9 Reaction mechanisms Part (iii) Bioorganic enzyme-catalyzed reactions Nigel G. J. Richards Department of Chemistry University of Florida Gainesville FL 32611 USA 1 Introduction Progress in structural and mechanistic enzymology continued to be rapid in 1998 and so precludes a comprehensive description of recent developments in the understanding of the myriad catalytic mechanisms employed in primary and secondary metabolism. Continuing the philosophy of the last report in this series, this review seeks to provide an overview of enzyme mechanisms that have not been the subject of extensive literature reviews and in which chemically interesting strategies are employed in catalyzing the overall transformation. In addition two examples of mechanism-based enzyme inhibitors are discussed for which the chemical basis of inhibition has only recently been established.2 Fundamental ideas in enzyme catalysis Debate continues about the fundamental energetic factors underlying the large rate enhancements in enzyme catalyzed reactions. A superb introduction to many current conceptual problems has been provided by the publication of a series of minireview articles on the role of low-barrier hydrogen bonds, reorganization energy and electrostatic e.ects in transition state stabilization and catalysis. In particular the role of low-barrier hydrogen bonds (LBHBs) in catalysis as pointed out in an earlier review, remains controversial with di.ering interpretations of theoretical and experimental results.— The primary issue in the debate concerns the amount of covalent character that is present in the LBHB within the active site relative to that in the cognate hydrogen bond in solution. Opponents argue that LBHBs can only be catalytically e.ective when formed in non-polar environments but that such hydrogen bonds will be less e.ective at stabilizing charged transition states than regular hydrogen bonds acting via electrostatic interactions. An additional energetic penalty must be paid when polar substrates are desolvated on entering the non-polar environment from aqueous solution which might also increase the activation energy for the reaction.Attention has therefore turned to establishing the existence of very short hydrogen bonding interactions using structural methods.Nuclear magnetic resonance (NMR) spectroscopy is often employed to identify protons that participate in LBHBs 299 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 and an excellent review of the advantages in using this technique has been published. Very high-resolution X-ray crystal structures also provide experimental insight into the existence and role of LBHBs. Thus electron density from hydrogen atoms can be observed in the structure of subtilisin solved at 0.78Å resolution, and a very short hydrogen bond between the histidine and aspartic acid residues in the Ser—His—Asp catalytic triad originally assigned by NMR measurements, appears to be present. The hydrogen is shared equally between the heteroatoms in this unusual hydrogen bond. With the exception of reactions involving electron transfer, discussions of enzyme catalysis are couched in terms of transition state theory and describe reacting nuclei using classical models.Contributions to catalysis from the quantum mechanical (QM) properties of particles are usually ignored due to the relatively short de Broglie wavelengths associated with heavy atoms of su.cient energy to surmount the kinetic energy barriers present in enzyme-catalyzed reactions. The situation is less clear in the case of hydrogen transfer from donor to acceptor atoms in the transition state. For a protium atom (H) with an energy of 10 kJ mol the de Broglie wavelength is 0.5Å which is similar in magnitude to distances anticipated in many biological hydrogen transfer reactions.This is also true for enzymes employing radical intermediates in order to overcome chemical problems associated with heterolytic bond cleavage. The extent of the contribution ofQMnuclear tunneling to catalysis has therefore been explored for a number of enzymes including dehydrogenases, amine oxidases and lipoxygenase. Evidence for tunneling in hydrogen transfer reactions is provided by the observation of anomalous kinetic isotope e.ects (KIEs) on substitution of a proton in the substrate by deuterium or tritium. As a result not only must the height of the energy barrier for a chemical step be considered in rationalizing enzyme catalysis but also the width given that this parameter determines the tunneling probability of the hydrogen nucleus.The motional properties of the residues within the active site also modulate tunneling since the barrier to reaction links the potential energy wells describing reactants and products. Two models have been proposed for the coupling of enzyme dynamics and hydrogen tunneling e.ects. In the .rst classical thermal .uctuations of the enzyme mediate hydrogen transfer by shortening the tunneling distance. In the alternate model a .uctuating double well system is employed in which there is a speci.c thermally excited protein mode that alters barrier width and shape allowing the particle to tunnel to the well describing the product state. The wavefunctions’ coherency (which enables tunneling) is then destroyed by active site dynamic motions that trap the system in the product state. Both models .t the known KIE behavior in bovine serum amine oxidase but predict di.erent dependence of the KIEs on temperature.Experiments to test which model (if any) correctly describes experimental observations over a range of temperatures are underway. On the other hand the contribution of tunneling e.ects to catalysis remains controversial because the extent of enzyme catalysis is evaluated by comparing the rates of the enzymecatalyzed and uncatalyzed reactions. If extensive nuclear tunneling takes place in the uncatalyzed reaction then its e.ects will cancel out when k and k are compared. Recent calculations on carbonic anhydrase suggest that the tunneling contribution to the reaction rate is almost identical in the reaction catalyzed by carbonic anhydrase and the non-catalyzed process in aqueous solution. Good experimental data to support such calculations for non-catalyzed systems are however hard to .nd.In this 300 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 regard additional studies of KIEs associated with well-chosen model chemical reactions will be necessary to resolve this issue. 