14 Biological Chemistry Part (iv) Enzyme Chemistry By M. C. SUMMERS Department of Biochemistry University of Cambridge Tennis Court Road Cambridge CB2 IQW and 0.C. WILLIAMS Department of Biochemistry Trinity College Dublin 2 Ireland 1 Irreversible Enzyme Inhibitors Irreversible enzyme inhibitors constitute a specific type of affinity labelling in which the inhibitor because of its similar structure to the natural substrate binds to the enzyme and subsequently becomes covalently attached to it.’ One class of irreversible inhibitor is the substrate analogue which carries a reactive functional group and by virtue of its structural similarities to the natural substrate binds at the enzyme active site.* Accordingly the effectiveness of this class of inhibitor depends on the binding affinity of the inhibitor for its target enzyme and the availability of reactive amino-acid residues at the enzyme active site.Another class of irreversible enzyme inhibitor is the k,, inhibit~r,~ where the enzyme substrate(s) carries a masked chemically reactive group and only after enzymic transformation is a reactive molecule produced. The product depending on its reactivity and availability of a suitable nucleophile either dissociates from the enzyme or becomes covalently attached. Inhibitors of this class have also been euphemistically referred to as ‘Suicide inhibitor^'.^ Irreversible inhibitors can provide information on the amino-acid residue(s) involved in the inactivation and the peptide sequence of the region enclosing the position of inactivation.In addition inhibitors of this type may be used to deter- mine the reactive pK of an amino-acid group involved in the ina~tivation.~ ’ Methods in Enzymology Vol. XLVI. ‘Affinity Labelling’ ed. W. B. Jakoby and M.Wilchek Academic Press N.Y.and London 1977; E. Shaw in ‘The Enzymes’ ed. P. D. Boyer Academic Press N.Y. and London 3rd ed. 1970 vol. 1 Ch. 2. * (a) M. Akhtar and D. C. Wilton Ann. Reports (B) 1973,70,98; (b)M. Akhtar and D. C. Wilton Ann. Reports (B),1971,68 167. (a) R. R. Rando Science 1974 185 320; (b) R. R. Rando. Biochem. Pharmacol. 1975 24 1153; (c) R. R. Rando Accounts Chem. Res. 1975,8 281. R. H. Abeles and A. L. Maycock Accounts Chem. Res. 1976,9 313. ’ (a)D. E. Schmidt and F.H. Westheimer Biochemistry 1971 10 1249; (b) J. R. Knowles Crit. Rev. Biuchem. 1976,4 165; (c)K. Brocklehurst and H. B. F. Dixon Biochem. J. 1977,167,859. 432 433 Biological Chemistry-Part (iv) Enzyme Chemistry 2 Acetylenic Irreversible Enzyme Inhibitors The original discovery by Bloch et d6that acetylenic substrate analogues are effective irreversible inhibitors of their respective target enzymes has led to the synthesis and study of a large number of inhibitors of this type. In recent years acetylenic substrate analogues of diverse structure have been found to inhibit irreversibly As-3-keto-steroid isomerase (see later),’ flavin-linked oxidases,8 Cu2+- amine oxidases,’ y-cystathionase,” L-alanine aminotransferase,” thiolase,” and 4-aminobutyric acid aminotransferase (see below).Two of the flavin-linked oxidases which are inhibited by acetylenic substrate analogues have been investigated with regard to the structure of the flavin-inhibitor adduct viz. L-lactate oxidase (EC.1.13.12.4) and mitochondria1 monoamine oxi- dase (EC.1.4.3.4). L-Lactate oxidase from Mycobacterium smegrnatis catalyses the oxidative decar- boxylation of L-lactate to give acetic acid and C02.2“ Abeles and co-w~rkers~~ using 2-hydroxybut-3-ynoate (l) which is both a substrate and an irreversible inhibitor of the enzyme characterized the structure of the isolated flavin-inhibitor adduct as (2) and using a combination of spectroscopic and chemical methods presented evidence for the structure of the initially formed enzyme-bound flavin- inhibitor complex as (3) (Scheme 1).R R R I I I NH N 0 + H HCrC-C-CO2H I 0 R = -CH2-(CHOH)3-CH2-OPO3-H H a; chemical modification and isolation (1) Scheme 1 6 (a) G. M. Helmkamp R. R. Rando D. J. H. Brock and K. Bloch J. Biol. Chem. 1968,243,3229; (6) K. Bloch Accounts Chem. Res. 1969,2,193; (c)K. Endo and G. M. Helmkamp J. Biol. Chem. 1970 245,4293; (d)K. Bloch in ‘The Enzymes’ ed. P. D. Boyer Academic Press N.Y. and London 3rd ed. 1971 vol. 5 p. 441. 7 F. H. Batzold and C. H. Robinson J. Amer. Chem. SOC.,1975,97,2576. 8 (a) L. Hellerman and V. G. Erwin J. Biol. Chem. 1968,243,5234;(b)A. L. Maycock R. H. Abeles J. L. Salach and T. P. Singer Biochemistry 1976 15 114; (c) J.L. Kraus and J. J. Yaouanc Mol. Pharmacol. 