzymes no direct evidence has as yet been obtained. This inhibitoryprocess indicates that it should be possible to find organophosphorus com-pounds which act as substrates, the phosphorylated intermediate beingsufficiently unstable. This has been partially achieved with dimethyl-phosphorus inhibitors and true cholinesterase. Although the factors of" fit " (reaction l), stability of inhibitor, and stability of the esterified inter-mediate (reaction 4) are well appreciated for organophosphorus compounds ,they could be more fully considered for substrates. There is no cleardemarcation between substrates and inhibitors of this type and, betweenthese two extremes, there is a spectrum of behaviour.The catalysis of the hydrolysis of DFP by histidine and various copperchelate compounds probably fits into the above reaction scheme.There isalso much circumstantial evidence that histidine participates in the hydro-lytic process of esterases.On degradation of a variety of inhibited esterases the phosphorus isfound to be attached to serine. All attempts to demonstrate phosphorusattached to other groups have so far failed. Treatment of cholinesterasewith nucleophilic reagents has shown that the inhibited enzyme changes froma reactivatable (reaction 4) to an irreversible stage (reaction 3). Furtherwork will undoubtedly tell us of the properties of this irreversible inhibitedenzyme and if such a transformation occurs with other esterases.W.N. A.4. METABOLISM OF STEROIDS.A tremendous volume of work continues to be published in this field.Much deals with experimental techniques-a fact significant of the diffi-culties of the analytical investigation of minute amounts of substances intissues, blood, and excreta. There is no lack of reviews, and attention maybe directed to Zimmermann's booklet on chemical analytical methods, tothe general articles by Lieberman and TeichJ2 by Hechter and Pincus? repre-senting the Worcester Foundation, by Roberts and %ego: and by Rosen-kilde and S~hroeder,~" and to articles on special subjects, such as thebiosynthesis of ch~lesterol,~ hormone metabolites in human urine,6 and1 W.Zimmermann. " Cbemische Bestimmungsmethoden von Steroidhormonm inKorperfliissigkeiten," Springer-Verlag, Berlin, 1955. * S. Lieberman and's. Teich, Pharmacol. Rev., 1953, 5, 285.3 0. Hechter and G. Pincus, Physiol. Rev., 1954, 34, 459.4 S. Roberts and C. M. Szego, Ann. Rev. Biochem., 1955, 24, 543.4a H. Rosenkilde and W. Schroeder, 2. Vitamin-, Hormon-, u. Fermentforsch., 1956,6 R. T. Dorfman and F. Ungar, " Metabolism of Steroid Hormones," Burgess Publ.8, 132.Co., Minneapolis, 1954.J. W. Cornforth, Rev. Pure Appl. Chem., 1954, 4, 216306 BIOLOGICAL CHEMISTRY.steroid hormones and cancer.’ The enzymic hydroxylation of steroids wasreviewed in these Reports in 1955. Reports of conferences also allowthe investigations and speculations of the main centres of research to befollowed.General Trend of Investigation.-Interest in steroid-hormone metabolismhad its origin in the hope that, by analysis of the urine, there might beobtained an index of the hormone levels in the body, and hence assistance begiven to medical diagnosis in suspected endocrine disease.In fact onlygross deviations from the normal levels of excretion have diagnostic value, inmost cases simply confirmatory of clinical indications of under- or over-function of the adrenal cortex or of defects in catabolism due to disease ofthe liver or kidneys. Routine assays are currently of value in the differenti-ation of Cushing’s syndrome of adrenocortical or hypophyseal origin or intests of corticoid excretion before and after administration of adrenocortico-trophin to determine whether apparent adrenocortical insufficiency is primaryor secondary to pituitary failure.l* Application to the diagnosis of neo-plastic growth not affecting specific endocrine organs has been disappointing(cf. Schubert ’).To cite one example only, 3 : ll-dihydroxytestan-l-one, once thought to be a urinary constituent found only in cases ofcancer, is now, with more refined methods of separation, found in theurine of normal subjects and recognised as one of the usual metabolites ofcortisol.llA profound reorientation in the study of metabolism of steroids, par-ticularly in the live animal, but also in vitro, has come about with the useof steroids labelled with radioactive isotopes, particularly those incorporatinglac, which has been introduced into the 3- or the 4-position in cholestenone,testosterone, progesterone, deoxycorticosterone, and cortisone, in the16-position in oestrone and related compounds, in the 21-position in pro-gesterone, and in the 24-, 25-, and 26-positions in cholesterol.In addition,earlier work with deuterated steroids has been succeeded by work with theanalogous tritiated compounds. The work in the hormone field, the firstphases of which were discussed at the Laurentian Hormone Conferencein 1952,12 has recently been summarised by two of the principal research7 K. Schubert, “ Steroide und Krebs,” Steinkopf, Dresden, 1966.* Cf. particularly, Recent Progr. Hormone Res., 12, Proceedings of the LaurentianHormone Conference, 1955, Academic , F s s , New York, 1956 ; containing “ Biogenesisof the Sterols and Steroid Hormones by R.D. H. Heard, E. G. Bligh, M. C. Cann,P. H. Jellinck, V. J. O’Donnell, B. G. F, and J. L. Webb, “ Some Aspects of theBiogenesis o:, Adrenal Steroid Hormones by M. Hayano, N. Saba, R. I. Dorfman, and0. Hechter, Enzymatic Mechanisms of Hormone Metabolism. I. Oxidation-Reduc-tion of the Steroid Nucleus” by G. M. Tomkins, and Enzymatic Mechanismsof Hcrmone Metabolism. Mechanism of Hormonal Glucuronide Formation ” byK. J. Isselbacher.10 A. C . Crooke, Lancet, 1955, ii, 1045; F. T. G. Prunty; Brit. Mad. J., 1956, ii,615, 673.11 L. 0. Plantin and G. Birke, Acta Endocrinot., 1965, 19, 8.19 R. D. H. Heard, R.Jacobs,V. J. O’Donnell, F.G. Peron, J. C . Saffran, S. S. Solomon,L. M. Thompson, H. Willoughby, and C. H. Yates, Aeccnt Progr. Hormone Rcs.,1954, 9, 383; T. F. Gallagher, H. L. Bradlow, D. K. Fukushima, C. T. Beer,T. H. Kritchevsky, M. Stokem, M. L. Eidenoff, L. Hellman, and K. Dobriner, ibid.,p. 411.Ann. Refiorts, 1965, 52, 316.11CALLOW : METABOLISM OF STEROIDS. 307groups.l3~l4 The obvious advantages of these new techniques are the useof “ physiological ” amounts of material, the possibility of assigning alabelled product to a labelled precursor, the ease with which the presence oflabelled products can be detected in fractions separated by some chemicalor physical procedure, and, in particular, the possibility of isolating difficultlyseparable substances present in small amount by adding a non-radioactivecarrier and then separating the substance, albeit in poor yield, admixedwith the radioactive product.Also, the proportion of precursor which isrecovered in various fractions can now be accurately measured ; the variationbetween individuals and from time to time in this proportion is now disclosed,and attention is directed to the fact that in every instance a relatively largeproportion of the material is catabolised by paths which have not beenexplored because hitherto no crystalline compounds have been isolated.Further development in this direction may be expected, and it seems notunlikely that in the near future the contribution of the endocrinologicalchemist to medical diagnosis may be that of investigating the idiosyncraciesof patients in the metabolism of isotopically labelled hormone administeredin a suitable dose.The Biosynthesis of Cholesterol.-The existence of a route of synthesis,involving squalene, from acetic acid to cholesterol in animals, including man,is established, but we are far from knowing all the steps, or of being assuredthat they are all obligatory.In this and other types of steroid synthesis itmay be that the course of metabolism has so many alternative routes thatit is more properly compared with a grid system of electricity supply ratherthan with an arterial road. Popjkk l5 discussed the quantitative aspect ofthe transformation :[ l-14C,]acetate --+ [14C]squalene + [14C]cholesterolin ovarian tissues of the hen, and concluded that the “ squalene ” hypothesiscould only be reconciled with experimental observations by assuming eitherthat squalene had only a transitory existence as an enzyme-substratecomplex, or that some other scheme was operative, such as :acetate ---t isoprenoid unit + X ---t cholesterol!IsqualeneThese considerations still apply.In the very first stages of the biosynthesisthe important discovery has been made l6 that p-hydroxy-p-methyl-8-valerolactone, “ divalonic acid ’’ (l), is converted virtually completely into13 L. Hellman, K. S. Rosenfeld, D. K. Fukushima, H. L. Bradlow, T. F. Gallagher,R. G. Gould, and G. V. Le Roy, ‘‘ Peaceful Uses of Atomic Energy, Proceedings of theInternational Conference at Geneva, August 1955,” United Xations, New York, Vol.XII, p.532.14 G. J. Alexander, E. Bloch, R. I. Dorfman, C. A. Fish, 0. Hechter, G. Pincus,E. Romanoff, N. Saba, K. Savard, E. Schwenk, D. Stevens, D. Stoqe, T. H. Stoudt,and F. Ungar, ibid., p. 539.Is G. Popjkk, Arch. Biochem. Biophys., 1964, 48, 102.16 P. A. Tavormina, M. H. Gibbs, and J. W. Huff, J . Amer. Chews. SOL, 1956, 78,4498; P. A. Tavormina and M. H. Gibbs, ibid.. p. 6210308 BIOLOGICAL CHEMISTRY.cholesterol in cell-free, rat-liver homogenates and is a much better sourcethan B-hydroxy- @-met hylglutaric or 8 p-dimethylacrylic acids. 8-H ydroxy-P-methyl-8-valerolactone either is an important biological precursor of iso-prene units, or is converted by some relatively minor biochemicaltransformation into an " active isoprene '' unit.The completion of one, more strictly chemical, line of investigation canbe recorded.When cholesterol is formed from acetic acid, the acetic acidunits are built up according to a fixed pattern. This has been elucidatedby supplying labelled acetic acid, e g . , CH,-14C0,H or 14CH,*C0,H to rat-liver slices and degrading the resultant cholesterol in such a fashion thatindividual carbon atoms can be recognised in the products. The carbonatoms can be assigned to the methyl- (m) or carboxyl- (c) carbon atom ofthe acetic acid used as source. The last step in this process has been reportedby Cornforth and his co-workers,17 and C(8), C(91, C,,,, C(12), and c(14) cannow be assigned to c, m, c, c, and c, respectively, grving as the final con-clusion the formula (2) for the cholesterol skeleton.3, Squalene. 