3 Co-factors biosynthesis catabolism and model systems Enzymes catalyzing the biosynthetic transformations leading to redoxactive cofactors continue to be characterized. A recent interesting example has been provided by studies on 4-(-ribofuranosyl)aminobenzene 5-phosphate (-RFAP) synthase which mediates a ce ntral step in the cellular synthesis of methanopterin 1 a modifed folate that is employed in C-1 metabolism by methanogenic archea.This reaction is unique in that the phosphoribosyl transfer yields a C-glycoside with concomitant loss of carbon dioxide. There is evidence that PLP may be present within the active site although the co-factor does not apparently play a catalytic role in the condensation reaction. In common with other PRTases,¡X the kinetic mechanism involves sequential binding of the two substrates to the enzyme to form a ternary complex. The proposed mechanism for the reaction involves an interesting modulation of reactivity in which the aromatic -electrons attack PRPP 2 via either an S1 or S2 mechanism (Scheme 1). Loss of CO from the resulting intermediate 3 then yields the nal product in a step that has chemical precedent. -Alanine 4 is an essential component in the biosynthesis of pantothenate 5 and is produced in Escherichia coli by decarboxylation of aspartic acid.This reaction is catalyzed by the enzyme aspartate decarboxylase but does not require pyridoxal phosphate as a co-factor. Instead there is a pyruvoyl residue at the active site that participates in imine formation with aspartate. Loss of CO from the resulting intermediate 6 then takes place to give -alanine after protonation and hydrolysis (Scheme 2). The catalytic pyruvoyl residue is produced in an interesting intramolecular cleavage reaction of the inactive pro-protein between residues Gly-24 and Ser-25 an example of N¡XO acyl transfer (Scheme 3). The mechanism for the activation step probably involves attack of the Ser-25 side chain on the adjacent peptide bond to yield an ester intermediate 7.-Elimination of the ester gives an enamine 8 which is then hydrolyzed to the catalytically active pyruvoyl moiety. Indirect evidence for this protein splicing reaction has been reported but the likely existence of the ester has now been demonstrated from the crystal structure of aspartate decarboxylase at 2.2 301 Annu. Rep. Prog. Chem. Sect. B 1999 95 299¡X334Scheme 1 Scheme 2 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 302 Scheme 3 resolution. Chemical models suggest that protonation of the amine is necessary to favor formation of the ester 7 at equilibrium. Signi.cant progress continues to be made in characterizing the pathways for formation and degradation of the essential co-factor thiamin 9. In prokaryotes 9 is hydrolyzed to yield the thiazole 11 and the pyrimidine 12 in a reaction catalyzed by the enzyme thiaminase-1 (Scheme 4).The biological function of this activity remains unknown. At present there are no protein sequences that are homologous to that deduced for thiaminase-1 on the basis of the sequence cloned for the gene encoding the enzyme in Bacillus thiaminolyticus. Thiaminase-1 will accept a number of nucleophiles as substrates including amines (aniline quinoline pyridine) thiols (cysteine dithiothreitol) and water. Although co-factor breakdown could proceed via direct attack of the nucleophile on 9 several lines of evidence suggest that the cleavage reaction involves a double displacement mechanism.— Site-directed mutagenesis studies implicate the Cys-113 thiolate as the enzyme-based nucleophile, a hypothesis that is supported by the X-ray crystal structure of thiaminase-1 as its covalently modi.ed form after reaction with 13. The mechanism of co-factor breakdown is therefore proposed to proceed by thiolate attack on the pyrimidine ring to yield a zwitterion that can expel the thiazole leaving group to give the modi.ed enzyme intermediate 10 (Scheme 4).Generation of the thiolate may involve general base catalyzed deprotonation of Cys-113 by the carboxylate of Glu-241. Subsequent attack of a nucleophile then proceeds to regenerate an anion from which the thiolate can be released. This proposal is consistent with the kinetic mechanism (ping-pong) and the stereochemistry of the overall reaction.303 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 4 4 Free radicals in enzyme-catalyzed reactions There is an increasing appreciation of the role of free radical intermediates in extending the repertoire of bioorganic transformations, despite the need to control the inherent reactivity of such species in order to prevent side reactions. Enzymes demonstrated to employ transient protein-based radicals in their catalytic mechanism generally perform reactions that proceed only under extreme conditions in model systems. For example heterolytic cleavage of the bond between two adjacent carbonyl groups does not proceed readily in the absence of covalent catalysis presumably because an acyl anion must be formed as an intermediate. Pyruvate formate lyase (PFL) an enzyme that plays a central role in anaerobic glucose fermentation can catalyze the interconversion of pyruvate and coenzyme A into acetyl-CoA and formate (Scheme 5). This reaction is reversible and proceeds by a ping-pong kinetic mechanism evidence for which has been provided by the isolation of the acetyl-PFL intermediate. C—C bond cleavage is accomplished using a homolytic mechanism although the details of the reaction remain controversial.PFL is synthesized as an inactive enzyme that is activated by another enzyme in an oxygen-dependent reaction. The active form of PFL has been shown to possess a protein-based radical located at Gly-734. The oxidation of glycine to a glycyl radical is chemically unprecedented although one model study has been reported. No structure is yet available for PFL but mutagenesis studies have implicated both Cys-418 and Cys-419 as residues that participate in 304 Annu.Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 5 catalysis. Two mechanisms have been proposed for the reaction both of which are not completely consistent with all experimental observations, and which lack chemical precedent for many of the individual steps. In the simplest of these, the glycyl radical generates a thiyl radical in the Cys-419 side chain that can then react with pyruvate to yield a radical intermediate 14 (Scheme 6A). Homolytic bond cleavage then gives a thioester intermediate and formate radical. The protonation state of the carboxylic acid in the enzyme has not been established although the cleavage of -ketoesters by Fenton’s reagent suggests that reaction takes place via the neutral acid. Hydrogen abstraction from Gly-734 regenerates the protein-based radical with concomitant production of formate.A series of acetyl transfers in which Cys-418 has been shown to participate then give the free enzyme and acetyl-CoA. Gly-734 and Cys-419 must therefore be in close proximity if hydrogen abstraction is to yield the thiyl radical. Evidence to support the proximity of Gly-734 and Cys-419 has recently been provided however by interesting EPR studies of the mechanism by which PFL is inactivated by oxygen. Remarkably oxygen inactivates PFL by causing breakdown of the protein into two fragments of 82 and 3 kDa the cleavage site being located at Gly-734. By monitoring the radical species formed by incubation of wild type PFL with oxygen at 77 K a long-lived sul.nyl radical (RSO·) was detected that was formed by oxidation of Cys-419.Assignment of Cys-419 as the radical site was accomplished by demonstrating that the same radical was formed by oxygen treatment of the C418A PFL mutant. Incubation of the C419A PFL mutant in which Cys-419 was replaced by an alanine residue with oxygen at 77 K however gave a di.erent species that was assigned as a peroxyl radical (ROO·) located at C of Gly-734. One plausible mechanism that is consistent with these data involves initial addition of oxygen to the Gly-734 radical in the free enzyme followed by reaction of the resulting peroxyl radical with the Cys-419 thiol and cleavage of the resulting adduct to yield the hydroxylated Gly-734 and the sul.nyl radical 17 (Scheme 7). C—Nbond cleavage can then take place to give the C-amidated 82 kDa fragment and the aldehyde 18.Although alternative mechanisms can be proposed that yield 17, all of these require that Gly-734 and Cys-419 be separated approximately by the length of molecular oxygen. A second mechanistic proposal however suggests that the key thiyl radical that initiates C—C bond cleavage is located on Cys-418 and avoids the formation of formate radical anion as an intermediate. Thus after the initial addition of the Cys-419 thiolate anion radical to the protonated form of pyruvate thiyl radical attack yields a cyclic intermediate that can undergo cleavage to the acetylated enzyme derivative 15 (Scheme 6B).Hydrogen migration followed by loss of formate then regenerates the thiyl radical which abstracts a hydrogen atom from Gly-734. Subsequent reaction of coenzyme A with the thioester gives acetyl-CoA and the free enzyme. This proposal is based on the observation that treatment of PFL with hypophosphite a formate analog in the presence of acetyl-CoA inactivates the enzyme by formation of the dead end product 16. This result is not easily accommodated by the .rst mechanism. 305 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 6A Galactose oxidase (GAO) is another enzyme for which protein-based radicals appear to play an important catalytic role. At .rst glance this is a suprising observation as the enzyme catalyzes the two-electron oxidation of the sugar substrate to the aldehyde 19 with the concomitant reduction of oxygen to hydrogen peroxide (Scheme 8).The involvement of a protein-based radical in the catalytic mechanism was .rst postulated on the basis of the unusual EPR spectrum of the redox-activated form 306 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 6B of GAO. As the enzyme also contains a Cu() metal center these spectroscopic properties suggested that a protein-based radical was anti-ferromagnetically coupled to the metal center. Subsequent EPR analysis of the protein radical obtained by oxidation of the metal-free apoenzyme showed that the radical resided on an aromatic ring.The most likely candidate is an unprecedented Cu() ligand containing a thioether bond between the sulfur atom of Cys-228 and the aromatic moiety of 307 Annu. Rep. Prog. Chem. Sect. B 1999 95 299¡X334Scheme 7 Scheme 8 Tyr-272 20 that was revealed in the recent crystal structure of GAO at 1.7Å resolution. Whether the observed structure represents the active form of the enzyme or inactive one-electron reduced GAO remains controversial. Kinetic isotope e.ect (KIE) measurements have therefore been employed in pre-steady state experiments to probe the mechanism by which galactose is oxidized. The presence of deuterium at C-6 of galactose gives rise to a dramatic H/D KIE of approximately 22 on anaerobic reduction of the enzyme by substrate. An H/D KIE of 8 persists in the oxygendependent reaction suggesting that the isotope is involved in a rate-limiting step for hydrogen peroxide formation.In an interesting observation the temperature dependence of this large KIE is consistent with the hypothesis that hydrogen tunneling takes place during catalysis. A mechanism that is consistent with these and other steadystate data has been proposed in which proton transfer from the substrate bound to the Cu(..) center occurs followed by hydrogen atom and electron transfer to give the reduced catalytic center (Scheme 9). As suggested in early studies using heterogeneous enzyme preparations containing protein in a variety of oxidation states, recent work has con.rmed that GAO exhibits ping-pong kinetics. Therefore oxygen-binding 308 Annu.Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 9 takes place after release of the aldehyde product from the active site and subsequent release of hydrogen peroxide restores the active form of the free enzyme. There is considerable evidence to support the existence of catalytically important protein-based radicals especially from studies of ribonucleotide reductase (RNR). — Amine mono-oxygenases also use protein-based radicals,— but a variety of complexco-factors including pyrroloquinoline quinone (PQQ) 21, and lysine tyrosylquinone (LTQ) 22, are present in the catalytic sites. Direct detection of substrate-based radical intermediates during RNR catalysis however has remained an elusive goal. New studies on the mechanism of RNR inhibition by the .uorinated 309 Annu.Rep. Prog. Chem. Sect. B 1999 95 299—334 nucleotide analog 23 (Scheme 10) have now provided the .rst indirect evidence for the validity of the hypothesis that the initial step in the chemistry catalyzed by RNR is hydrogen abstraction at C-3 of the ribose ring. Incubation of 23 the phosphorylated derivative of a potential new anti-cancer agent, with Escherichia coli RNR yields an inactivated enzyme species in which the tyrosyl radical is lost. EPR spectroscopic analysis however indicates that a new radical species is formed the signal for which is altered if the inhibitor is deuterated at C-6. Two structures for the radical intermediate 24 and 25 in which .uoride has been lost from the molecule are consistent with the EPR observations. These structures are formed by initial transformation of 23 into the unsaturated ketone 26 (Scheme 10), which can then react with an enzymebased nucleophile.Hydrogen abstraction by one of two possible thiyl radicals then yields inactivated RNR and either 24 or 25. Regardless of which of these alternative structures is actually formed during RNR inactivation the initial step leading to their formation must involve hydrogen abstraction at C-3 of the nucleoside analog providing further evidence in support of the generally accepted mechanism of nucleotide reduction. 5 Nitrogen metabolism The oxygen-dependent oxidation of urate 27 is important in the nitrogen metabolism of plants animals and bacteria. Indeed the urate oxidase from Aspergillus .avus has been used as a drug in the treatment of hyperuricemic disorders. The high nitrogen content of bicyclic ring systems such as that of 27 gives rise to unexpected patterns of reactivity as observed in recent studies on the enzyme urate oxidase. Although early work that is still cited in many text books suggested that urate oxidase converts 27 to allantoin 28 and carbon dioxide, recent NMR studies have shown that the true products of the enzyme-catalyzed conversion are 5-hydroxyisourate 29 and hydrogen peroxide. — The product of the reaction then undergoes a series of nonenzymatic transformations that yield CO and allantoin 28 (Scheme 11).Urate oxidase is of mechanistic interest given that it catalyzes the redox reaction without the involvement of transition metals or co-factors such as .avins. The similarity of the urate ring-system to that of .avins has lead to the proposal that the reaction may proceed via similar hydroperoxide intermediates to those that have been demonstrated in .avin-dependent enzymes and recent studies using O-labeled oxygen and water have shown that the oxygen atoms in the hydrogen peroxide formed during the reaction are both derived from molecular oxygen. Furthermore the hydroxy substituent at C-5 is derived from water. These observations are consistent with a mechanism in which hydrogen peroxide is eliminated from a hydroperoxide intermediate 30 to yield an unstable intermediate 31 that can react with water to give the .nal 310 Annu.Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 10 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 311 Scheme 11 product (Scheme 12). This proposal is supported by stopped .ow spectroscopic studies in which a series of intermediates can be observed whose absorbance characteristics are consistent with those predicted by the mechanistic scheme. In the absence of detailed computational studies it is interesting to speculate on the unusually high reactivity at C-5 in the proposed intermediate 31 which may be associated with the antiaromatic character of the 8-electron system evident in one of the resonance structures 32 that can be drawn for 31. Progress in characterizing the structures and mechanisms of glutamine-dependent amidotransferases continues to be made.— As discussed previously, these enzymes usually possess two active sites that participate in (i) catalyzing the hydrolysis of glutamine to ammonia and glutamate and (ii) binding and/or activating an electrophilic species that can react with ammonia.Structural evidence suggests that all amidotransferases possess a ‘‘channel’’ through which ammonia can pass from one 312 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 12 active site to the other sequestering this critical intermediate from solvent during the nitrogen transfer reaction. Carbamoyl phosphate synthetase (CPS) which catalyzes the formation of carbamoyl phosphate 33 from glutamine bicarbonate and ATP is a remarkable amidotransferase possessing three active sites. CPS from Escherichia coli is composed of two subunits the smaller of which contains a Cys—His—Glu catalytic triad of residues responsible for catalyzing glutamine hydrolysis, placing this enzyme in the family of Class I amidotransferases. Chemical modi.cation and site-directed mutagenesis experiments identi.ed the Cys-269 thiolate as the active site nucleophile. This prediction was con.rmed by the X-ray crystal structure of CPS, which also revealed that the fold of this glutaminase domain was similar to that observed in / hydrolases. The mechanism of glutamine hydrolysis was therefore proposed to be similar to that in thiol proteases in which histidine acts to protonate the nitrogen leaving group yielding a thioester intermediate after attack of the thiolate anion on the side chain amide of the substrate.Support for the existence of a thioester as an intermediate in the activity of the CPD small subunit was obtained in biochemical studies of CPS and