Perturbing the hydrophobic pocket of mandelate racemase to probe phenyl motion during catalysis

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Crystal structures of MR reveal that the phenyl group of all ground-state ligands is located within a hydrophobic cavity, remote from the site of proton abstraction. MR forms numerous electrostatic and H-bonding interactions with the alpha-OH and carboxyl groups of the substrate, suggesting that these polar groups may remain relatively fixed in position during catalysis while the phenyl group is free to move between two binding sites [i.e., the R-pocket and the S-pocket for binding the phenyl group of (R)-mandelate and (S)-mandelate, respectively]. We show that MR binds benzilate (K(i) = 0.67 +/- 0.12 mM) and (S)-cyclohexylphenylglycolate (K(i) = 0.50 +/- 0.03 mM) as competitive inhibitors with affinities similar to that which the enzyme exhibits for the substrate. Therefore, the active site can simultaneously accommodate two phenyl groups, consistent with the existence of an R-pocket and an S-pocket. Wild-type MR exhibits a slightly higher affinity for (S)-mandelate [i.e., K(m)(S)(-)(man) < K(m)(R)(-)(man)] but catalyzes the turnover of (R)-mandelate slightly more rapidly (i.e., k(cat)(R)(-->)(S) > k(cat)(S)(-->)(R)). Upon introduction of steric bulk into the S-pocket using site-directed mutagenesis (i.e., the F52W, Y54W, and F52W/Y54W mutants), this catalytic preference is reversed. Although the catalytic efficiency (k(cat)/K(m)) of all the mutants was reduced (11-280-fold), all mutants exhibited a higher affinity for (R)-mandelate than for (S)-mandelate, and higher turnover numbers with (S)-mandelate as the substrate, relative to those with (R)-mandelate. (R)- and (S)-2-hydroxybutyrate are expected to be less sensitive to the additional steric bulk in the S-pocket. Unlike those for mandelate, the relative binding affinities for these substrate analogues are not reversed. These results are consistent with steric obstruction in the S-pocket and support the hypothesis that the phenyl group of the substrate may move between an R-pocket and an S-pocket during racemization. These conclusions were also supported by modeling of the binary complexes of the wild-type and F52W/Y54W enzymes with the substrate analogues (R)- and (S)-atrolactate, and of wild-type MR with bound benzilate using molecular dynamics simulations.     

Esters of mandelic acid as substrates for (S)-mandelate dehydrogenase from Pseudomonas putida: implications for the reaction mechanism

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes (S)-mandelate to benzoylformate. In this work, we show that the ethyl and methyl esters of (S)-mandelic acid are substrates for MDH. Although the binding affinity of the neutral esters is 25-50-fold lower relative to the negatively charged (S)-mandelate, they are oxidized with comparable k(cat)s. Substrate analogues in which the carbonyl group on the C-1 carbon is replaced by other electron-withdrawing groups were not substrates. The requirement of a carbonyl group on the C-1 carbon in a substrate suggests that the negative charge developed during the reaction is stabilized by delocalization to the carbonyl oxygen. Arg277, a residue that is important in both binding and transition state stabilization for the activity with (S)-mandelate, is also critical for transition state stabilization for the esters, but not for their binding affinity. We previously showed that the substrate oxidation half-reaction with (S)-mandelate has two rate-limiting steps of similar activation energies and proceeds through the formation of a charge-transfer complex of an electron-rich donor and oxidized FMN [Dewanti, A. R., and Mitra, B. (2003) Biochemistry 42, 12893-12901]. This charge-transfer intermediate was observed with the neutral esters as well. The observation of this electron-rich intermediate for the oxidation of an uncharged substrate to an uncharged product, as well as the critical role of Arg277 in the reaction with the esters, provides further evidence that the MDH reaction mechanism is not a concerted transfer of a hydride ion from the substrate to the FMN, but involves the transient formation of a carbanion/ene(di)olate intermediate.     

Understanding the mode of action of ThDP in benzoylformate decarboxylase

The mechanism of all elementary steps involved in the catalytic cycle of benzoylformate decarboxylase (BFD, E.C. 4.1.1.7) to generate the acyloin linkage is investigated by extensive molecular dynamics simulations. Models involving different charge states of amino acids and/or mutants of critical residues were constructed to understand the involvement of the catalytically active residues and the reactivity differences between different substrates in this reaction. Our calculations confirm that H70, S26, and H281 are catalytically active amino acids. H281 functions as a base to accept H_donor in the first nucleophilic attack and as an acid in the second, to donate the proton back to O_acceptor. S26 assists H281 in deprotonation of the donor aldehyde and protonation of the acceptor aldehyde. In both the first and second nucleophilic attacks, H70 interacts with O_aldehyde and aligns it toward the nucleophilic center. H70 has been found to have an electrostatic effect on the approaching aldehyde whose absence would block the initiation of the reaction. The reactivity difference between benzaldehyde (BA) and acetaldehyde (AA) is mainly explained by the steric interactions of the acceptor aldehyde with the surrounding amino acids in the active center of the enzyme. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 32–46, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Exploring the active site of benzaldehyde lyase by modeling and mutagenesis