4, Lanosterol.5, Desmosterol. 6, Cholesterol.This constitution is in complete accord with the hypothesis of the bio-genesis of cholesterol from the squalene molecule condensed as suggested byWoodward and Bloch.18 The stages between squalene and cholesterol are17 J. W. Cornforth, I. Youhotsky Gore, and G. PopjBk, Biochem. J., 1966, 64, 3 8 ~ ;18 R. €3. Woodward and K. Bloch, J . Amer. Ckm. SOC., 1963, 75, 2023.1957.65, 94CALLOW : METABOLISM OF STEROIDS. 309still under investigation : the work of Clayton and Bloch l9 which showedthat lanosterol is formed in rat-liver “ homogenate,” and that it is convertedinto cholesterol, combined with the work of Stokes et aL2* on the “ high-counting companions” which can be isolated from the sterol fraction ofchick embryos from fertile eggs injected with [l-l*C]acetate, suggests thesequence shown in formula (3)-(6).An ionic mechanism for the biological cyclisation of squalene to steroidshas been outlined by Ruzicka.21 In yeast the biosynthesis of sterols wouldseem to follow similar lines, with zymosterol asa precursor of ergosterol and other unidentified‘‘ highcounting companions ” in the sterolfraction.At least some of the intermediatesbetween acetic acid and the sterols in the animaland in the plant are identical and interchange-able, and zymosterol (7) is converted in the ratinto cholesterol (Schwenk et aLZ2). An approachto a purely enzymic synthesis of cholesterol from acetate has been made byRabinowitz and his co-workers23 who claim to have prepared squalene ofhigh specific activity by incubation of labelled acetate with mitochondria1enzymes from rat-liver supplemented with supernatant fluid. The resultingsqualene yielded radioactive cholesterol when incubated with a mito-chondrial extract combined with supernatant fluid.Cholesterol Balance.-The subject of the balance of cholesterol in the bodyhas received much attention recently because of its importance in medicine.Atherosclerosis, a condition in which cholesterol is deposited on the arterialwalls, is associated with hypertensive disease, and the relation of hyper-tension, high blood cholesterol, and atherosclerosis has been the subject ofmany discussions as to which is cause and which effect.Thinking on thesubject was for a long time dominated by the old observation that athero-sclerosis was produced in rabbits by administering cholesterol-a substancethat does not normally form part of its food. The pendulum then swungthe other way, and emphasis was put on the endogenous production ofcholesterol in man and other animals. Here again, the use of isotopicallylabelled materials is beginning to clarify the situation. The synthesis inthe liver of cholesterol from acetate, and migration of the cholesterol tothe plasma, and the incorporation of dietary cholesterol which enters byway of the chyle through the liver into the circulation have been studied 13*24925with [2-14C]acetate, tritiated cholesterol, and [4-14C]cholesterol.Significantdifferences are found between normal and diseased subjects, and the in-vestigations are full of promise but detailed discussion is beyond the scopel9 R. B. Clayton and K. Bloch, J . B i d Chem., 1956, 218, 305, 319; cf. referencesquoted in ref. 8.2o W. M. Stokes, W. A. Fish, and F. C. Hickey, ibid., 1956, 2m, 415.23 E. Schwenk, G. J. Alexander, C. A. Fish, and T. H. Stoudt, Fed. Proc., 1965,14,43 J. L. Rabinowitz. F. Dituri, F. Cobey, and S. Gurin, ibid., p. 760.e4 M. W. Biggs, ref. 13, p. 526.HO &L. Ruzicka, Experientia, 1953, 9, 357.762.N. E. Eckles, C. B. Taylor, D. J. Campbell, and R. G. Gould, J . Lab. Clin. Med.,1955, 46, 359; R. G. Gould, G.V. Le Roy, G. T. Okita. J. J. Kabara, P. Keegan, andD. M. Bergenstal, ibid., p. 372310 BIOLOGICAL CHEMISTRY.of this Report. Investigations with rats by Chevallier 26 show that[4-l4C]cholesterol levels can be brought to a steady state after a daily dose of 5mg. for 8 days, and he has introduced the conception of " cholesterol space."Catabolism of Cholesterol.-Siperstein, Chaikoff, and their co-w~rkers,~'and Bergstrom and his co-workers,28 with isotopically labelled compounds,have mapped out the main course of cholesterol breakdown in the body. Itis now clear that, entirely contrary to what was once the general opinion,coprostanol is but a minor product of cholesterol metabolism; rather, mostof the body cholesterol is converted into bile acids and, moreover, all the bileacids are probably derived from cholesterol. In man, as also in the rat,cholic acid is the chief end-product.In man it is present as the glycineconjugate, whilst the rat forms taurocholic acid. Siperstein and Murray 29have found enzyme systems in guinea-pig liver which form cholyl-coenzymeA (the first activated steroid in a biological system) and transform this, byFIG. 1.HO frihydroxy -acid tincubation with taurine, into taurocholic acid. It is probable that hydroxyl-ation of the nucleus takes place before the degradation of the side chain iscompleted and it is suggested that the hydroxylation may be the rate-determining step. As at present elucidated the scheme of degradation ofcholesterol (8) is shown in Fig. 1.In the rat, deoxycholic acid (10) yieldsa6 F. Chevallier, Avch. Sci. Physiol., 1966, 10, 249.1 7 M. D. Siperstein and I. L. Chaikoff, Fed. Proc., 1956, 14, 767; M. D. Sipersteinand A. W. Murray, J . Clin. Invest., 1965, 34, 1449; earlier references are given in thesepapers.a * For references see S. Bergstriim and B, Borgstriim, Ann. Rev. Biochem., 1956,25, 177.2. 11. D. Siperstein and A. W. Murray, Science, 1956, 123, 377CALLOW METABOLISM OF STEROIDS. 311cholic acid (11). Lithocholic acid (12) is formed, and converted into cheno-deoxycholic acid (13) which, in turn, yields a trihydroxy-acid which is notcholic acid.Fredrickson 3O found that liver mitochondria converted [4-14C]cholesterolinto acids and a 25- or 26-hydroxycholesterol.The conversion of cholesterolinto coprostanol (14) occurs in the gut as a result of microbial action, andanaerobic bacteria have been found in human faeces which carry out ther e d ~ c t i o n . ~ ~ Incubation of [3-2H]cholesterol with faeces 32 yields deuteratedcoprostanol, the deuterium in the product being situated at CQ) and ator or both.The results indicate that a direct stereospecific reduction of the doublebond occurs. In the course of this some of the cholesterol must be used asa deuterium donor for other cholesterol molecules accepting deuterium atthe 5 : 6-double bond.A clue to the degradation of cholesterol to C,,-steroids, discussed below,is given by works showing that material from ox adrenals, testes, andovaries yields an enzyme stable to precipitation with ammonium sulphateand dialysis, which splits off isohexanoic acid from cholesterol.An inter-mediate stage is probably 20-hydroxycholesterol, and the residue is describedas a hydroxypregnenone-like substance with C,,,, “ involved or blocked.”Biogenesis of C,,-Steroids-In their latest review l4 the WorcesterFoundation group give Fig. 2 to summarise their conclusions in the field of“ corticosteroidogenesis. ’ ’The pathways from cholesterol (8) to corticosterone (17) and cortisol (19)can be demonstrated stage by stage in cell-free homogenates prepared fromadrenal glands, but there remains evidence for an alternative pathwaythrough an uncharacterised material “ X,” and the interrupted lines indicatethe alternative possibilities.The origin of aldosterone (18) is still a matterof debate, and conflicting results have been reported. Wettstein and hisco-workers 34 found that deoxycorticosterone (16) could be converted intoaldosterone (18) by adrenal homogenates, but progesterone (15) or cortico-sterone (17) could not. The Worcester Foundation by perfusingcalf adrenal gland, obtained aldosterone-like material from progesterone butdoubtfully from corticosterone and not at all from deoxycorticosterone.Ayres et aZ.,36 on the other hand, by using capsule strippings of ox adrenals,which are mainly zona-glomerulosa tissue, were able to demonstrate thatthere was a route of aldosterone biosynthesis through progesterone, deoxy-corticosterone, and corticosterone, though this was not necessarily the onlyso D.S. Fredrickson, J. Bid. Chew., 1956, 222, 109.81 A. Snog-Kjaer, I. Range, and H. Dam, J. Gen. Microbid., 1956, 14, 266.9% R. S. Rosenfeld, D. K. Fukushima, L. Hellman. and T. F. Gallagher, J. Bid.Chcm., 1954, 211, 301; R. S. Rosenfeld, L. Hellman, and T. F. Gallagher, ibid., 1956,222, 321.33 W. S. Lynn, jun., E. Staple, and S. Gurin, J. Awer. Chem. SOL, 1954, 76. 4048;Fed. Proc., 1955, 14, 783; E. Staple, W. S. Lynn, jun., and S. Gurin, J. Bid. Chem.,1956, 219, 845.34 R. Neher, F. W. Kahnt, and A. Wettstein, Experientia, 1955, 11, 446.8s E. Rosemberg, G. Rosenfeld, F. Ungar, and R. I. Dorfman, Endocrinology, 1956,58. 708.36 P. J. Ayres, 0. Hechter, N. Saba, S. A. Simpson, and J. F. Tait, Biochem.J.,1957, 65, 2 2 ~ 312 BIOLOGICAL CHEMISTRY.pathway. These results exemplify the occasional discrepancies which areencountered when even minor differences in mechanical treatment of tissuesprecede enzymic reactions in vitro and the difficulty in assigning particularII ' I co$H,OH CH2OH '\%I co I \ t o I '\ IIII FHjOHco$HIOHco J. FH3 coenzyme activities to specific cellular 37 although attractive andingenious speculations may be made. It may be relevant to the questionof genesis of aldosterone that there is physiological evidence 38 of stimulationof the secretion of aldosterone but not of glucocorticoids, in heat stress.The total number of compounds which have been isolated from extractsof adrenal cortex of the ox and pig has now reached 41, of which 34 arepregnane deri~atives.