the C269S CPS mutant in which the cysteine residue is replaced by serine. In an unexpected result the thioester 34 (Fig. 1) has now been observed in the X-ray structure of the H353N CPS mutant crystallized in the presence of glutamine. This mutant in which His-353 is substituted by asparagine had been shown to bind glutamine although it exhibited no detectable glutaminase activity. The original crystallography experiments were therefore aimed at determining the mode of glutamine-binding but on solving the structure it was evident that the glutamylated enzyme had been crystallized. The hydrolytic stability of the thioester was proposed to result from the inability of the mutant enzyme to activate water su.ciently for nucleophilic addition using general base catalysis.In contrast to the mechanism of glutamine activation which is apparently identical in known amidotransferases even though the glutaminase active site structures of the Class I and Class II families di.er in important respects, — there is substantial 313 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Fig. 1 variation in the reactions used in the synthetase domains of these enzymes. Structural characterization of the synthetase domains throughout Class I and Class II amidotransferases is of interest given the issue of whether structures for channeling ammonia from the glutaminase sites have evolved from a common precursor or many times.New insights into the structural basis for synthesis and activation of substrates for reaction with ammonia have been obtained in recent studies especially for NAD synthetase and glutamine fructose-6-phosphate amidotransferase (GFAT), which are both Class II amidotransferases. 314 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 13 NAD synthetase catalyzes the .nal step in NAD biosynthesis converting the deamido-NAD 35 into the .nal form of the co-factor 36. Mutants of Bacillus subtilis with severely a.ected cellular metabolism were shown to possess NAD synthetase with reduced activity. Evidence supporting a role for this enzyme in stress-response has also been reported. Interest in NAD synthetase has recently increased with the observation that there is a decrease in NAD in tubercule bacilli grown in the presence of isoniazid, opening a new avenue for developing drugs against strains of Mycobacterium tuberculosis that are multi-drug resistant. In this regard the gene encoding NAD synthetase in this organism has recently been cloned and expressed.In common with several other amidotransferases, — peptide synthetases, luciferase and tRNA synthetases, NAD synthetase catalyzes the initial formation of an NAD-adenylate 37 which is then attacked by either free ammonia as in Bacillus subtilis, or ammonia released from glutamine in an adjacent GAT-domain. The key chemical issue in employing such a strategy is to prevent hydrolysis of the adenylated intermediate before its reaction with ammonia thereby avoiding futile ATP breakdown.Insight into the mechanisms by which this problem is solved in Bacillus subtilis NAD synthetase has been provided by a recent crystal structure of the enzyme complexed to the NAD-adenylate intermediate 37. On the basis of structural homology NAD synthetase appears to be evolutionarily 315 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 related to GMP synthetase and phosphadenyl sulfate (PAPS) reductase. Since structures for the free enzyme and its complexwith ATP are available, conformational changes in the protein structure on formation of 37 are evident from this new structure. Cleavage of the ATP — bond appears to be mediated solely by two Mg ions that (i) increase the electrophilic character of the AMP and (ii) neutralize the negative charges on the pyrophosphate leaving group.No side chains from any of the protein residues are positioned so as to stabilize the transition state leading to the pentacovalent intermediate in the .rst step of NAD-adenylate formation. This catalytic strategy is in sharp contrast to the results of previous studies on Class II aminoacyl tRNA synthetases and asparagine synthetase in which evidence for the involvement of arginine side chains in catalyzing the formation of acyl-AMP intermediates has been obtained.— The crystal structure of the E·ATP·Tl complex obtained in the same study, has revealed an ammonium ion binding site adjacent to the carboxylate side chain of Asp-173.It is proposed that this site is occupied prior to formation of 37 allowing in situ delivery of nitrogen after deprotonation of the ammonium ion by Asp-173. As a result the nitrogen transfer reaction can take place in the presence of the aqueous environment. In the case of GFAT the synthetase active site must not only activate fructose-6- phosphate 38 to nucleophilic attack by ammonia released from glutamine in the N-terminal GAT-domain, by opening the furanose ring of the sugar phosphate to the corresponding linear form 39 but also subsequently act as an isomerase to convert the adduct 40 to glucosamine 6-phosphate 41 (Scheme 13). The crystal structures of a C-terminal domain proteolytic fragment of Escherichia coli GFAT which exhibits isomerase activity, complexed to 41 and a transition state analog 2-amino-2-glucitol-6-phosphate 42 have de.ned the catalytic role of key active site residues (Scheme 13).After ring-opening to yield the linear form of the substrate a step that is proposed to involve general acid/base catalysis by His-504 Lys-603 activates C-2 to attack by ammonia released in the GAT-domain of the enzyme. Independent evidence for the catalytic importance of Lys-603 was obtained in earlier site-directed mutagenesis studies. Nucleophilic attack on the Schi.s base intermediate then yields an imine from which the carboxylate of Glu-488 can abstract the C-1 proton to give the hydroxyenamine 43 an analog of the presumed enediolate intermediate in the reaction catalyzed by triose phosphate isomerase. Reprotonation at C-2 by Glu-488 then yields the linear form of 41 that can cyclize in a reaction that is also likely catalyzed by His-504.It has also been claimed that the structure of the C-terminal