1977,13,378; (d)J. L. Kraus J. J. Yaouanc and G. Stratz EuropeanJ.Med. Chem. 1975 5 507; (e) C. T. Walsh A. Schonbrunn 0.Lockridge V. Massey and R. H. Abeles J. Biol. Chem. 1972,247,6004 (f)T. H. Cromartie J. Fisher G. Koczorowski R. Laura P. Marcotte and C. T.Walsh Chem. Comm. 1974 597; (g) P. Marcotte and C. T. Walsh Biochemistry 1976 15 3070; (h) K. Horiike Y. Hishina Y. Migake and T. Yamano J. Biochem. (Japan) 1975,78,57; (i)T.H. Cromartie and C. T. Walsh Biochemistry 1975 14 3482. 9 (a) R. C. Hevey J. Babson A. L. Maycock and R. H. Abeles J. Amer. Chem. SOC.,1973,95,6125; (6) R. R. Rando and J. DeMairena Biochem. Pharmacol. 1974,23,463. 10 R. H. Abeles and C. T. Walsh J.Amer. Chem. SOC., 1973,95,6124. 11 P. Marcotte and C. T. Walsh Bioche-Biophys. Res. Comm. 1975,62 677. 12 P. C. Hollan M. G. Clark and D. P. $loxham Biochemistry 1973,12,3309. 13 A. Schonbrunn R. H. Abeles C. T. Walsh S. Ghisla H. Ogata and V. Massey Biochemistry 1976,15 1798. 434 M. C.Summers and D. C. Williams In the case of mitochondria1 monoamine-oxidase studies aimed at elucidating the structure of the flavin-inhibitor adduct have been undertaken using model ~ystems'~"~ and partially purified enzyme preparations.16 In both types of study when propargylamines e.g. 3-dimethylamino-prop-1-yne (4) are. used as inhibi- tors the same flavocyanine flavin-inhibitor adduct has been characterized. The characterized adduct (5) (Scheme 2) is the result of attachment of the C-1 atom of (4)to N-5 of the flavin nuc1eus.l' Several mechanisms were proposed by Abeles et al.to account for the structure of the flavocyanine (5) reaction between R R' ! R' I NH N 0 '0 + H/ C+CH Me / HC-C-CH2N \ Me Me (4) (5 ) R = -CH2-(CHOHh -CH2 -0-(P03-)2 -adenosine R = 8a-cysteinyl peptide Scheme 2 oxidized flavin and allenic carbanion; Michael addition of reduced flavin with oxidized substrate; and radical-pair formation of flavin and substrate with subsequent collapse and protonation.to give a stable covalent adduct. However a recent report by Krantz and Lipkowitz" indicates that the mechanism of inactiva-tion may be more complex than had previously been proposed.'6 These workers studied the irreversible inhibition of monoamine oxidase using the acetylenic inhibitor (6) and the allenic isomer (7) and observed distinct spectral properties for the respective flavin-inhibitor adducts with only the former inhibitor (6) giving a spectrum typical of a flavocyanine.Krantz and Lipkowitz18 argued that if one of Me /Me H / Me-C=C-CH2-N HZC=C=C-CH2-N \CH2Ph \CH2Ph (6) (71 l4 (a)B. Gartner and P. Hemmerich Angew. Chem. Internat. Edn. 1975,14 110; (6) A. L. Maycock J. Amer. Chem. Soc. 1975 97 2270. Is B. Gartner P. Hemmerich and E. A. Zeller European J. Biochem. 1976.63 211. 16 A. L. Maycock R. H. Abeles J. I. Salach and T. P. Singer Biochemistry 1976,15 114. 17 For recent reviews of the chemistry of flavins and flavoenzymes see (a)P.Hemmerich Fortschr. Chem. org. Naturstoffe 1976 33 451; (b)T. C. Bruice Prog. Bioorganic Chem. 1976 4 1; (c) 'Flavins and Flavoproteins' ed. T. P. Singer Elsevier Amsterdam 1976; P. Hemmerich V. Massey and H. Fenner F.E.B.S.Letters 1977,84,5. 18 A. Krantz and G. S. Lipkowitz J. Amer. Chem. SOC.,1977 99 4156. Biological Chemistry -Part (iu)Enzyme Chemistry 435 the three mechanisms proposed by Abeles et al. is correct then the mechanism of inactivation of inhibitors (6) and (7) should be very similar and give rise to the same flavin-inhibitor adduct. However this appears not to be the case. At the moment the structure of the flavin-allene inhibitor adduct is unknown. Apart from the use of acetylenic substrate analogues to inhibit flavin-linked enzymes they have also been used successfully irreversibly to inhibit pyridoxal phosphate-dependent enzymes.The mechanism of inactivation of a pyridoxal phosphate enzyme is illustrated by the work of Washtien and Abeles with 'y-cystathi~nase.'~ The enzyme is inhibited irreversibly by propargylglycine (8; 2-aminopent-4-ynoic acid); the inactivation displays pseudo-first order kinetics and is accompanied by the covalent attachment of inhibitor to the enzyme. In addition when radio-labelled (8) was used it was possible to isolate an inhibitor-enzyme adduct which on treatment with aqueous acid yielded (2S)-[2-'4C]-2-amino-4- ketopentanoic acid (9). The proposed mechanism of inactivation is shown in Scheme 3. From indirect evidence it was suggested that the active site nucleophile H H+ fl J HCrC-CH2-C-CO2H 4HYC-CHKCO,H + I NH2 HX (8) HN+ + PpI Py-CHO PY CH -CH-COY Py-CHO = Pyridoxal phosphate H3CK0 +AH3 Scheme 3 (9) (X) involved in covalent bond formation is either tyrosine or cysteine.Recently Washtien et d2*have shown that y-cystathionase catalyses exchange of the a-and l9 W. Washtien and R. H. Abeles Biochemistry 1977 16 2485. 