~~ The latest compounds are derived from previouslyknown corticoids by hydroxylation in the 6p- or the 19-position, by reductionof the 4 : &double bond or the 2O-oxo-group, and by oxidation of the angular13-methyl group to carboxyl.It is clear that the evidence provided by thechemical examination both of large-scale adrenal-cortical extracts and ofhuman adrenal and peripheral venous efiuent-Pincus and Romanoff 4037 N. Saba and 0. Hechter, Fed. Proc., 1955.14, 775.38 K. Hellman. K. J. Collins, C . H. Gray, R. M. Jones, J. B. Lunnon, and J. S.Weiner, J . Endocrinol., 1956, 14, 209.39 R. Neher and A. Wettstein, He1v;'Chirn. Acta. 1956, 39, 2062.40 G. Pincus and E. B. Romanoff, Ciba Foundation Colloquia on Endocrinology,Vol.VIII. The Human Adrenal Cortex," Churchill, London, 1966. p. 97CALLOW : METABOLISM OF STEROIDS. 313found some M ) substances in this material-must be sifted carefully if asatisfactory, simple, general theory of corticosteroidogenesis is to be con-structed. For one thing, differences between species of animal are onlyrecently receiving much attentionjlBiosynthesis of C,, Steroids.-Both the testis and the adrenal cortex con-tribute to the C,, compounds in the body. However, it seems clear that thetestis is the sole source of testosterone (21) (unless it is essential to supposethat testerone is the precursor of estrogens in the ovary). It appears, onthe one hand, that acetate may be converted into testosterone by testistissue from pig, rabbit, or man in d r o by a path which does not includecholesterol 42 and, on the other, that progesterone may be converted intotestosterone by rat-testis tissue * shown in Fig.3.FIG. 3.CholesterolAcer a t,e \+ H y drox ypr egne none + Frog e s t e ron eHydmx yprogesteroneThe genesis of dehydroepiandrosterone (22) is still a matter for conjecture.It has long been known that in human urine it is of adrenal origin; morerecently 44 it has been isolated from human blood, in which, like androsterone,it is present in conjugated form, probably as the s ~ l p h a t e . ~ ~ It has beendemonstrated 46 that human adrend slices can synthesise dehydroepiandro-sterone from [carboxy-14C]acetate, but only a very low yield seems tohave been obtained.The possibility remains that it is a secondary degrad-ation product of an as yet unidentified adrenal compound. Adrenal venousblood yielded androst-4-ene-3 : 17-dione (20) and the 11 (3-hydroxy-derivative .Catabolism of C,, and C,, Steroids.--“ Paradoxical as it may seem, moreinformation is available about the catabolic fate of the steroid hormonesthan about any other phase of steroid chemistry. This is so, in spite of thefact that the catabolic fate of the hormones may be completely unrelated totheir unique biological functions.” These sentences, witten by Liebermanand Teich three years ago, still adequately summarise the position, but withI. E. Bush, Schweiz. med. Wochenschr., 1955,85, 645; F. G. Hofmann, Endocrino-logy, 1956, 59, 712.48 R.0. Brady, J . Biol. Chem., 1961, 193, 145; cf. K. Savard, R. I. Dorfman, andE. Poutasse, J . Clin. Endocrinol., 1952, 12, 935.43 W. R. Slaunwhite, jun., and L. T. Samuels, J . Biol. Chern., 1966, am, 341.44 C. J. Migeon and J. E. Plager, ibid.. 1954, 209, 767; C. J. Migeon, ref. 40, p. 141.46 Idem, J . Biol. Chem., 1966, 218, 941.p6 E. Bloch, R. I. Dorfman, and G. Pincus, A Y G ~ . Biochm. Biophys., 1966, 61, 245.47 E. B. Romanoff, P. Hudson, and G. Pincus, J . Clin. E ~ f o c r i ~ o l . , 1953, 13, 1546,and ref. 40314 BIOLOGICAL CHEMISTRY.the reservation that the quality of the information available and the validityof the generalisations based on it are under rather critical consideration.The postulation of progesterone as a key compound in the biogenesis ofadrenal corticoids or of androgens has not led to the corollary that adminis-tered progesterone can serve as a source of the other hormones.In fact,injected progesterone disappears rapidly from the blood and the onlyproducts recognised as metabolites are pregnane-3a : 20a-diol, allopregnane-3a : 20a-diol, pregnane-3a-ol-20-oneJ aZZopregnane-3a-ol-20-oneJ and d o -pregnane-3P : 20a-di01.~~ Klopper and Michie 49 have reviewed previouswork in this field; they found rather less than 20% of injected progesteronein the form of pregnane-3a : 20a-diol in the urine, irrespective of sex, stageof the menstrual cycle, pregnancy, or period of dosage. The time seems ripefor investigation with labelled progesterone, e.g., [16-3H]progesterone.50 Inother animals there is clearly a field for enquiry, for, although progesterone,assumed to be essential for the maintenance of pregnancy, has been isolatedfrom the placentz of woman and mare, it has not been found in the late-termplacentE of cow, ewe, sow, or b i t ~ h .~ 1 Moreover, these specific differencesrecall the unanswered question of the origin of the pregnane derivativesfound by Pearlman and his co-workers 52 in cow bile. Some observationshave been recorded that are not in line with the orthodox hypotheses.Kaiser 53 observed low excretion of pregnanediol in a pregnant womanwith Addison’s disease, suggesting that the source of urinary pregnanediolis normally the adrenal cortex and compounds other than progesterone, andthe conversion of deoxycorticosterone into progesterone in the kidneys andadrenal glands has been rep0rted.5~ Taylor 55 found allopregnane-3 : 20-dione, 3a- and 3p-hydroxyaZZopregnan-20-one, 3a-hydroxypregnan-20-one,and pregnane-3a : 20a-diol in the products of incubation of progesterone withrabbit liver, and pregnane-3 : 20-dione and 3a-hydroxypregnan-20-one inthe products of incubation of pregane-3a : 20a-diol with rabbit liver.It seemed at one time that a clue through the maze of urinary meta-bolites and glandular precursors in man was given by Dorfman’s 56 general-isation on the type of reduction undergone by steroids in the course ofHO.HzC0 0 @ (25)(23) (2 41metabolism.(23) and Sp-(testane) (24) forms in essentially equal proportions.4 8 E.J. Plotz and M. E. Davis, Acta Endocrinol., 1966, 21, 259.A. Klopper and €3. A. Michie, J . Endocrinol., 1956, 13, 360.W. H. Pearlman, Biochem. J., 1966. 64, 5 4 ~ .61 R. V. Short, Nature, 1956, 178, 743.62 W. H. Pearlman and E. Cerceo, J . Biol. Chem., 1948, 176, 847.63 I. H . Kaiser, J . Clin. Endocrinol., 1956, 16, 1251.54 E. A. Lazo-Wasem and M. X. Zarrow, EndocrinoZogy, 1966, 56, 511.6 6 W. Taylor, Biochem. J., 1956, 60, 380; 1966, 62, 332.S 6 Ti. I. Dorfman in Recent. Prop,. Hormone Res., 1954, 9, 5.According to this, C,,O, steroids are reduced to 5a-(androstane)C,, steroidCALLOW : METABOLISM OF STEROIOS. 315possessing oxygen functions at C(ll) (in effect this means adrenosterone, orandrost-4-ene-3 : 11 : 17-trione) are reduced primarily to the 5a-forms whilethe presence of the C, side chain, with or without oxygen at orients thereduction of the 4 : &double bond predominantly to the Sb-(pregnane) form.Stimulating as this hypothesis has been, and indicative of some basic patternof reduction, it now seems clear that the modification of the ratio 5B : 5 aby age, sex, or physiological status of the individual, which Dorfmanpostulated from the beginning, is an important and overriding factor.Fukushima and Gallagher and their co-~orkers,~~ using deuteratedtestosterone, found wide variation in the ratio 5p : 5a at different timeintervals after administration and a variation over the range 2.5 to 0 5 indifferent subjects.Engel and his co-workers 58 reported that corticosteroneyielded as much of 5a- as of 5p-compounds.Bush 59 has referred to worknot yet published in detail showing that the 5a-compound 3a : ll(3 : 17a : 21-tetrahydroxyallopregnan-20-one is a normal and fairly plentiful urinaryproduct after cortisone administration and also that administeredadrenosterone gave in one subject, a healthy young man, the expected5p : 5a ratio : in another, an adrenalectomised orchidectomised maleaged 58 with cancer of the prostate the ratio found was the reverse of the‘‘ expected.”It appears that prednisone and prednisolone (17a : 21-dihydroxypregna-1 : 4-diene-3 : 11 : 20-trione and the llp-hydroxy-compound), which are notnatural hormones, are to some extent excreted unchanged or reduced, in thecase of prednisone,60 to the llp-hydroxy-compound, or in both cases to the20p-hydroxy-compounds. 61 Ring A is not immediately affected, and itmay be that this escape from the degradative machinery of the body is atleast partly responsible for the high activity of these compounds.Theearlier view, which would seem to have inspired much of the work on hormonemetabolites, that drugs are metabolised in the organ where they act, is nolonger tenable; 62 metabolic degradation is perhaps most obviously, in thecase of steroid hormones, a regulatory and detoxicatory mechanism fordisposing of any excess. No studies of metabolism of the 9ct-halogenatedcorticoids have appeared : it may be that a different explanation is to befound for the high activity of these compounds, and that it is derived frominfluence on the oxidation-reduction of the oxygen at C(ll), as suggested byBush.63 Fried and his colleagues,@ independently, made a similar sugges-tion, and supported it by showing that 12~~-halogenated corticoids also hadenhanced activity which could be regarded as due to inductive (-1) effecton the C(,,,-substituent.The intense, and purely glucocorticoid, activity67 D. K. Fukushima, K. Dobriner, and T. F. Gallagher, J . Biol. Chem., 1954. 208,845; D. K. Fukushima, H. L. Bradlow, K. Dobriner, and T. F. Gallagher, ibid., p. 863.68 L. L. Engel, P. Carter, and M. J. Springer, Fed. Proc., 1954.13, 204.69 I. E. Bush and M. Willoughby (unpublished), and I. E. Bush (unpublished),quoted by I.E. Bush, ref. 63.80 A. Vermeulen, J . Clin. Endocrinol., 1966, 16, 163.dl C. H. Gray, M. A. S. Green, N. J. Holness, and J. B. Lunnon, J . EndocrinoE.,1956, 14, 146; A. Vermeulen, Actu Endocrinot., 1956, 23, 113.68 B. B. Brodie, J . Pharm. Pharmacol., 1956, 8, 1.63 I. E. Bush, Experiential 1956, 12, 326.64 J. E. Hem, J. Fried, and E. F. Sabo, J . Amar. Chem. Soc., 1966, 78, 2017316 BIOLOGICAL CHEMISTRY.of 16a : 21-diacetoxy-9a-fluoro-ll : 17a-dihydroxypregna-1 : 4diene-3 : 10-dione 66 demands some additional explanation.Biosynthesis of the C,, (Estrogenic Steroids.