fragment is consistent with the existence of an ‘‘ammonia channel’’ linking the synthetase and glutaminase sites as observed in CPS and the ‘‘active’’ conformation of glutamine 5-phosphoribosylpyrophosphate amidotransferase (GPA). Direct sup- 316 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 14 port for this assertion awaits the determination of the structure of the intact enzyme by X-ray crystallography.There seems little doubt however that all glutamine-dependent amidotransferases will employ some form of channel structure for sequestering ammonia released from glutamine. The observation that indole another neutral small molecule intermediate is channeled between active sites in tryptophan synthase raises the question of whether this molecular solution is used more generally especially for passing reactive intermediates between di.erent enzymes. A particularly good example of such a situation is provided in fact by 5-phosphoribosylamine 44 which is the product of the reaction catalyzed by GPA and is the .rst intermediate in the biosynthetic pathway used for the de novo synthesis of purines. In the second step of the pathway 44 reacts with the acyl-phosphate 45 to yield glycinamide ribonucleotide 46 in a reaction catalyzed by the enzyme glycinamide ribonucleotide synthetase (GRS) (Scheme 14). In vitro kinetic studies have shown that 44 has a half-life of 5 seconds at 37 °C in aqueous solution suggesting that this reactive intermediate is passed directly from GPA to GRS in a multi-enzyme complex.E.orts to obtain kinetic evidence for the 317 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 15 existence of such a complex did not provide de.nitive support for this hypothesis. The three-dimensional structure of GRS therefore represents a major step in resolving this question. Molecular modeling studies indicate that GRS and the active conformation of GPA have complementary surfaces and hence transfer of 44 between the two enzymes via a substrate channel is structurally feasible.Kinetic studies which con.rm this hypothesis are eagerly awaited. 6 -Lactam synthesis and degradation The continued rise in bacterial resistance to -lactam antibiotics has stimulated 318 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 interest in delineating the biosynthetic pathways involved in the production of such compounds,— and the mechanisms of enzymes underlying the observed drug resistance particularly -lactamases. Clavulanic acid 49 continues to be a widely used -lactamase inhibitor and signi.cant e.orts have been made to de.ne the enzymes involved in its biosynthesis. As a result the central transformations in the pathway leading to clavulanate appear well understood.One surprising feature in the production of 49 is that clavaminate acid synthase (CAS) an -ketoglutarate-dependent (-KG) iron(..)-containing monooxygenase, catalyzes three separate transformations on the pathway each involving a di.erent substrate. Another intriguing observation and in contrast to the construction of the bicyclic system in penicillins, is that the synthesis of the -lactam moiety in 49 is not catalyzed by CAS. Instead the -lactam is formed by cyclization of 47 to 48 in an ATP-dependent reaction (Scheme 15). The details of the transformations that yield the acyclic precursor 47 remain to be established although arginine and pyruvate are almost certainly used in its construction.In mechanistic terms the chemical activation of 47 by ATP and subsequent intramolecular cyclization with the -amino group are identical to the reaction catalyzed by asparagine synthetase (AS). It was therefore predicted that the enzyme responsible for mediating the conversion of 47 to 48 -lactam synthetase (BLS) would be homologous to AS assuming that chemistry is the principal determinant of active site structure. Sequence analysis of the gene cluster encoding the enzymes involved in clavulanate biosynthesis suggested that one open reading frame ORF3 displayed approximately 25% identity over 434 of 513 residues including a motif associated with pyrophosphatase release from ATP. Cloning and expression of the enzyme encoded by the ORF gene in Saccharomyces clavuligerus then yielded pure BLS which was demonstrated to catalyze the ATP-dependent cyclization of 47 in the presence of Mg using an HPLC-based assay. A subsequent report con.rmed this assignment of enzyme function and made the intriguing mechanistic speculation that the -lactam 50 might be an intermediate in AS-catalyzed asparagine synthesis. In the absence of structural data on AS and BLS however the evolutionary relationship between the two enzymes remains to be established.319 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 16 The emergence of plasmid encoded metallo-lactamases in some bacteria has conferred simultaneous resistance to available -lactam antibiotics on these pathogens.Crystal structures,¡X in combination with metal binding and other spectroscopic studies have indicated that the active site contains two Zn() ions bridged by a deprotonated water molecule. It has been proposed that the metals perform two roles in catalysis. First coordination of the carbonyl group of the -lactam to Zn() activates the substrate to nucleophilic attack by water. Second the metals facilitate deprotonation of the water. Recent work employing nitrocen 51 has allowed the direct spectroscopic observation of an intermediate in the -lactamase catalyzed hydrolysis reaction the properties of which are consistent with the novel acyl-enzyme intermediate 52. Breakdown of 52 is rate-limiting with this substrate the anionic intermediate being stabilized to an unusual degree.In addition to extensive charge delocalization throughout the conjugated double bond system the histidinerich Zn() ion likely stabilizes the intermediate electrostatically together with residues from the substrate-binding pocket such as Lys-171 and Trp-36. The direct interaction of the second active site Zn() with the nitrogen anion excludes an apical water ligand eliminating a potential proton source from the local environment. 