2o W.Washtien A. J. L. Cooper and R. H. Abeles Biochemistry 1977,16,460. M. C. Summers and D. C. Williams &protons of (2S)-amino-acids that are unable to undergo y-elimination and yet are competitive inhibitors of the normal enzyme-catalysed reaction. The high specificity of k,, inhibitors makes them useful agents for the study of the in vim function of enzymes and in particular in the investigation of enzyme synthesis and degradation; e.g.Abeles and Walsh" found that when mice were injected i.p. with propargylglycine a condition similar to the genetic defect cysta- thionuria was induced. A particularly useful target enzyme in this respect is y-aminobutyrate-a-ketoglutarate transaminase [GABA-T (EC.2.6.1.19)l. The putative inhibitory neurotransmitter y-aminobutyric acid (10; GABA) is degraded via transamination with 2-oxoglutarate by pyridoxal phosphate- dependent GABA-T. Irreversible inhibitors of the enzyme include ethanolamine- O-sulphate,21 4-amino-hex-5-ynoic acid,22 4-amino-hex-5-enoic acid (1 1)23and the naturally occurring neurotoxin gabaculine (12; cyclohexa-l,3-dienyl-5-amino-carboxylic acid).24 The mechanism of inactivation of GABA-T by gabaculine involves Schiff base formation with pyridoxal phosphate tautomerization and finally aromatization to give m-carboxyphenyl pyridoxamine phosphate (1 3).Chemical studies by Rando and Bangerter25 support the mechanism outlined above and in particular that the structure of the inactivator adduct has the struc- ture shown and not the other possibility resulting from Michael addition at C-3. Interestingly the dihydro analogue (14) does not irreversibly inhibit the enzyme.25 DC02H /-fco2H (13) (14) Both (1 1) and (12) appear to be very specific k,, inhibitors of GABA-T. Thus i.p. injection of gabaculine solution leads to a dose-dependent loss of GABA-T activity and an increase in GABA levels in mouse brain.Other pyridoxal phosphate- dependent enzymes such as glutamate decarboxylase ornithine decarboxylase aspartate aminotransferase and alanine aminotransferase are not inhibited irre- ~ersibly.~~' Similarly 4-amino-hex-5-enoic acid (11) when administered peripherally causes both a dose-dependent irreversible inhibition of GABA-T and an increase of brain GABA in mice.26 The tlI2of GABA-T in mouse brain was *' L. J. Fowler and R. A. John Biochem. J. 1972,130 569. 22 M. J. Jung agd B. W. Metcalf Biochem. Biophys. Res. Comm. 1975,67 301. 23 B. Lippert B. W. Metcalf M. J. Jung and P. Cassara European J. Biochem. 1977,74,441. 24 (a) R. R. Rando and F. W. Bangerter J. Amer. Chem. SOC. 1976 98 6762; (6) K. Kobayashi S.Miyazawa and A. Endo F.E.B.S. Letters 1977 76 207; (c) R. R. Rando and F. W. Bangerter Biochem. Biophys. Res. Comm. 1977,76 1276. 25 R. R. Rando and F. W. Bangerter J. Amer. Chem. SOC.,1977,99,5141. 26 M. J. Jung B. Lippert B. W. Metcalf P. Bohlen and P. J. Schechter J. Neurochem. 1977 29 797. Biologica 1 Chemistry -Part (iv) Enzyme Chemistry 437 estimated to be 3.4 days. Also the inhibitor has little or no effect on L-glutamate decarboxylase (cf. 4-amino-hex-5-ynoic acid ref. 27) succinic semialdehyde dehydrogenase aspartate aminotransferase and alanine aminotransferase.26 3 Steroid Isomerases Rose,” in a review of aldose-ketose isomerases emphasized the importance of investigating the mechanism of action of enzymes catalysing a-hydroxyketone-a- hydroxyaldehyde interconversions which do not use sugar phosphates as substrates.Monder et a[.’’ have described an enzyme from hamster liver that catalyses the isomerization of the ketol side-chain of 11-deoxycorticosterone (DOC) to the corresponding hydroxy aldehyde (isoDOC). The proposed mechanism for the isomerization is shown in Scheme 4. Initial results obtained by Monder’s group indicate that the enzyme has both similar and different properties to the more extensively studied sugar phosphate i~omerases.’~*~~ The enzyme catalyses intramolecular proton transfer between C-21 and C-20 of the steroid side-chain; this is a common feature of other isomerases e.g. glucosamine-&phosphate iso- mera~e,~~ and triose phosphate is~merase.~~ glucose-6-phosphate i~omerase,~’ The generally accepted mechanism for intramolecular proton transfer to and from adjacent carbon atoms is that a single base (-B; Scheme 4)removes a proton 0 HB DOC OH HB-i isoDOC Scheme 4 from the substrate to give a cis-enediol intermediate and protonated enzyme (B-H) this is followed by protonation of the cis-enediol on the adjacent carbon 27 M.J. Jung B. Lippert B. W. Metcalf P. J. Schechter P. Bohlen and A. Sjoerdsma J. Neurochem. 1977 28 717. 28 I. A. Rose Adv. Enzymol. 1975 43,491. 29 (a) A. K. Willingham and C. Monder Endocrin. Res. Comm. 1974,l; 145; (b)C. Monder B. Zumoff H. L. Bradlow and L. Hellman. J. Clin. Endocrin. Metabolism 1975,40,86; (c)K. 0.Martin S.W. Oh H.J. Lee and C. Monder Biochemistry 1977,16,3803. 30 I. A. Rose Brookhaven Symp. Biol. 1962,15 293. 