-The conversion of [carboxy-14C]acetate to oestrone and oestradiol has been observed in perfused sowovaries,66 in minced bitch and in perfused human placenta.68Labelled cholesterol was found in the first two instances, but it was notpossible to decide whether it was an intermediate in the synthesis of cestro-gens.On the other hand, Heard and his co-workers are quite convincedthat cholesterol is not an intermediate. From a series of experiments inwhich pregnant mares were given [carbo~y-~~C]acetate, [4-14C]cholesterol,[16-14C]oestrone, and [4-14C]testosterone they deduce a scheme (shown inFig. 4) in which acetate is converted independently into oestrone (26), testo-FIG. 4.sterone (21), equilin (27), and equilenin (28), but testosterone can be con-verted into cestrone and there is also a very doubtful possibility of a deriv-ation of equilenin (28) from testosterone (21) by way of cestrane-3 : l7-diokand 3-hydroxyoestra-5 : 7 : 9-trien-17-one.Slices of human ovary can alsoaccomplish the transformation of testosterone (21) not only to oestradiol 70(29) but also, it is reportedJ71 to oestrone (as), and cestriol (30). Ovariecto-mised adrenalectomised women excrete cestrone and cestradiol after adminis-tration of testosterone. 72 19-Hydroxyandrost-4-ene-3 : 17-dione (25) is a66 S. Bernstein, R. H. Lenhard, W. S. Allen, M. Heller, R. Littell, S. M. Stolar,L. I. Feldman, and R. H. Blank, J . Amer. Chem. Soc., 1966, 78, 6693.o6 N. T. Werthessen, E. Schwenk, and C. Baker, Science, 1953, 117, 380.67 J. L. Rabinowitz and R. M. Dowben, Biochim. Siophys. Ada, 1966, 16, 96.68 H. Levitz, G. P. Condon, and J. Dancis, Fed. Proc., 1986, 14, 246.69 R. D.H. Heard and V. J. O'Donnell, Endocrinology, 1954, 54, 209; see ref. 9 for70 B. Baggett, L. L. Engel, K. Savard, and R. I. Dorfman, J . Biol. Chem., 1956,921,7l H. H. Wotiz, J. W. Davis, H. M. Lemon, and M. Gut, ibid., 1966, 332, 487.72 C. D. West, B. L. Damast, S. D. Sarro, and 0. H. Pearson, ibid., 1966, 218, 409.urther references.931CALLOW : METABOLISM OF STEROIDS. 317probable intermediate in this transformation, for its conversion into oestronehas been demonstrated 73 in human placenta or, to a lesser extent, in cowfollicular fluid or adrenal gland. Moreover, it has been isolated from oxadrenal gland by mat to^.^^The investigation of urinary metabolites of cestrone and estradiol byorthodox chemical methods, with the aid of new analytical techniques ofchromatographic or countercurrent separation, has led to the recognitionthat there are other metabolites, the presence of which was suspected manyyears ago,75 in the shape of 16-e$icestriol (31) 76 and 16a-hydroxycestrone(32).77 It may be mentioned incidentally that to the natural 16-hydroxy-compounds previously listed 78 there may be added 17-oxoandrost-5-ene-38 : 16a-diol from urine of men 79 and 3p : 1 6 ~ - and 3p : 16p-dihydroxy-androstanes from urine of pregnant mares.80 The occurrence in human urineof 16-oxo-17p-cestradio1 81 and 16-oxooestrone 82 has been reported but,whilst artificial formation during manipulation, of the one by isomerisationand of the other by oxidation of 16a--hydroxycestrone, seems to be excluded,confirmatory evidence would be welcome.A hypothetical scheme ofestrogen catabolism 83 is shown in Fig. 5. The stage 16a-hydroxyestrone+ estriol has been experimentally confirmed recently in man.84FIG. 5.onOHIt has been pointed out 83 that, with the recognition that a hithertounknown major metabolite occurs in human urine, methods of chemical73 A. S. Meyer, BiocAim. BwFhys. Acta, 1956, 17, 441.74 V. R. Mattox, Proc. Mayo Clin., 1955, 30, 180.G. F. Marrian, Bull. N.Y. Acad. Med., 1939,15, 27.7% G. F. Marrian and W. S. Bauld, Biochem. J., 1955, 59, 136; E. J. D. Watson and77 G. F. Maman, K. H. Loke, E. J. D. Watson, and M. Panattoni, quoted in ref. 79;79 K. Fotherby, A. ColQs, S. M. Atherden, and G. F. Marrian, BiocAem. J., 1956, 64,80 R.V. Brooks and W. Klyne, aid., 1966, 63, 2 1 ~ .81 M. Levitz, J. R. Spitzer, and G. H. Twombly, J . Biol. Chem., 1956, 2%, 981.82 W. R. Slaunwhite, jun., and A. A. Sandberg, Avch. Biochem. Biophys., 1956, 63,83 G. F. Maman, public lecture at University College, London, December 9th, 1956.84 G. F. Marrian and J. B. Brown (personal communication).G. F. Maman, ibid., 1856, 68. 64.G. F. Marrian, E. J. D. Watson, and M. Panattoni, Biochem. J., 1957, 65, 12.6 0 ~ .Ref. 8, p. 326.478318 BIOLOGICAL CHEMISTRY.analysis must be revised. In any case, work with labelled estrogens 85 hasshown that, as is the case with other steroid hormones, no inconsiderableproportion of administered material remains to be accounted for in un-charact erised forms.R.K. C .5. SULPHATASES.The sulphatases are a group of hydrolytic enzymes which are capableof liberating sulphuric acid from various monoesters of sulphuric acid.They are widely distributed in Nature but their physiological functionremains obscure. Studies of these enzymes before 1947 have been ade-quately summarized but only certain aspects of developments since thisdate have been r e ~ i e w e d . ~ ~ ~Until recently, four different sulphatases of clearly contrasting specificitywere recognized : arylsulphatase (phenolsulphatase) , capable of hydrolysingaryl sulphates * such as potassium phenyl sulphate ; myrosulphatase,capable of liberating sulphate from potassium myronate (sinigrin) andsimilar mustard-oil glycosides ; chondrosulphatase, capable of desulphatingchondroitin sulphate ; and gluco-(glyco-)sulphatase, capable of hydrolysingglucose 6-sulphate * and certain other simple sulphated carbohydrates.This list has now been extended by the discovery of a highly specificenzyme which hydrolyses certain steroid ~ulphates.~-~ There is also apossibility that two enzymes present in extracts of a flavobacterium may besulphatases of hitherto unknown types, in view of their ability to releasesulphate from heparin.8 The presence of a more general alkylsulphatasealso seems possible since Bacihs cereus var.mycoides, isolated from soil,converts inactive sodium 2-(2 : 4-dich1orophenoxy)ethyl sulphate into theactive herbicide 2 : 4-dichlorophenoxyacetic acid, presumably by hydro-lysis of the ester sulphate linkage followed by oxidation of the primaryalcohol formed.gThe Preparation of Substrates-Both synthetic and naturally-occurringsulphate esters have been used for the study of sulphatases.The preparationof most aryl sulphates is readily accomplished by treatment of the appro-85 C. T. Beer and T. F. Gallagher, J . Biol. Chews;, 1966,214, 336, 361 ; C. Heusghemand W. Verly, I1 Furmaco (Sci.), 1956, 11, 404; Actes de la 3me Reunion d’Endo-crinologie,” 1955, p. 139; A. M. Budy, J . Pharpnacol., 1956, 116, 10.1 C . Neuberg and E. Simon, Ergebn. Physiol., 1932, 34, 896 ; C. Fromageot, Ergebn.Enzymforsch., 1938, 7, 50; T. Soda in “Die Methoden der Ferment Forschung,” byE. Baumann and K. Myrback, Academische Verlag, Leipzig, 1940, Vol.2, p. 1696;C. Fromageot in “ The Enzymes,” by J. B. Sumner and K. Myrback, Academic PressNew York, 1950, Vol. 1, p. 517.2 K. S. Dodgson, A. B. Roy, and B. Spencer in “ Biochemie de Soufre,” ed. byC. Fromageot, Centre National de Recherche Scientifique, Paris, to be published.3 K. S. Dodgson and B. Spencer in ‘‘ Methods in Biochemical Analysis,” ed. byD. Glick, Academic Press, New York, 1957, Vol. IV.R. Henry and M. Thevenet, Bdl. SOC. Chim. biol., 1962, 34, 886.R. Henry, M. Thevenet, and P. Jarrige, ibid., p. 837.S . R. Stitch and I. D. K. Halkerston, Nature, 1953, 172, 398.7 Idem, J , Endocrinol., 1963, 9, xlfxvi:8 A. N. Payza and E. D. Kom, Btochzm. Biophys. Acla, 1966, 20, 596.9 A. J. Vlitos, Contribn. Boyce Thompson Inst., 1953, 17, 127.* Most of these acid sulphates are prepared and used as potassium salts.of the cation is therefore omitted unless it is different from potassium.The namDODGSON AND SPENCER : SULPHATASES. 310priate phenol with chlorosulphonic acid in the presence of diethylaniline orpyridine 10 or with pyridine-sulphur trioxide. 11-13 Occasionally otherprocedures have been employed including direct sulphation with sulphuric l4or chlorosulphonic acid15 a t low temperatures and the use of pyro-sulphate.16* 1' Some aryl sulphates containing amino-groups in the aromaticring have been prepared by the reduction of the corresponding nitro-compound^.^^-^^ The formation of sulphuric esters as intermediates in theElbs persulphate oxidation has led to the use of alkaline persulphate for thepreparation of various o-amino-phenyl sulphates,20 which are formed irre-spective of the orientating influence of other functional groups, and for thepreparation and use of the mono(hydrogen sulphates) of nitroquinol andnitrocatechol.19Dipotassium 2-hydroxy-5-nitrophenyl sulphate, prepared by per-sulphate oxidation of p-nitrophenol, has recently been extensively used asa substrate for arylsulphatases. However, when prepared according toRoy's directions 21 the product is contaminated with nitropyrogallol di-(hydrogen sulphate) 22*23 from which it may be freed by recrystallization asthe monopotassium salt 24 or by paper ionophoresis.22y 24Aryl sulphates in aqueous solution tend to decompose spontaneously andshould be stored in the dark at 0".In the case of the mononitrophenylsulphates, the initial breakdown is photochemically accelerated, the reactionbeing non-sensitized and independent of pH.25 Autocatalytic hydrolysisthen presumably occurs 26 leading eventually to complete decomposition.Apart from analysis, aryl hydrogen sulphates and their alkali salts arenot readily characterized by melting point. In some cases the fi-toluidine z7and 9-bromoaniline 28 salts are fairly insoluble but usually show indefinitemelting points.29 A number of aminoquinoline and aminoacridine deriv-atives form salts with aryl hydrogen sulphates but again the salts do nothave distinct melting points.29 Eufl avin, safranine, and 5-aminoacridinehydrochloride form particularly insoluble salts from which the parent arylhydrogen sulphate can be regenerated.29 These heterocyclic bases can be10 G.N. Burkhardt and A. Lapworth, J., 1926, 684.l1 P. Baumgarten, Bsr., 1926, 59, 1976.la Inorg. Synth., 1946, 2, 173.13 A. E. Sobel and P. E. Spoerri, J . Amer. Chem. SOC., 1941, 63, 1259.l4 H. Fraenkel-Conrat and J. Fraenkel-Conrat, Bioclzim. Biofihys. Acta, 1950, 5, 98.G. N. Burkhardt, J., 1933, 337.16 E. Baumann, Ber., 1878, 11, 1907.17 S. Bernstein and R. W. McGilvery, J . Biol. Chem., 1952, 198, 195.18 G. N. Burkhardt and H. Wood, J., 1929, 141.Is J. N. Smith, J., 1961, 2861; D. Robinson, J. N. Smith, B. Spencer, and R. T.2O E. Boyland, D. Manson, and P. Sims, J., 1953, 3623; E. Boyland and P. Sims,21 A. B. Roy, Biochem. J., 1953, 58, 12.22 Idem, ibid., 1956, 62, 3 5 ~ .23 A.B. Roy and L. M. H. Kerr, Nature, 1956, 178, 376.24 K. S. Dodgson and B. Spencer, Biochim. Biophys. Acta, 1956, 21, 175.2S A. E. Havinga, R. 0. DeJongh, and W. Dorst, Rec. Trav. chim., 1956, 75, 378-26 G. N. Burkhardt, W. G. K. Ford, and E. Singleton, J., 1936, 17.27 A. D. Barton and L. Young, J . Amer. Chem. Soc., 1943, 65, 294.28 D. H. Laughland and L. Young, Trans. Roy. SOC. Canada, 1942, 111, 36, 166.