7 Saccharide biosynthesis Enzymes that utilize phosphoenolpyruvate continue to be of medicinal and mechanistic interest. These systems are also of mechanistic interest because reactions involving this high-energy metabolite occur through the chemically unusual scission of the C¡XO bond to give phosphate.Two types of pathways are known for PEP-utilizing enzymes in which (a) substitution occurs to give an enol ether or (b) condensation with an electrophile such as an aldehyde takes place with concomitant hydrolysis to give a keto-acid product (Scheme 16). Reactions proceeding by the second pathway are observed for DAHP synthase a key enzyme in the shikimate pathway and Kdo8P synthase which catalyzes the formation of the complexsaccharide 3-deoxy- manno-2-octulosonate-8-phosphate 53 (Kdo8P) (Scheme 17). This eight-carbon sugar is an important constituent of the cell wall in most gram negative bacteria and therefore represents an important target for the development of novel antibac- 320 Annu. Rep. Prog. Chem. Sect. B 1999 95 299¡X334Scheme 17 terial agents. Two mechanisms can be envisaged for the catalytic mechanism of Kdo8P synthase. The .rst of these involves the formation of a carbocationic intermediate that is trapped by water to form 54 (Scheme 17A).Elimination of phosphate from 54 then yields an -ketoacid which can cyclize to give the product (Scheme 17A). This mechanism is therefore analogous to that determined for EPSP synthase now that an alternative proposal (discussed in the previous review in this series) has been invalidated by new solid-state NMR experiments. The second possible catalytic mechanism for Kdo8P formation (Scheme 17B) involves a concerted step in which a cyclic intermediate is formed in the .rst step from which phosphate is removed through nucleophilic attack by a water molecule.Transient kinetic experiments using P- or C-radiolabeled aldehyde demonstrated (i) that the reaction is not reversible and (ii) the absence of covalent intermediates in which substrates become attached to 321 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Kdo8P synthase. No evidence was obtained to support the existence of cyclic intermediate 55 in the reaction mechanism an authentic sample of which was only a modest inhibitor of Kdo8P synthase activity. While acyclic bisphosphate 54 is most likely a reaction intermediate as proposed in early studies, the inherent instability of the hemiacetal moiety in 54 probably precludes its isolation and/or chemical synthesis. Indirect evidence for this hypothesis is provided however by the observation that acyclic tertiary amine 56 is a potent Kdo8P synthase inhibitor. Thus the intrinsic reactivity of PEP remains unchanged in the two mechanistic pathways employed for C—O bond cleavage by both classes of enzymes that employ this substrate.Branched chain sugars are an important class of carbohydrates many of which are present in molecules involved in cell signalling and antibiotics. The biosynthesis of these materials is thought to involve the modi.cation of simple sugar derivatives. Yersiniose 57 is found in the O-antigen of Yersinia pseudotuberculosis. Although simple methyl substituents are derived from S-adenosylmethionine pyruvate has been proposed to be the origin of the two-carbon unit present in 57. Early experiments employing cell-free extracts of the organism however failed to yield evidence supporting this hypothesis. Sequence analysis of the gene cluster encoding enzymes in the biosynthetic pathway leading to 57 has now resolved this issue.Thus the deduced amino acid sequence of the enzyme encoded by the YerE gene appeared homologous to acetohydroxyacid synthase (AHAS) a TPP-dependent enzyme catalyzing the condensation of two pyruvate molecules in the initial step of branched chain amino acid biosynthesis. AHAS is a .avoprotein in which the bound .avin is catalytically inactive apparently playing a structural role as a vestigial co-factor due to the evolution of the enzyme from pyruvate oxidase. Expression puri.cation and characterization of the YerE gene product con.rmed the ability of the enzyme to catalyze the addition of pyruvate to the ketosugar 58 using TPP as a co-factor (Scheme 18).In accordance with the predictions from sequence analysis the recombinant enzyme possesses a bound .avin that is not employed in catalysis. 322 Annu. Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 18 8 Glycosyl hydrolases Glycosyl hydrolases have functions that range from modulating viral invasion and the control of cell—cell interactions to the simple hydrolysis of polysaccharides. Structural diversity in substrates for these enzymes is mirrored by the number of sequencedistinct families of glycosyl hydrolase. Their reaction mechanisms have been the focus of numerous studies given the potential of inhibitors in the treatment of viral infections and their usefulness as model systems for elucidating the role of general acids and bases in enzyme catalysis. In particular studies on -galactosidase which hydrolyzes a wide range of -galactopyranosyl derivatives 59 via a covalent intermediate in a two-step mechanism, have provided new data on the hypothesis that enzymatic Brønsted acid/base catalysis must be enhanced relative to that in aqueous solution. New structural insights into the conformational changes in the enzyme that take place during glycosyl hydrolysis and the relocation of key catalytic side chains have recently been provided in an elegant study of all of the stable states of Bacillus 323 Annu.Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 19 agaradhaerens Cel5A along the reaction coordinate. Cel5A catalyzed glycoside hydrolysis proceeds with identical stereochemistry as observed for the reaction mediated by -galactosidase and the active site structures of the two enzymes are similar, suggesting an identical catalytic mechanism (Scheme 19).In addition to that of the free enzyme structures are reported for the Michaelis complexwith unhydrolyzed substrate 60 a catalytically competent glycosyl-enzyme intermediate 61 and the enzyme—product complex 62. These structures yield insight not only into the spatial location of groups involved in acid/base catalysis and stabilization of positively charged transition states but also into the use of binding energy in distorting substrate structure as a mechanism for lowering activation energy barriers in the reaction.9 Nucleotide hydrolysis and repair Enzymes catalyzing the speci.c cleavage and/or repair of phosphoester linkages in DNA and RNA have important cellular functions — and are of chemical interest for the development of synthetic catalysts for phosphoester hydrolysis. Topoisomerases are a particularly important class of DNA-manipulating enzyme, that solve the topological problems that are associated with DNA replication recombination and chromosome segregation. These enzymes are important clinical targets of several antibacterial and anti-cancer agents including camptothecin 63. An important step in understanding the mechanism by which these enzymes create reversible scissions in DNA using a conserved tyrosine residue, has been the recent 324 Annu.Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 20 determination of the structures of Y723F human topoisomerase I mutant in covalent and non-covalent complexes with DNA. This work has therefore a.orded snapshots of the active site region before and afterDNAcleavage. Molecular modeling indicates that the tyrosine oxygen is positioned for nucleophilic attack on the phosphodiester group and that the pentacovalent intermediate 64 is probably stabilized by two proximal arginine residues. P—O bond cleavage is then promoted by general acid catalysis involving the His-632 side chain to yield the covalently modi.ed enzyme 65 325 Annu. Rep. Prog.Chem. Sect. B 1999 95 299—334 (Scheme 20). One interesting feature of the crystal structure is the lack of protein residues that might act to facilitate nucleophilic attack by removing the proton from the Tyr-723 hydroxy group. It is possible that this role is played by an active site water molecule but the presence of the phenylalanine residue in place of Tyr-523 may cause substantial conformational rearrangement in the active site. The general base therefore remains to be identi.ed. Reversing the mechanism of phosphoester cleavage repairs the strand scission after topological rearrangement of the DNA. The molecular mechanism by which the DNA is rotated before product release remains ill-de.ned. DNA-photolyases represent another class of important DNA-manipulating enzymes which repair pyrimidine dimer lesions such as 66 that are formed on irradiation of double-strandedDNAwith UV light. Repair of the lesion requires electron Scheme 21 326 Annu.Rep. Prog. Chem. Sect. B 1999 95 299—334 transfer from a reduced deprotonated .avin co-factor and an 8-hydroxy-5-deaza.avin as a second co-factor that functions within the photolyase as a photoantenna transferring energy to the reduced .avin. Unexpectedly the separation of the two cofactors in the enzyme is approximately 17Å raising questions concerning the mechanism by which energy transfer takes place. The interesting chemical models 67 and 68 have been synthesized in an e.ort to study interactions between these .avin co-factors as a function of co-factor redoxand protonation state. Unfortunately an alternate mechanism of electron transfer interfered with the cleavage reaction in these model compounds presumably due to lack of control in the separation of the 327 Annu.Rep. Prog. Chem. Sect. B 1999 95 299—334 Scheme 22 .avin and deaza.avin moieties. Preventing such side-reactions may account for the large separation of these components in the DNA-photolyase structure. 10 Mechanism-based inhibitors Isoniazid 69 is one of the most widely employed and e.ective drugs for treating tuberculosis, but its mechanism of action and enzyme target were until recently unknown. Due to increasing isoniazid resistance exhibited by strains of Mycobacterium tuberculosis the molecular mechanism by which 69 exerts its e.ects has been delineated as an aid in developing new therapeutic agents. The biological target of 69 is an enoyl-acyl carrier protein reductase (InhA) that catalyzes the NADH-dependent reduction of unsaturated precursors of mycolic acids such as 70 and 71 which are C —C long chain -branched fatty acid components of the mycobacterial cell wall. Isoniazid 69 does not interact directly with InhA however but must be oxidized by a catalase-peroxidase katG before inhibiting the enzyme. Recent crystallographic studies have now established that the activation process yields isonicotinic acyl-NADH 73 that is bound tightly within the co-factor binding site of 328 Annu.Rep. Prog. Chem. Sect. B 1999 95 299—334 InhA. The potent inhibitor 73 is probably formed by reaction of isonicotinic acyl radical 72 obtained in katG-catalyzed isoniazid oxidation, and the NAD radical (Scheme 21).An ionic pathway involving modi.cation of NAD appears less likely as isoniazid-dependent InhA inhibition occurs fastest in the presence of NADH. Cycloserine 74 is a broad-spectrum antibiotic that has also been used in the treatment of tuberculosis and has been shown to inhibit pyridoxal phosphate (PLP) dependent enzymes such as -aminobutyric acid (GABA) aminotransferase and .-amino acid aminotransferase (.-aAT). The latter enzyme is involved in formation of the .-alanine and .-glutamate components required for cell wall biosynthesis. A number of mechanisms have been proposed for the process by which cycloserine inactivates PLP-dependent enzymes in all of which the initial adduct between 74 and PLP is transformed in the active site to an electrophilic species capable of covalently modifying the enzyme.— Recent studies on GABA aminotransferase employing radiolabeled cycloserine and isolation of radioactive inactivated adducts revealed an inhibition mechanism in which enzyme-catalyzed aromatization takes place to give a covalently modi.ed PLP co-factor 75 that is tightly bound within the active site (Scheme 22). The crystal structure of the cycloserine-inactivated form of .-aAT con.rms this proposal and clearly shows the key non-covalent interactions between 75 and the enzyme. 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