31 C. F. Midelfort and I. A. Rose Biochemistry 1977,16 1590 32 I. A. Rose and E. L. O’Connell J. Biol. Chem. 1961,236 3086. 33 J. M. Herliky S. G. Maister W. J. Albery and J. R. Knowles Biochemistry 1976 15 5601. 0- M. C. Summers and D. C. Williams atom. There appears to be a time-dependent stereoselective exchange of the C-21 protons. However Lee and M~nder~~ have evidence of a C-20 epimerase in their isomerase perparation and it is possible that the stereochemistry at C-20 deter-mines the stereochemistry of proton removal at C-21. Another isomerase which uses a steroid as substrate is A5-3-ketosteroid iso- merase and in this case the reaction catalysed is the isomerization of a A’-3- ketosteroid to the corresponding A4-3-ketosteroid e.g.(15) -+ (16) in Scheme 5. (15) R=O (17) R =H -CH(Me)-(CH2)3-CHMe2 Scheme 5 The enzyme from Pseudomonas testosteroni has been studied in detail and in fact was the first example found of an enzyme catalysing an intramolecular proton tran~fer.~’ However reinvestigationsf the mechanism of proton transfer for the P. testosteroni enzyme indicates that the proton transfer may not be as specific as had previously been assumed36 Vige? and Marquet have now shown that during enzyme-catalysed isomerization of A’-androstendione (15) there is not only 46 -D 6p hydrogen transfer but dso exchange of the 4p hydrogen. It appears therefore that the assumed dienol intermediate may be generated by removal of either the a or proton at C-4; at the moment there are no accurate data on the relative contribution of the various exchange processes in the overall mechanism i.e.46 +6p and 4a -D 6a proton migration; 4p and 4a proton exchange. It was mentioned earlier that P. testosteroni A’-3-ketosteroid isomerase is inhibited by 34 H. J. Lee and C. Monder Biochemistry 1977,16,3810. ’’ P.Talalay and V. S. Wang Biochim. Biophys. Acta 1955,18,300. 36 A. Viger and A. Marquet. Biochim. Biophys. Acta 1977,485,482. Biological Chemistry-Part (iu) Enzyme Chemistry acetylenic substrate analogues such as (19) in Scheme 6. In 1973 Martyr and Benisek3! reported that the enzyme is also inactivated irreversibly by light at wavelengths greater than 300 nm in the presence of A4-3-ketosteroids.It has now been shown that under the conditions of photoinactivation aspartic acid-38 in the polypeptide chain is decarboxylated to alanir~e.~* R &_ &L Inactivation (19) R=O (20) R = H -CH(M~)-(CHZ)~-CH(M~)~ Scheme 6 Cholesterol oxidase from Nocardia erythropolis has mechanistic similarities to the A5-3- ketosteroid isomerase described above. The enzyme oxidizes cholesterol (18) to cholest-4-en-3-one (16) via cholest-5-en-3-one (17) with hydrogen peroxide as the other product. Smith and Brooks using cholesterol stereospecifically deu- teriated at C-4 showed that the enzyme catalysed 4p +60 proton transfer and 4a-proton exchange.39 In addition the enzyme is inhibited irreversibly by the acetylenic substrate analogue (20).40 The mechanism of inactivation of the As-3-ketosteroid isomerase and cholesterol oxidase is most probably the same and involves proton loss from the a-carbon atom to give an allene (21) which reacts with an active-site nucleophile.This is outlined in Scheme 6. Whereas the bacterial AS-ketosteroid isomerase has been studied extensively the same cannot be said of the corresponding enzyme-catalyzed process of animal origin Benson and Talalay41 observed that human and rat liver cytoplasm contains As-ketosteroid isomerase activity which was stimulated specifically by reduced glutathione (GSH). They also noted that the molecular and physical properties of the partially purified isomerase were similar to a group of basic proteins isolated from rat (6 proteins designated AA A B C D and E) and human (5 proteins designated a,p 'y 6 and E) liver and referred to as glutathione-S-transfera~es.~~ The GSH-transferases catalyse the transfer of GSH to a wide range of small organic molecules such as epoxides and halobenzenes to give the corresponding thioether.Also it transpires that one of the GSH-transferases from rat liver is identical to ligandin (GSH-transferase B) an intracellular binding protein so termed because of its ability to 37 (a)R. J. Martyr and W. F. Benisek Biochemistry 1973,12 2172; (6)R. J. Martyr and W. F. Benisek J. Biol Chem. 1975 250. 1218. J. R. Ogez W. F. Tivol and W. F. Benisek J. Biol.Chem. 1977 252 6151. 39 A. G. Smith and C. J. W. Brooks Biochem. SOC.Trans. 1977 5 1088. 40 A. G. Smith and C. J. W. Brooks Biochem. J. 1977,167 121. 41 A. M. Benson and P. Talalay Biochem. Biophys. Res. Comm. 1976,69 1073. 42 W. B. Jakoby and J. H. Keen Trends Biochem. Sci. 1977,229. 