*v K. S. Dodgson, F. A. Rose, and B. Spencer, Natuw, 1956, 174, 599; Biochem. J .Williams, Biochem. J . , 1952, 51, 202.J . , 1954, 980.1955, 60, 346320 BIOLOGICAL CHEMISTRY.used to precipitate some aryl hydrogen sulphates from urine with little inter-ference from other urinary constituents. 5-Aminoacridine hydrochloride,for example, has been used to isolate 4-chloro-2-hydroxyphenyl hydrogensulphate from the urine of rabbits which had been fed with chlorobenzene or4-chlorocatechol.29 Similar procedures may be applicable to the biosyntheticpreparation of useful substrates for arylsulphatase assay (e.g., phenol-phthalein monosulphate) which are not readily obtained by direct chemicalsynthesis.A convenient method for the preparation of steroid sulphates30 and anew method for the isolation of potassium myronate from mustard seedshave been reported.31, 32The impure nature of potassium glucose 6-sulphate when prepared bydirect sulphation of glucose33 has been confirmed.= The main impurityappears to be an isomer, the extent of the contamination being less whenSoda's chlorosulphonic acid method 35 is used than with the pyridine-sulphur trioxide method.= Although glycosulphatase hydrolyses both theglucose 6-sulphate and the contaminant, only the latter is rapidly andcompletely hydrolysed by hydrazine.=*= Small amounts of a second con-taminant have been detected chromatographically in the glucose 6-sulphateprepared by direct sulphation with pyridine-sulphur trioxide.= Con-tamination can be reduced considerably by repeated recrystallization ofglucose 6-sulphate as the corresponding brucine salt 33*34 and in this wayEgami 33 obtained a final preparation which was " hardly hydrolysed byhydrazine." The impure nature of glucose 6-sulphate when prepared bydirect sulphation of glucose appears to have escaped the notice of someworkers.36 Glucose 3-sulphate can be unequivocally prepared by sulphationof diisopropylideneglucose followed by removal of the residues but , althoughit is readily hydrolysed by glycosulphatase, it is unsuitable as an assay sub-strate owing to the labile nature of the ester sulphate group.= This com-pound is completely hydrolysed by hydrazine but is not identical with eitherof the impurities present in preparations of the glucose 6-sulphate obtainedby direct sulphation of glucose.M Adenosine-5' (hydrogen sulphate), whichis also hydrolysed by glycosulphatase , has recently been synthesi~ed.~'Three different types of chondroitin sulphate are now known to bepresent in mammalian tissues 38 and it seems likely that preparations usedin the past for the study of chondrosulphatase 399 40 were mixtures of the3 0 A.B. Roy, Natuve, 1956, 62, 41.sf 0. E. Schultz, R. Gmelin, and A. Keller, 2. Natwrfovsch., 1953, 8b, 14.8s F. Egami, J . Chem. SOC. Japan, 1938, 59, 1034; 1940, 61, 692; 1942, 63, 763.8' K. S. Dodgson and B. Spencer, Biochem. J., 1964, 57, 310.86 T. Soda, Bull. Chem. SOC. Japan, 1933, 8, 37.86 R. B. Duff, J., 1949, 1597; H. L. Wolfrom and R. Montgomery, J . Amer. Chem.87 *F. Egami and N. Takahashi, Bull. Chem. SOC. Japan, 1955, 25, 666.8 8 K. Meyer and M. M. Rapport, Science, 1961, 118, 696; K. Meyer, E. Davidson,8 ) C. Neuberg and E. Hoffman, Natuvwzss., 1931, 19, 484; Biochem. Z., 1931, m,( 0 K. S. Dodgson. A. G.Lloyd, and B. Spencer, Biochem. J., 1967, 85, 131.Cf. J. Gadamer, Arch. Pharm.. 1897,235,44; H. Herisseyand R. Boivin, J . Pharm.Chim., 1927, 6, 337.Soc., 1950, 72, 2869.A. Linker, and P. Hoffman, Biochiun. Biqhys. Acta, 1966, 21, 606.345DODGSON AND SPENCER : SULPHATASES. 321various types. The great difference in the relative activity of bacterialchondrosulphatase towards the polymerized and depolymerized forms ofchondroitin sulphate 41 suggests that it will now be important to devisestandard methods for the isolation of sulphated oligosaccharides fromdepolymerized chondroitin sulphate. The chemical nature of charoninsulphate, a possible substrate for molluscan chondrosulphatase, has beenin~estigated.~~ This glucan polysulphate, which is foufid in the marinegastropod Charonia Zumflas (Triton .nodiferus),43 has been shown to containboth cellulose- and amylose-type structures.The choice of substrates and their use for the assay of sulphatases havebeen recently re~iewed.~Ary1sulphatases.-Although the availability of numerous convenientmethods for the assay of arylsulphatases has led many workers to investigatethese enzymes, their physiological function remains obscure.showed that the arylsulphatases of fungi, molluscs, and mammals werespecific for the sulphates of phenols hut other properties of the enzymeswere not extensively examined.Two types of arylsulphatases are nowrecognized.2* The first (Type I) possesses a7preciable activity and affinitytowards 9-acetylphenyl and p-nitrophenyl sulphates but shows little activitytowards 2-hydroxy-5-nitrophenyl sulphate (nitrocat echo1 hydrogen sulphate) .They are inhibited by cyanide but are not affected markedly by sulphateand phosphate ions.The second type (Type 11) has the converse specificityto the Type I enzymes, showing high activity towards 2-hydroxy-5-nitro-phenyl sulphate but low affinity and activity towards 9-acetylphenylsulphate and 9-nitrophenyl sulphate. They are strongly inhibited bysulphate and phosphate ions but are unaffected by cyanide.Mammalian tissues contain one enzyme of Type I and two of Type II.44s 45The Type I enzyme, arylsulphatase C, is extensively distributed throughoutthe animal body 45* 46 and, in rat liver, is exclusively localized in the micro-somes of the liver cells.@V 47 The enzyme appears to form part of an insolublelipid-protein complex which becomes soluble when incorporated into themicelles of surface-active agents,48 but which resumes its insoluble natureon removal of the detergent.The enzyme can be brought into true solutionby treatment of the insoluble comple:; with crude pancreatic lipase prepar-ations in the presence of detergent.49 The two Type I1 enzymes, aryl-sulphatases A and B, are probably present in all tiss~ies.~j Their distri-bution within the individual cell has been studied in rat 5* and mouse 51Early studies41 K. S. Dodgson and A. G. Lloyd, Biochem. J., 1957, 65, 4 ~ .42 F. Egami, T. Asahi, N. Takahashi, S. Suzuki, S. Shikata, and K. Nisizawa, Bull.Chem.SOC. Japan, 1965, 28, 685; K. Nakanishi, N. Takahashi, and I?. Egami, ibid.,1956, 29, 434.43 T. Soda, J . Chem. SOC. Japan, 1936, 57, 981; T. Soda and F. Egami, Bull. Chem.SOC. Japan, 1938, 13, 652.44 K. S. Dodgson, B. Spencer, and J. Thomas, Biochem. J . , 1955, 59, 29.b5 K. S. Dodgson, B. Spencer, and C. H. Wycn, ibid., 1955, 62, 500.46 K. S. Dodgson, B. Spencer, and J. Thomas, ibid., 1953, 53, 452.4 7 Idem, ibid., 1954, 56, 1 7 7 ; R. Gianetto and R. Viala, Science, 1955, 121, 801.4 8 K. S. Dodgson, F. A. Rose, B. Spencer, and J. Thomas, Biochem. J . , in the press.K. S. Dodgson. F. A. Rose, and B. Spencer, ibid., in the press.8o A. B. Roy, Biochim. Biophys. Acta, 1964, 14, 149.61 Idem, Biochem. J.. 1963, 53, 12.REP .-VOL. LIT1 322 BIOLOGICAL C H E M ISTKY.livers.They occur mainly in the mitochondria of the liver cell althoughappreciable activity also occurs in the microsomes and the soluble materialof the cytoplasm. Arylsulphatases A and B are readily obtained in solutionby any method which ruptures the mitochondria1 membrane uv 511 52 andcan be separated by fractional precipitation with acetone and ammoniumsulphate or by paper electrophoresis.44? 