440 M. C.Summers and D. C. Williams bind hydrophobic substrates such as bilirubin bromosulphophthalein drugs cortisol metabolites and azocar~inogens.~~.~~ Remarkably Benson et al.45have now shown that the same rat and human liver proteins are responsible for the GSH-activated As-3-ketosteroid isomerase activity. In rat liver the isomerase activity is associated mainly with ligandin (GSH-transferase B) while in humans all five GSH-transferases have isomerase activity but transferase y 6 and E are considerably more active than a and p.4 Biotin-requiring Enzymes Biotin acts as a CO carrier in a number of enzyme reactions where it functions either to accept (decarboxylases) to donate (carboxylases) or to transfer (trans- carboxylases) C0246,47 There are nine known biotin-requiring enzymes and the list includes transcarboxyla~e,~~ and acetyl-CoA carboxyla~e.~~ pyruvate carb~xylase,~~ The biotin is covalently linked to the &-amino group of a lysine residue to give a biotin carboxyl carrier protein (22) and this subunit transfers CO between two 0 A HN NH 0 (CH2)4-C-N-(CH2)4-lysyl peptide (22) active sites. Amino-acid sequence data of the biotin carboxyl carrier-protein of transcarboxylase acetyl-CoA carboxylase and several pyruvate carboxylases shows a high degree of conservation particularly around the biotin atta~hment-site.~~”~ However whether the amino-acid sequence serves as a recognition factor for the attachment of biotin to the apoenzyme or a conformational or catalytic role is not known.In general the reaction catalysed by a biotin enzyme is shown in Scheme 7. The overall reaction consists of two partial reactions first carboxylation of biotin by 43 (a) G. Litwack B. Ketterer and I. M. Arias Nature 1971 234 466; (b) G. J. Smith K. Huebrer and G. Litwack Biochem. Biophys. Res. Comm. 1977,76 1174; (c) A. Grahnen and I. Sjoholm European J. Biochem. 1977,80,573. 44 (a) A. J. Levi Z. Gatmaritan and I.M. Arias J. Clin. Invest. 1969 48 2156; (6) B. Ketterer P. Ross-Mansell and J. K. Whitehead Biochem. J. 1967 103,316; (c) K. S. Morey and G. Litwack Biochemistry 1969 8 4813. 4s A. M. Benson P. Talalay J. H. Keen and W. B. Jakoby Proc. Nut. Acad. Sci. U.S.A.,1977,74 158. 46 (a) H. G. Wood and R. E. Barden Ann. Rev. Biochem. 1977 46 385; (b) J. Kaappe Ann. Rev. Biochem. 1970,39 757. 41 J. Moss and M. D. Lane Adu. Enzymol. 1971,35,757. 48 (a) H. G. Wood and G. K. Zwolinski Crit. Reu. Biochem. 1976 4 47; (b) H. G. Wood in ‘The Enzymes’ ed. P. D. Boyer Academic Press N.Y. and London 3rd ed. 1972 vol6 p. 83. 49 (a) M. Scrutton and M. R. Yount in ‘The Enzymes’ ed. P. D. Boyer Academic press N.Y. and London 3rd ed. 1972 vol 6 p. 1; (6) M.F. Utter R. E. Barden and L. B. Taylor Ado. Enrymol. 1975,42 1. 50 (a)A. W. Alberts and P. R. Vagelos in ‘The Enzymes’ ed. P. D. Boyer Academic Press N.Y. and London 3rd ed. 1972 vol 6 p. 37; (b) M. D. Lane J. Moss and S. E. Palakis Current Topics Cell Regul. 1974,8 139. 51 (a)D. B. Rylatt D. B. Keech and J. C. Wallace Biochern.SOC.Trans. 1977,5 1544; (b)M. R. Sutton R. R. Fall A. M. Nervi A. W. Alberts P. R. Vagelos and R. A. Bradshaw J. Bid. Chem. 1977 252 3934. 441 Bio logica 1 Chemistry -Part (iu ) Enzyme Chemistry ATP +HC03-E-biotin xaccep tor-C02 X ADP +Pi E-biotin-COZ acceptor Scheme I HC03- and ATP and second transfer of CO to an acceptor to give carboxylated product. The decarboxylases carry out the second partial-reaction in reverse.The following discussion concerns recent studies on transcarboxylase (EC.2.1.3.1) which catalyses the transfer of C02 from methylmalonyl-CoA to biotin to give carboxybiotin. Consequently the enzyme does not require HC03-and ATP. The second partial-reaction is the carboxylation of pyruvate to oxaloacetate by carboxybiotin (Scheme 8). coz-c02-I x Me-C-COSCoA E-biotin &H2-:-CO2H H II Me-CH2COSCoA E-biotin-C02 Me-C-C02H II 0 Scheme 8 The enzyme is found in the propionic acid bacteria and it provides the mechanism by which propionate is formed in fermentation. Studies on the quater- nary structure of the 26s form of the enzyme have been published recently; the early studies being carried out on smaller more stable forms of the enzyme complex (18s form).The 26s form of transcarboxylase is composed of a central hexameric subunit of MW 360 000 (12s) and to this are attached two sets of three subunits on opposite sides of the central subunit. Each outside subunit is a dimer of MW 120 000 (5.8s) and each dimer is attached to the central subunit by two biotin carboxyl carrier proteins of MW 12 000 (1.3s). The 26s form of the enzyme is therefore made up of thirty individual proteins to give a complex of MW 1210 000. At acid pH the 26s form is stable but as the pH is raised selective dissociation occurs and as the pH is raised even higher only the central hexameric subunit remains intact.52 The methylmalonyl-CoA and pyruvate binding sites are on different subunits and as the enzyme displays two-site Ping-Pong kinetics the biotin ‘carries’ the CO from one binding site and delivers it to the The actual mechanism of C02 transfer from methylmalonyl-CoA to biotin and from biotin-C02 to pyruvate is not completely understood although a plethora of data has been obtained from kine ti^,'^ ~tereochernical,~~ subunit ~repla~ement,~~ ’* (a) H.G. Wood Fed. Proc. 1976 35 1899; (6) H. G. Wood J. P. Chiao and E. M. Poto J. Biol. Chem. 1977 252 1490; (c) N. G. Wrigley J. P. Chiao and H. G. Wood J. Biol. Chem. 1977 252 1500; (d)E. M. Pot0 and H. G. Wood Biochemistry 1977.16 1949. 53 (a) D. B. Northrop J. Biol. Chem. 1969 244 5808; (b) D. B. Northrop and H. G. Wood J. Biol. Chem. 1969,244,5820. 54 Y.F. Cheung C. H. Fung and C. T.Walsh Biochemistry 1975,14,2981. ” (a)H. G. Wood H. Lochmueiler C. Riepertinger and F. Lynen Biochem. Z. 1963,337,247; (6) M. Chueng F. Ahman B. Jacobson and H. G. Wood Biochemistry 1975.14 1611. 442 M. C. Summers and D. C. Williams isotope,56 and n.m.r. studies.” A plausible mechanism for CO transfer is shown in Scheme 9. The mechanism incorporates a pertinent observation by Rose et ul? who observed that transcarboxylase not only catalyses CO transfer between methylmalonyl-CoA and pyruvate but also proton transfer. When [3-3Hl]pyru- vate and unlabelled methylmalonyl-CoA were incubated together with trans-carboxylase a small amount of pyruvate-derived tritium was located in the pro-2R position of propionyl-CoA. Conversely incubation of (2R)-[2-3Hl]propiony1-CoA and unlabelled oxaloacetate resulted in the transfer of a small amount of tritium to pyruvate.Rose et al. considered biotin as the most likely proton transfer agent. Thus during transfer of CO to [3-3Hl]pyruvate via a cyclic mechanism as shown in Scheme 9 some tritium is transferred to the ureido oxygen of biotin which *H\ 0 NANH +X SCoA SCoA I / 0’/c ;c\ H* Me H + Scheme 9 is then converted to carboxybiotin by another molecule of methylmalonyl-CoA. The carboxylation of biotin by methylmalonyl-CoA must be sufficiently fast that complete exchange of the tritium with solvent does not occur and some of it ends up in propionyl-CoA. A modified mechanism to the one above has been proposed by Cleland.” However the problem of a concerted or stepwise mechanism for biotin-dependent carboxylation reactions is unresolved.60 5 Adenylate Cyclase Receptor Complexes Adenylate cyclase catalyses the conversion of adenosine-5’-triphosphateinto cyclic 3’,5’-adenosine monophosphate and plays an important role in the cellular modu- lation of regulatory signals.It is generally accepted that the extracellular messengers hormones bind to a receptor protein on the external surface of the cell membrane and this binding process activates the adenylate cyclase.61 Fundamental 56 I. A. Rose E. L. O’Connell and F. Solomon J. Biol. Chem. 1976 251,902. 57 H. G. Wood Trends Biochem. Sci. 1976,4. ”A. S. Mildvan Accounts Chem. Res. 1977,10,246. 59 W. W. Cleland Adv. Enzymol. 1977 45 273.6o J. Stubbe and R. H. Abeles J. Biol. Chem. 1977 252 8338. 6’ See M. F. Greaves Nature 1977 265,681. Biological Chemistry -Part (iu) Enzyme Chemistry 443 to the understanding of the mechanism of adenylate cyclase is the elucidation of the structural and functional organization of the receptor and cyclase molecules. Using the technique of radiation-enhanced inactivation using an electron beam the component sizes of the glucagon receptor-cyclase system from rat liver plasma membranes have been determined62 in the absence and presence of glucagon. The technique neatly demonstrated that in the absence of hormone the receptor and cyclase have molecular weights corresponding to their individual molecular weights and thus are probably unlinked.In the presence of glucagon associatian of receptor and cyclase takes place and the complex appears to be in the dimeric form with two molecules each of receptor and cyclase. The interaction between receptors and adenylate cyclase from different cells using the technique of cell fusion has also been The catecholamine receptor from turkey erythrocytes has been shown to activate and thus interact with adenylate cyclase from mouse erythroleukaemia cells within minutes of cell fusion. Recently it was dern~nstrated~~ that the catecholamine receptor can be donated and coupled to adenylate cyclase from a number of diverse cell types e.g. erythrocytes containing receptor have been fused with mouse adrenal tumour cells; rat glioma cells containing receptor have been fused with mouse erythroleukaemia cells.Thus evidence has been produced which supports the ‘mobile-receptor’ hypo- thesis that