45p 51* 53,The properties of arylsulphatases A and B have been investigated j3-55and the anomalous kinetics shown towards 2-hydroxy-5-nitrophenyl sul-phate by the arylsulphatase A of ox liver have been interpreted by Koy 53as a function of the polymerization of the enzyme. Dodgson and Spencer 56working with the corresponding hunian enzyme have shown that thisexplanation is untenable, the basic anomaly being that the reaction is notof zero order.The anomaly is not due to the presence of an impurity,nitropyrogallol di~ulphate,~~ in the substrate preparation as later suggestedby Roy,57 since anomalous kinetics are still obtained when purified sub-strate is used. 56 Molluscan, fungal, or bacterial arylsulphatases do notshow the anomaly which must be regarded therefore as peculiar tomammalian arylsulphatase A.58Arylsulphatase activity has been demonstrated in a number of bacteria.When phenolphthalein disulphate 599 6o is incorporated into culture media,the presence of arylsulphatase-producing bacteria may be detected byexposing the culture plates to gaseous ammonia and observing the redcolour of the liberated 61* 62 A large number of organismshave been tested by this and similar techniques 63 and it has been concludedthat, apart from rnyc~bacteria,~~~ 64 comparatively few organisms possessthe enzyme.These conclusions may not be valid, however, since the screen-ing procedures have usually failed to take certain factors into account. Forinstance, many bacteria can rapidly metabolize the phenolphthaleinliberated,62 whilst others, known to possess arylsulphatase activity towardsother substrates, are inactive towards phenolphthalein d i ~ u l p h a t e . ~ ~ More-over, Harada and Kono 66 have shown that some bacteria produce aryl-52 R. Viala and R. Gianetto, Canad. J . Biochem. Physiol., 1955, 33, 839.63 A.B. Roy, Biochem. J., 1953, 55, 653.64 Idem, ibid., 1954, 5'7, 465.LS Idem, ibid., 1955, 59, 8.5 6 K. S. Dodgson and B. Spencer, ibid., 1956, 62, 3 0 ~ .5 7 A. B. Roy, ibid., p. 3 5 ~ .68 K. S. Dodgson and €3. Spencer, Biochim. Biophys. A d a , 1956, 21, 175.59 J. E. M. Whitehead, A. R. Morrison, and L. Young, Biochem. J., 1952, 51, 585.61 L. Young, A. R. Morrison, and J . E. M. Whitehead, Nature, 1952, 169, 711.62 K. S. Dodgson, T. H. Melville, B. Spencer, and K. Williams, Biochem. J., 1954,58, 182.63 K. L. Arora, A. T. Dudani, and C. R. Krishnamurti, J . Sci. Ind. Res. (India),1953,12, B, 502; M. Barber, B. W. L. Brooksbank, and S. W. A. Kuper, J . Path. Bact.,1951, 63, 57; M. Chauncey, F. Lionetti, R. A. Winer, and V. F. Lisanti, J .Dent. Res.,1954,33, 321 ; T. Ishikawa, Med. J . Chiba Univ. (Jafian), 1943, 21, 700; G. C. Shrivas-tava, K. L. Arora, and S. S. Bhatanagar, Experientia, 1954,10, 493; R. Hare, P. Wildy,IF. S. Billet, and D. N. Twort, J . Hyg., 1952, 50, 295.64 J. E. M. Whitehead, P. Wildy, and H. C . Engbaeck, J . Path. Bact., 1953, 65, 451.6 5 T. Harada, K. Kono, and K. Yagi, Mem. Inst. Sci. Ind. Res., Osaka Univ., 1955,11, 193 ; T. Harada and K. Kono, J . Agric. Chew. Soc. Japan, 1954, 28, 608.(i6 T. Harada and K. Kono, Mem, Inst. Sci. Ind. Res., Osrrka Univ., 1956, 12, 183.M. Pantlitschko and F. Kaiser, Monatsh., 1952, 83, 1140DODGSON AND SPENCER : SULPHATASES. 323sulphatase only if a certain factor, identified as tyramine,6i is present inthe peptone of the culture medium. Arylsulphatase activity of a fusiformbacillus is similarly dependent on accessory factors which can be providedby addition of cystine or sterile raw potato to the culture medium.68The arylsulphatases of Aerobacter aerogenes 66 and Alcaligenes metal-caligenes 62a 69 show properties which classify them as Type I.From in-vestigations of the variation with pH of the Michaelis constant of theA lcaligenes enzyme acting on 9-acetylphenyl, $-nitrophenyl, and 2-hydroxy-5-nitrophenyl sulphates, it has been concluded that two ionizing substrate-binding groups (pK 8.2 and 9.4, respectively) are present in the enzymemolecule.69 Application of Dixon’s rules 70 to the results obtained hasindicated that the group with pK 8.2 must gain a positive charge (or lose anegative charge) on “ desubstration,” whereas the converse is true of thegroup with pK 9.4.The chemical nature of the former group is obscure,but that with pK 9.4 may be an a- or E-amino-group, and some support forthis suggestion has been obtained by studying the effects of various group-specific protein reagents on enzyme a~tivity.6~ As a result of these in-vestigations, Dodgson et al.69 suggested that the enzyme contains positively-and negatively-charged substrate-binding groups which reactTf with substrate in the form shown inset, where the sulphurArO-s-0- atom, by virtue of its semipolar bonds, possesses some ‘+) degree of positive charge. Further information has beenobtained by examining the eflect on enzyme activity of the introduc-tion of substituents into the benzene ring of phenyl hydrogen sulphate.’lBoth the affinity of the enzyme for the substrate and the rate of hydrolysisshow a progressive increase with increasing electrophilic nature of the sub-stituent group. As a result of these various findings it has been possible tosuggest the following reaction mechanism for the hydrolysis of arylsulphates by the Alcaligenes enzyme, where X and Y are the nucleophilicand electrophilic groups present at the active sites of the enzyme.Theintroduction of electrophilic substituents into the aromatic ring is presumedto facilitate combination of enzyme and substrate by withdrawing electronsfrom the sulphate group. This would lead to an increase in net charge onthe sulphate group, since the loss in negative charge on the ionized oxygenatom would be less than the gain in positive charge on the sulphur atom,the latter being nearer to the benzene ring. An increase in affinity ofenzyme for substrate would be expected to result from this increase in netcharge and this agrees with the experimental findings.i1 The electrophilic6 7 T.Harada and C. Hattori, Bull. Agvic. Chem. SOC. Japan, 1956, 20, 110.6 8 S . D. Schultz-Handt and H. D. Scherp, J . Bact., 1955, 69, 665.69 K. S. Dodgson, B. Spencer, and K. Williams, Biochem. J . , 1956, 61, 374.i o M. Dixon, ibid., 1953, 55, 161.K. S. Dodgson, B. Spencer, and K. Williams, ibid., 1956, 64, 216324 BIOLOGICAL CHEMISTRY.hydroxonium ion is presumed to be responsible €or the breakdown of theenzyme-substrate complex since the acidic hydrolysis of aryl sulphates,which is known to be mediated in this manner,26 is very similar to theenzymic hydrolysi~.~~ The increase in the rate of enzymic hydrolysis whichfollows the introduction of an electrophilic substituent is probably due toincreasing stabilization of the product ArO: formed by rupture of the 0-Sbond.The other product, -O*S(O,) (+), is presumably stabilized by com-bination with the enzyme. It would appear that before a sulphuric estercan be hydrolysed by the Alcaligenes arylsulphatase there must be a strongelectron-withdrawing influence on the sulphate group. The absence ofstrong electrophilic groups in glucose, ethyl, and chondroitin sulphates mightexplain the inactivity of the Alcaligenes enzyme towards these and similarsulphuiic esters.Although the arylsulphatases show an absolute specificity towards thesulphates of phenols (or phenolic-like compounds, cf.the hydrolysis of kojicacid disulphate by glyco- plus aryl-sulphatase '9, the relative specificitytowards various aryl sulphates varies greatly.71 Indeed the arylsulphatasesof human urine (arylsulphatases A and B 73) and Aspergillus oryzae do notattack many o-aminoaryl ~ u l p h a t e s . ~ ~ It has been suggested 74 that thisis related to the ability of these aryl hydrogen sulphates to form zwitterionsat pH's where the enzymes would normally exhibit maximum activity. Therate of hydrolysis of o-, m-, and 9-aminophenyl sulphates by the Alcaligenesenzyme is also very low but the reason for this lies in the nucleophilic natureof the amino-group rather than in the presence of zwitterions since at theoptimum pH (8.75) of this enzyme there is little tendency for zwitterionformation.In any case there are reasons 71 for suspecting that the orthoeffect, rather than zwitterion formation, may be responsible for the resist-ance of o-aminoaryl sulphates to hydrolysis by the enzymes of Aspergilhsand urine, I t is clear irom the observations of Dodgson et aL71 and Boylandet ~ 1 . 7 ~ that the failure of an arylsulphatase to hydrolyse an unknown phenolicconjugate (e.g., Clarke et does not necessarily mean that the conjugateis not an aryl sulphate.Myrosulphatase.-This enzyme has been little studied in recent years.The best known source is mustard seed where it is found in association withits substrates, the mustard-oil glycosides.A thioglycosidase is also presentin the seed and the two enzymes together degrade potassium myronate toglucose, ally1 mustard oil, and inorganic s ~ l p h a t e . ~ ~ The enzyme has beenfound in bacteria 39 and marine molluscs 77 whilst its presence in horse andrabbit tissues has been However, recent work suggests thatniyrosulphatase does not occur in mammalian tissues to any appreciable72 T. Soda, T. Katsura, and 0. Yoda, J . Chem. SOC. Japan, 1940, 61, 1227.73 K. S. Dodgson and B. Spencer, Clin. Chim. Acta, 1956, 1, 478.74 E. Boyland, D. Manson, P. Sims, and D. C . Williams, Biochem. I., 1956, 62, 68.7 5 W.G. Clarke, R. J. Akawie, R. S. P6grund, and T. A. Geissman, J . Pharmacol.,76 C. Neuberg and 0. Schoenbeck, Biochem. 2.. 1933, 265, 223; Nalurwiss., 1933,77 M. Ishimoto and J. Yamashina, Symp. Enzyme Chem. (Japan), 1941, 2, 36.7 a C. Neuberg and J. Wagner, Z. exp. Med.. 1927, fi6, 334.79 H. Baum and K. S. Dodgson, Nature, 1967, 179, 312.1951, 101, 6.21, 404; C. Neuberg and J. Wagner, Biochem. Z., 1926,174, 467DODGSON AND SPENCER : SULPHATASES. 325;extent and has demonstrated that the enzyme is quite distinct from bothTypes I and I1 arylsulphatases.Chondr0sulphatase.-The early observation that certain putrefactivebacteria were able to utilize chondroitin sulphate for growth 80 led to thepreparation of extracts of Pseudomonas JEuomcens non-liquifaciens whichwere able to liberate inorganic sulphate from chondroitin s ~ d p h a t e .