receptor and cyclase are freely dissociable in cell membranes. On agonist-receptor binding receptor-cyclase interaction is locked causing activation of cyclase and thus formation of CAMP to perform its regulatory functions within the cell. 6 Multienzyme Complexes Multi-enzyme complexes are aggregates of enzymes which catalyse two or more steps in a metabolic sequence the most-widely known example being fatty acid synthase a cluster of seven enzymes which catalyses the biosynthesis of fatty acids from acetyl- and malonyl-CoA. Many enzymes have been implicated in multi- enzyme complexes and the advantages of association of enzymes for metabolic pathways has been An extreme example of a multienzyme complex is a multifunctional polypeptide a single protein chain having more than one catalytic activity.Multifunctional polypeptides are common in the biosynthetic pathway to the aromatic amino-acids tyrosine phenylalanine and tryptophan; examples being; chorismate mutase-pre- phenate dehydrogenase of Escherichia tryptophan synthase of Neurospora cra~sa,~’ indol-3-yl glycerol phosphate synthase of E. coli,68 and 3-deoxy-~- arabino-heptulosonate 7-phosphate synthase-chorismate mutase of Bacillus 62 M. D. Housley J. C. Ellery G. A. Smith T. R. Hesketh J. M. Stein G. B. Warren and J. C. Metcalfe Biochim. Biophys. Acta 1977,467 208. 63 J. Orly and M. Schramm Proc.Nut. Acad. Sci. U.S.A.,1976,73 4410. 64 M. Schramm J. Orly S. Eimerl and M. Korner Nature 1977,268 310. See for instance L. J. Reed and D. J. Cox in ‘The Enzymes’ (Student Edition) ed. P. D. Boyer Academic Press New York 1970 vol. 1 Ch. 4. 66 B. E. Davidson E. H. Blackburn andT. A. A. Dopheide J. Biol. Chem. 1972,247,4441. 67 W. H. Matchett and J. A. DeMoss J. Biol. Chem. 1975 250,2941. “ T. E. Creighton and C. Yanofsky J. Bid. Chem. 1966 241,4616. 444 M. C.Summers and D. C. Williams s~btilis,~~ which all contain bi-functional polypeptides. A trifunctional polypeptide anthranilate synthase from N. crassa has also been de~cribed.~' The urom multienzyme complex of N. crassa has been shown'l to catalyse five consecutive steps in the aromatic amino-acid biosynthetic pathway the conversion of 3-deoxy-~-arabino-heptulosonate-7-phosphate (23) into 5-enoylpyruvoyl-shikimate-3-phosphate (28),the immediate precursor of chorismic acid which is itself the common precursor of tyrosine phenylalanine and tryptophan.The pathway proceeds via 3-dehydroquinate (24) 3-dehydroshikimate (25) D-shik- imate (26),and 3-phospho-shikimate (27) as shown in Scheme 10. CO,H HO CO,H CO2H HO 'O@OH* OQOH-OOOH C0,H CO,H POQOtCOZH CH OH OH -poooH (28) (27) P= Po3= Scheme 10 Recently a new procedure for the purification of the arom complex from N. crassa has been reported7* which minimized possible proteolytic damage to the enzyme. A homogeneous enzyme was obtained MW 270000 which under dis- sociating conditions produced apparently identical subunits of 165 000.Evidence was presented that proteolysis leads to progressive degradation of the complex into smaller subunits which retain enzymic activity thus accounting for the confusing subunit compositions previously obtained. It was concluded that the arom complex consists of two identical subunits each composed of a single polypeptide-chain capable of catalysing all five reactions. It would appear that discrete folding domains exist for each catalytic activity and that the domains have become connected as a result of fusion of the five structural genes coding for the enzymes and indeed it has been that the five genes occur as a cluster. The co-synthesis of these five activities thus provides an economy of synthesis of the enzymes of the biosynthetic pathway.69 L. Huang A. L. Montoya and E. W. Nester J. Biol. Chem. 1974,249,4473. '* F. M.Hulett and J. A. DeMoss J. Biol. Chem. 1975,250,6648. 71 N. H.Giles M. E. Case C. W. H. Partridge and S. I. Ahmed Proc. Nut. Acad. Sci. U.S.A.,1967,58 1453. '' J. Lumsden and J. R. Coggins Biochem. J. 1977,161,599. 73 S.R.Gross and A Fein Genetics 1960,45 885. 445 Biological Chemistry -Part (iu)Enzyme Chemistry Pyruvate dehydrogenase multienzyme complex74 from E. coli catalyses the reac- tion according to the generally accepted mechanism shown in Scheme 11. Pyruvate +NAD++CoA + Acetyl-CoA +NADH +H++C02 CoASH MeCOC02H r NADH Lip =Lipoic acid TPP =Thiamine pyrophosphate I NAD+ Scheme 11 The complex has a large molecular weight (-8 X lo6) and is composed of multiple copies of three different subunits having pyruvate decarboxylase (El) lipoate acetyltransferase (E2) and lipoamide dehydrogenase (E3) activity respec- tively.There has been some confusion as to the stoicheiometry of the subunits in the complex and the symmetry of the complex (see78). Evidence has been pr~vided~~.’~ for the involvement of a lipoyl-lysine ‘swinging arm’ which can transfer the ‘acyl’ moiety between subunits but the mechanism cannot be simple since each E2 subunit carries two functionally active lipoyl residues.77 Recent studies7’ on the self-assembly of the complex have demonstrated a 2 :1 stoicheiometry of El :E2and shown that the overall catalytic activity is proportional to the state of assembly of the complex.In the presence of [2-14C]pyruvate and absence of CoA the lipoic acid residues of the complex become acetylated and the reaction ceases. Thus a partial reaction involving El and E2 subunits can be studied while excluding that involving E3. It has been shown7’ that the depen- dance of acetylation of the E2 subunit on E1:E2 ratio (partially re-assembled complexes) is not as predicted by a simple El :E2 ratio of 2 :1 but rather by an El:EZratioof 24*2:1. Thus it would appear that each EZsubunit can be ‘catalytically serviced’ by 12 El dimers. On this basis a complex sub-structure is postulated consisting of a central E2 core a cube with octahedral symmetry comprising 24 polypeptide chains.Since each El dimer can acetylate half of the total E2 subunits and must perform this 74 L. J. Reed Accounts Chem. Res. 1974,7,40. 75 M. C. Ambrose and R. N. Perham Biochem. J. 1976,155,429. 76 H. J. Grande H. J. Van Telgen and C. Veeger European J. Bidchem. 1976,71 509. 77 M.J. Danson and R. N. Perham Biochem. J. 1976,159,677. 78 D. L.Bates M. J. Danson G. Hale E. A. Hooper and R. N. Perham Nature 1977,268 313 446 M C,Summers and D. C. Williams reaction indirectly (for direct acetylation large distances would have to be spanned) transacetylation reactions between E2subunits have been postulated. An El dimer bound to one of the E2subunits could cause direct acetylation of either 3 (or 4) E2 subunits. Indirect acetylation (transacetylation) could then occur between a directly-acetylated EZsubunit and either 3 (or 2) other Ez subunits.Thus only 12 EZsubunits could be serviced by each El dimer. This ‘transacetylation’ reaction provides a novel functional connection between active sites; however the significance of the reaction is not yet apparent.78 The possibility of interaction between the glycolytic enzymes and the advantages of such interactions have been frequently Recently direct evidence has been produced in favour of the presence of such glycolytic complexes. Glycolytic enzymes from rat skeletal muscle have been shown” to exist as a complex with myosin. However this situation could be regarded as an exclusive case being a consequence of accommodating rapid anaerobic respiration during muscle action.Recently a large aggregate has been isolated from E. coli spheroplastss1*s2which demonstrates all the enzyme activities of glycolysis. Gel chromatography demon- strated the presence of a fraction with high mol. wt. (1.6 X lo6) containing all the enzyme activities as well as low mol. wt. fractions (corresponding to the individual enzymes). Reassembly of the individual enzymes was demonstrated on concen- tration of the fractions. Total flux through the glycolytic pathway from [U-14 Clglucose to pyruvate was demonstrated. The presence of unlabelled glycolytic intermediates reduced the specific radioactivity of the pyruvate (isolated as alanine) by a much smaller extent than that expected for free mixing of intermediates and thus a functional organization within the complex is indicated.Another glycolytic complex has been reported83 from the parasitic protozoan Trypanosoma brucei where all the glycolytic enzymes are associated with a rapidly sedimentable particle on sucrose density gradient centrifugation. Latency studies have indicated that the enzymes were contained in a microbody which has been named the ‘glycosome’. These organisms when in the morphological form found in the host bloodstream rely completely on a modified glycolysis scheme for energy production the respiratory chain and citric-acid cycle enzymes being absent. Glucose is converted to pyruvate and NADH reoxidized via coupled glycerol-3- phosphate dehydrogenase :glycerol-3-phosphate oxidase. It would appear that the glycosome has developed to optimize conditions for glycolysis with high substrate and enzyme concentrations being maintained inside the microbody.79 See for instance C. de Duve in ‘Structure and Function of Oxidation-Reduction Enzymes’ ed..A. Akeson and A. Ehrenberg Pergarnon Press Oxford and N.Y. 1972 pp. 715-728. F. M. Clarke and C. J. Masters Biochim. Biophys.Acta 1973,327,233; 1974 358 193. J. Mowbray and V. Moses European J. Biochem. 1976,66 25. 82 D. M. Gorringe and V. Moses Biochem. SOC. Trans. 1978,6 167. 83 F. R. Opperdoes and P. Borst F.E.B.S. Letters 1977,80 360.