~ ~ Com-plete hydrolysis of the substrate to acetic acid, sulphate, glucuronic acid,and galactosamine was achieved by such extractss1 The presence ofchondrosulphatase in Ps. juorescens has since been confirmed 82 and otherorganisms, including Proteus v z i l g a ~ i s , ~ ~ ~ 40* 82-86 Pseudomonas ae~uginosa,~~*Micrococczts pyogenes a u ~ e u s , ~ ~ Peizicillium spindosum a7 and organismsisolated from human gingival crevices,88 have also been shown to possessthe enzyme. Chondrosulphatase also occurs in certain rn0lluscs.8~~Some confusion exists at present as to whether chondrosulphatase occursin mammalian tissues. Early workers could not detect liberation of sul-phate from chondroitin sulphate by macerated tissues 81 and, more recently,negative results have been obtained by using more sensitive detectionmethods.g1 On the other hand, a comparatively rapid metabolic turnoverof the sulphate groups of chondroitin sulphate occurs in the animal body,92and the live rat appears to be able to liberate sulphate from injected chon-droitin sulphate containing isotopic sulphur, since significant amounts ofinorganic sulphate containing isotopic sulphur can be isolated from theurine.g3 There is some indication of the presence of a chondrosulphatase-type enzyme in mammalian pancreas as a result of studies on the degradationof elastic tissue by pancreatic elastase.Preparations of elastic tissue con-tain a metachromatic sulphated polysaccharide which, like chondroitinsulphate, loses its metachromatic properties after incubation with bacterial 86or molluscan g4 chondrosulphatase preparations.The metachromasia alsodisappears after treatment with pancreatic extracts possessing elastaseactivity.86 Other observations 95 have suggested the possibility of someenzymic liberation of sulphate from the sulphated polysaccharide of elastictissue by crude elastase preparations.Both bacterial and molluscan chondrosulphatases are associated with achondroitinase enzyme system which degrades the polysaccharide chain of8o C. Neubergand 0. Rubin, Biochem. Z., 1914, 67, 82.81 C. Neuberg and W. L. Cahill, Enzymologia, 1936, 1, 82.83 H. J.Buehler, P. A. Katzman, and E. A. Doisy, Proc. SOC. Exp. Biol. Med., 1951,83 H. Candelli and A. Tronieri, Bull. SOC. ztal. Biol. sper., 1951, 27, 651.84 W. A. Konetza, AT. J. Pelczar, and G. W. Burnett, Bact. Proc., 1964, 106.R 6 W. J. Pepler and F. A. Brandt, Brit. J . Exp. Path., 1854, 35, 41.87 R. Pincus, Nature, 1950, 166, 187.8 8 S. D. Schultz-Handt and H. W. Scherp, J . Dent. Res., 1956, 35, 229.89 T. Soda and F. Egami, J . Chem. SOC. Japan, 1938, 59, 1202.91 C. H. Dohlman and J. S. Friedenwald, J . Histochem. Cytochem., 1955, 3, 492.92 See, e.g., H. Bostrom, J . Biol. Chem., 1952, 196, 177.93 C. H. Dohlman, personal communication; D. D. Dziewiatkowski, J . Bid. Chew.,94 H. Hayashi, T. Funaki, K. Udaka, and Y . Kato, Mie Med. J., 1956, 4.143.78, 3.0. Reggianini, Bull. SOC. ital. BioE. sper., 1950, 166, 187.Y . Horiguchi and M. Mikaya, Bull. Jap. SOC. Sci. Fish., 1954, 19, 957.1966, 223, 239.D. A. Hall and J. E. Gardiner, Biochem. J., 1966, 69, 465326 BIOLOGICAL CHEMISTRY.chondroitin sulphate with consequent release of reducing substances.Several attempts have been made to determine the extent to which thesulphatase and chondroitinase activities are interdependent. Soda andEgami,89 using mollusc preparations, have succeeded in showing thatsulphatase activity can be completely inhibited without markedly affectingchondroitinase activity. More recently, a study of the effects of inhibitorson the ability of viable cultures of P. vulgaris to degrade chondroitin sulphatehas suggested that liberation of sulphate is secondary to the release ofreducing substances.@This problem of interdependence has now been resolved for the enzymesof P.vulgaris. Dodgson and Lloyd41 have succeeded in obtaining a pre-paration of the sulphatase which is completely free from chondroitinase.The activity of this sulphatase towards polymerized chondroitin sulphate isnegligible. On the other hand the enzyme is extremely active towards thesulphated oligosaccharides which are formed when chondroitin sulphate isexhaustively degraded by testicular hyaluronidase. Inhibitor studies haveconfirmed the earlier observation 89 that chondroitinase activity can pro-ceed independently of the sulphatase. It seems clear that the true substratesfor chondrosulphatase are to be found in the sulphated oligosaccharidesresulting from chondroitinase action.These findings may possibly throwlight on the failure of several workers 81*91 to detect chondrosulphataseactivity in mammalian tissues, and it would now be interesting to repeat thiswork using, as the assay substrate, chondroitin sulphate which has beendegraded by testicular hyaluronidase.It is not yet clear whether all three types of mammalian chondroitinsulphate 38 are substrates for bacterial chondrosulphatase and chondro-itinase. The three types have been separated and occur in different propor-tions in the various tissues.96 Preparations of chondroitin sulphate obtainedby simple extraction procedures will almost certainly contain more thanone type and it is worth noting that Dodgson et a1.,4O using extracts ofP.vulgaris, achieved complete release of sulphate from chondroitin sulphatewhich had been prepared in this way.The finding that Proteus chondrosulphatase, in the absence of chondro-itinase, shows negligible activity towards polymerized chondroitin sulphatereopens the question of the specificity of the enzyme. Enzyme concen-trates containing both sulphatase and chondroitinase are without action onthe polysaccharide sulphates, heparin, agar, carragheenin, fucoidin, Chondrusocellatus mucilage, or sulphated l a m i n a r i ~ ~ . ~ ~ However, since chondroitinaseactivity was not followed during these investigations, the failure to noterelease of sulphate may simply reflect the inability of chondroitinase to degradethese substrates. On the other hand, these compounds do not contain thesulphated acetylgalactosamine residues which are present in chondroitinsulphate.The failure 40 of Protezts concentrates to liberate sulphate fromuridine diphosphate-acetylgalactosamine ~ulyhate,~' or a mixture of uridinediphosphate and acetylgalactosamine sulphate derived from this compound21, 506.K. Meyer, E. Davidson, A. Linker, and P. Hoffman, Biochiw. Biophys. A&, 1966,97 J. L. Strominger, ibid.. 1966, 17, 283DODGSON AND SPENCER : SULPHATASES. 327is unexpected, however, in view of the suggested participation of the coni-pounds in the biosynthesis of chondroitin ~ u l p h a t e . ~ ~ Little work has beendone on the specificity of other chondrosulphatases and it is not certaintherefore whether the release of sulphateg8 from the sulphated poly-saccharideg9 of the jelly coat of sea-urchin's eggs by crude extracts ofCharonia lampas or sea-urchin sperm 100 is due to the enzyme.Some con-fusion also exists on the ability of crude mollusc preparations to hydrolysecharonin sulphate.42 This activity was attributed at first to glyco-sulphatase 101 but recent work suggests that chondrosulphatase isresponsible. l02The available evidence suggests that, whatever the true substrates ofchondrosulphatase may be, they are certainly carbohydrate in Nature. TheProteus enzyme is completely inactive towards simple alkyl sulphates andthe substrates of aryl- and myro-sulphatase and of steroid-s~lphatase.~~ 1 tis interesting also that glucose 6-sulphate is not hydrolysed by the enzymeand early claims that this compound 103 and potassium myronate 39 weresubstrates for chondrosulphatase presumably reflect the presence of specificsulphatases for these substrates in the crude bacterial extracts used.Theclaim lo4 that " cerebron sulphuric acid " (now known to contain galactose6-sulphate lo5) was hydrolysed by the chondrosulphatase of these prepar-ations should also be treated with caution.The reports l1 that extracts of a flavobacterium are able to degradeheparin are interesting since it has been tentatively suggested that a glycos-idase and two distinct sulphatases, an amino- and an alcohol-sulphatase, areinvolved. It will be important in this case also to see whether sulphataseactivity is dependent on preliminary chain degradation by the glycosidase.Probably other enzyme systems containing both sulphatase and chondro-itinase-like activity will be discovered in the future.The sulphatases ofsuch systems may well possess fairly narrow substrate specificities dictatedby the chemical nature of the individual saccharides present in the substratemolecule, their mode of linkage, and the position of the sulphate groups.Glycosu1phatase.-A sulphatase capable of hydrolysing glucose 6-sulphatehas been found in snails,lo6 tropical lo7 and temperate marine molluscs,bacteria,lo6* lo8 and fungi log and in the livers of certain fishes andrnammals.ll0 Apart from a series of studies from Soda's laboratoriesbetween 1931 and 1950, this enzyme has been almost neglected. Soda andhis co-workers studied the distribution of the enzyme in various marineH.Numanoi, Sci. Papers Coll. Gen. Educ., Univ. Tokyo, 1953,3, 55; ibid., p. 71.R9 See J. Runnstrom, Symp. SOC. Ex$. Biol., 1952, 6, 39.loo H. Numanoi, Sci. Pa$ers Coll. Gen. Educ., Univ. Tokyo, 1953, 3, 67.lo1 T. Soda and Y. Yamazaki, BuEZ. Chem. SOC. Japan, 1933, 8, 207 ; C. Hattori andH. Terasaki, J . Chem. SOC. Japan, 1936, 57, 981.lop F. Egami, personal communication.lo3 B. Tankb, Biochem. Z., 1932, 247, 486.lo* C. Neuberg and W. L. Cahill, ibid., 1934-35, 275, 328.loS S. J. Thannhauser, J. Fellig, and G. Schmidt, J . BioZ. Chem., 1955, 215, 211.lo6 T. Soda and C.Hattori, Bull. CJaem. SOC. Japan, 1931, 6, 258.107 T. Soda, J . Fac. Sci. Univ., Tokyo, 1936. 3, 149.lo* Idem, Chern. Res. (Japan), 1948, 1, 51.los J. Yamashina, J . Chem. SOC. Japan, 1951, 72, 124.11O T. Soda, personal communication328 BIOLOGICAL CHEMISTRY.organisms ll1 and described the purification, properties, and specificity ofthe glycosulphatase of Chznronia Zamflas.lo7* 112 The enzyme was separatedfrom aryl- lI3 and chondro-sulphatase 89 and was able to hydrolyse a numberof mono-, di-, and tri-sulphated niono- and di-saccharides. Recently,adenosine-5’ (hydrogen sulphate) has been shown to be a substrate for theenzyme 37 whilst the ability of limpet extracts to hydrolyse cortisone 21-sulphate is probably due to the glycosulphatase present in the extracts.30Synthetic substrates have been used for the study of glycosulphatase,and the natural substrates and physiological function of the enzyme areunknown.It is possible that, like bacterial chondrosulphatase, glyco-sulphatase is normally associated with a chondroitinase-like enzyme, the twoenzymes collectively being responsible for the degradation of an unknownpolysaccharide sulphate.Steroid-su1phatase.--The series of events leading to the recent discoveryof this enzyme are worth recording. The original stimulus came fromworkers interested in finding a specific enzyme for the hydrolysis of urinarysulphate esters before ketosteroid assay, It was considered that enzymichydrolysis of steroid sulphates would be more selective and less liable toartifacts than other available methods.6914 The sulphuric esters of phenolicsteroids (e.g., oestrone sulphate) are hydrolysed by arylsulphatase 115 butnon-phenolic steroid sulphates are not substrates for thisIn attempts to obtain sulphatases capable of hydrolysing the sulphatesof neutral 17-ltetosteroirls, Buehler et al.62 examined 23 different bacteria,but no sulphatase was found able to hydrolyse either androsterone or dehydro-efiiandrosterone sulphates. The high arylsulphatase activity of the snail,Helix pornatia,117 prompted Henry and Thevenet * to use the digestive juiceof this organism for the hydrolysis of the 17-ketosteroid conjugates of urine.Hydrolysis of dehydroefliandrosterone sulphate was observed, but it was notappreciated that an enzyme, hitherto unknown, was responsible.Extractsof the limpet, Patella vulgata, have high arylsulphatase activities similar tothose of the snail 118* 119 and Stitch and his co-workers 6* 73 lZo found thatsuch extracts released sufficiently large amounts of the neutral 17-keto-steroids, which were present in urine as conjugates, to indicate the presenceof a “ steroid alcohol sulphatase.” In other experiments the extracts werefound to hydrolyse dehydroefliandrosterone sulphate. A similar enzymewas subsequently found 121 in the African land snail, Otala punctata.A more complete examination of the purified limpet enzyme has recentlybeen made by R0y.30~ 122 The enzyme is very specific since it will hydrolysell4* 116111 T.Soda and F. Egami, J . Chem. Soc. Japan, 1933, 54, 1069.11$ T. Soda and A. Yoshida, ibid., 1948, 69, 119, 121 ; 1960, 71, 60.113 T. Soda and F. Egami, ibid., 1934, 56, 256.114 R. Henry, Rec. Trav. chim., 1955, 74, 442.115 A. Butenandt and H. Hoffstetter, 2. physiol. Chem., 1939, 259, 222.116 H. Cohen and R. W. Bates, Endocrinology, 1949, 44, 317 : 45, 86.117 P. Jarrige and R. Henry, Bull. SOC. Chim. biol., 1952, 34, 872.11* I<. S. Dodgson, J . I. M. Lewis, and B. Spencer, Biochem. J., 1953, 65, 253.l1S K. S. Dodgson and B. Spencer, ibid., p. 315.lZo S. R. Stitch, 1. D. K. Halkerston, and J. Hillman, ibid., 1956, 83, 706.lZ1 H. Savard, E. Bagnoli, and R. I. Dorfman, Fed. Proc., 1954.13, 289.122 A. B. Roy, Biochim. Biophys. Ada, 1964, 15, 300DODGSON AND SPENCER : SULPHATASES.329the 3P-sulphates of 5cc- and A5-steroids only, other isomeric 3-sulphates beingunattacked. The surprising finding 30 that the enzyme preparation,like that from Otaln,l21 could hydrolyse cortisone 2f-sulphate, neednot necessarily disturb this concept of high specificity since it can beexplained by assuming that the compound was hydrolysed by someother sulphatase present in the preparation. Roy suggests 30 that glyco-sulphatase, which is known to be present in Patella,= may be the enzyme inquestion.The ability of mammalian-liver preparations to hydrolyse dehydro-eeiandrosterone sulphate has recently been reported. 123 The specificity ofthis mammalian enzyme has not been thoroughly examined but it is probablysimilar to that of the molluscan enzyme since androsterone and testosteronesulphates are not attacked.The enzyme preparations also hydrolysedaestrone sulphate but this activity is probably due to arylsulphatase C,which would also have been concentrated during the preparative procedure.In direct contradiction to this explanation is the failure of the preparationsto hydrolyse phenolphthalein disulphate but it is possible that, in commonwith certain other aryl~ulphatases,~~ arylsulphatase C has little activitytowards this substrate.Although steroid sulphatase has been recommended in preference to acidfor the hydrolysis of urinary steroid sulphates?? 6, 114 the marked specificityof the enzyme severely limits its general appli~ation.~~ Lack of knowledgeof the specificity of the snail enzyme led Jayle and Beaulieu 124 to suggestthat a urinary 17-ketosteroid conjugate which was not hydrolysed by thesnail juice was neither an ester sulphate nor a glucuronide. As Roy30points out, it is more likely that the conjugate was androsterone or a similarsulphate, against which steroid sulphatase is inactive. Roy has suggested 30that the specific nature of the enzyme might be useful in determining thestructure of steroid sulphates and gives, as an example, the fact that ranolsulphate lZ5 is not hydrolysed by the limpet enzyme arid is not therefore a3p-sulphate of a 5a- or a A5-steroid. However, a more complete study of thespecificity of the enzyme is necessary before such conclusions can be regardedas unequivocal.Steroid sulphatases from all sources are inhibited by phosphate andsulphate ions,30>120y123 and, if the enzyme is to be used in the presence ofurine, these ions should be removed 126 as the insoluble barium salts 119in order to achieve reasonable enzyme activity.Biosynthesis of Ester Sulphates.-The physiological functions of thevarious sulphatases are obscure, and it is probable that in many cases theselection of substrates for the study of the enzymes in vitro has been fortuitousand the natural substrates have yet to be discovered. The evidence availablesuggests that the sulphatases are distinct enzymes which can be differentiatedfrom many other types of esterase. They are so widely distributed inNature that it seems reasonable to suppose that they fulfil some fundamentall Z 3 H. Gibian and G. Bratfisch, 2. physiol. Chem., 1596, 305, 265.124 M. F. Jayle and E. E. Beaulieu, Bull. SOC. Chim. biol., 1954, 36, 1391.lZ5 G. A. D. Haslewood, Biochem. J., 1952, 51, 139.l z 6 S. R. Stitch and I. D. K. Halkerston, ibid., 1966, 63, 710330 BIOLOGICAL CHEMISTRY.metabolic r81e.2 At the present time all suggestions as t o the nature of thisr81e are speculative.The most obvious suggestion is that, in vivo, the sulphatases catalysethe transfer of sulphate groups to hydroxyl acceptors. From a study iizzdro of the synthesis of arylsulphates by liver ~ l i c e s , l ~ ~ homogenates,128and particle-free solutions,129 it has been shown that the reaction requiresproduction of an activated form of sulphate by a mechanism involvingadenosine triphosphate (ATP), inorganic sulphate, and an activating enzymesystem, followed by sulphate transfer to a hydroxyl acceptor through theagency of a transferase. The activating system and a phenol-specifictransferring enzyme present in mammalian livers have been separated. 17* 130Hilz and Lipmann 131 have shown that Neurospora sitophila possesses astrong sulphate-activating system but no enzyme capable of transferringsulphate to P-nitrophenol. These workers used the activating systems oflamb’s liver and Neurospora to prepare “ active sulphate ” which wasidentified as adenosine-(3’ phosphate)-(5’ phosph~sulphate).~~~ Sega1,lSusing rat-liver preparations, showed that 3-3-34 moles of orthophosphatewere liberated per mole of sulphuric ester formed, suggesting that more thanone ATP molecule is involved. Furthermore, his results show that pyro-phosphate is involved in a reversible reaction during sulphate activation andit seems probable that the formation of “ active sulphate ” is at least a two-stage process. I t is therefore interesting that the sulphate-activating systemof yeast requires at least two separate heat-labile protein f r a ~ t i 0 n s . l ~ ~ Theyeast system will also activate selenate. The arylsulphate-synthesizingsystem of rat liver has been used by Japanese workers for studies in vitro onthe metabolism (detoxication) of various aromatic compounds. 135It is not certain whether adenosine-(3’ phosphate)-(5’ phosphosulphate)can donate sulphate groups for the sulphation of compounds possessing non-phenolic hydroxyl groups. So far no such transference has been observed,but Roy 136 has suggested that the sulphation of non-phenolic steroids bymammalian-liver preparations 137 may be brought about by the same systemas that responsible for the sulphation of phenols. It seems possible that therewill be found a series of specific sulphate-transferases responsible for thetransfer of sulphate from ‘ I active sulphate,” and the ability of a tissue tosynthesize a particular sulphuric ester will depend on the presence of bothactivating system and specific transference. Thus the hen’s oviduct is ablelz7 R. H. De Meio and K. I. Arnolt, J. Biol. Chem., 1944, 156, 577.I z 8 R. H. De Meio and L. Tkacz, Arch. Biochem. Biophys., 1950, 27, 242.lZ9 R. H. De Meio, M. Wizerkaniuk, and E. Fabiani, J. Biol. Chem., 1963, 203, 257.lSo R. H. De Meio, M. Wizerkaniuk, and I. Schreibrnan, ibid., 1956, 213, 439; H. I,.131 H. Hilz and F. Lipmann, Proc. Nut. Acud. Sci. U.S.A., 1955, 41, 880.132 P. W. Robbinsand F. Lipmann, J. Amer. Chem. SOC., 1956, 78, 2652.133 H. L. Segal, Biochim. Biophys. Acta, 1956. 21, 194.13* 1,. G. Wilson and R. S. Bandurski, Arch. Biochem. Bio$hys., 1956, 62, 503.136 T. Sato, M. Yamada, T. Suzuki, and T. Fukuyama, J. Biochem. ( J a p m ) , 1966.43, 25; T. Sato, T. Suzuki, T. Fukuyama, and H. Yoshikawa, ibid., p. 413, 421.136 A. B. Roy, Biochem. J., 1956, 63, 294.13’ R. H. De Meio and C. Lewycka. EwdocrinoZogy, 1955, 56, 489; R. H. De Meio,C. Lewycka, and M. Wizerkaniuk, Fed. P‘Yoc., 1956, 15, 241 ; J. J. Schneider and M. L.Lewbart, J. BioZ. Chem., 1956, 222, 787.Segal, ibid., p. 161DODGSON AND SPENCER SULPHATASES. 33 1to synthesize " active sulphate " but does not possess the required trans-ferase for the transfer of the sulphate group to p-nitrophenol, nor is thesulphate-nitrophenol transferase of liver able to transfer sulphate to anyuridine nucleotide.138The possibility that the sulphatases are actually sulphate transferaseshas not yet been directly investigated. However, some evidence has beenoffered which suggests that the sulphate-nitrophenol transferase of liver isnot identical with arylsulphatase A, B, or C.2K. S. D.B. S.W. N. ALDRIDGE.R. K. CALLOW.K. S. DODGSON.W. C. EVANS.B. SPENCER.R. T. WILLIAMS.J. L. Strominger, Abs. Amer.. Chem. SOC. Meeting, Diu. Carbohydrate Chem.,Sept. 1956, p. 1 8 ~