Benzaldehyde lyase (BAL) is a thiamin diphosphate-dependent enzyme, which catalyzes the breakdown of (R)-benzoin to benzaldehyde. In essence, this is the reverse of the carboligation reaction catalyzed by benzoylformate decarboxylase (BFD). Here, we describe the first steps towards understanding the factors influencing BFD to form a CC bond under conditions wherein BAL will cleave the same bond. What are the similarities and differences between these two enzymes that result in the different catalytic activities? The X-ray structures of BFD and pyruvate decarboxylase (PDC) were used as templates for modeling benzaldehyde lyase. The model shows that a glutamine residue, Gln113, replaces the active site histidines of BFD and PDC. Replacement of the Gln113 by alanine or histidine reduced the value of k(cat) for lyase activity by more than 200-fold. The residues in BFD interacting with the phenyl ring of benzoylformate have similarly positioned counterparts in BAL but Ser26, the residue known to interact with the carboxylate group of benzoylformate, has been replaced by an alanine (Ala28). The BAL A28S variant exhibited 7% of WT activity in the BAL assay but, in the most intriguing result, this variant was able to catalyze the decarboxylation of benzoylformate. Conversely, the BFD S26A variant was unable to cleave benzoin.     

(S)-Mandelate dehydrogenase from Pseudomonas putida: mutations of the catalytic base histidine-274 and chemical rescue of activity.

(S)-Mandelate dehydrogenase from Pseudomonas putida, an FMN-dependent alpha-hydroxy acid dehydrogenase, oxidizes (S)-mandelate to benzoylformate. The generally accepted catalytic mechanism for this enzyme involves the formation of a carbanion intermediate. Histidine-274 has been proposed to be the active-site base that abstracts the substrate alpha-proton to generate the carbanion. Histidine-274 was altered to glycine, alanine, and asparagine. All three mutants were completely inactive. The mutants were able to form adducts with sulfite, though with much weaker affinity than the wild-type enzyme. Binding of the inhibitor, (R)-mandelate, was not greatly affected by the mutation, unlike that of the substrate, (S)-mandelate, indicating that H274 plays a role in substrate binding. The activity of H274G and, to a lesser extent, H274A could be partially restored by the addition of exogenous imidazoles. The maximum rescued activity for H274G with imidazole was approximately 0.1% of the wild-type value. Saturation kinetics obtained for rescued activity suggest that formation of a ternary complex of imidazole, enzyme, and substrate is required for catalysis. pH-dependence studies confirm that the free base form of imidazole is the rescue agent. An earlier study of pH profiles of the wild-type enzyme indicated that deprotonation of a residue with a pK(a) of 5.5 in the free enzyme was essential for activity (Lehoux, I. E., and Mitra, B. (1999) Biochemistry 38, 5836-5848). Data obtained in this work confirm that the pK(a) of 5.5 belongs to histidine-274.     

Reaction intermediate analogues for mandelate racemase: interaction between Asn 197 and the alpha-hydroxyl of the substrate promotes catalysis

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelic acid, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, carboxylate-, phosphonate-, and hydroxamate-containing substrate and intermediate analogues were examined for their ability to inhibit MR. Comparison of the competitive inhibition constants revealed that an alpha-hydroxyl function is required for recognition of the ligand as an intermediate analogue. Two intermediate analogues, alpha-hydroxybenzylphosphonate (alpha-HBP) and benzohydroxamate, were bound with affinities approximately 100-fold greater than that observed for the substrate. Furthermore, MR bound alpha-HBP enantioselectively, displaying a 35-fold higher affinity for the (S)-enantiomer relative to the (R)-enantiomer. In the X-ray structure of mandelate racemase [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., and Petsko, G. A. (1994) Biochemistry 33, 635-643], the alpha-hydroxyl function of the competitive inhibitor (S)-atrolactate is within hydrogen bonding distance of Asn 197. To demonstrate the importance of the alpha-hydroxyl function in intermediate binding, the N197A mutant was constructed. The values of k(cat) for N197A were reduced 30-fold for (R)-mandelate and 179-fold for (S)-mandelate relative to wild-type MR; the values of k(cat)/K(m) were reduced 208-fold for (R)-mandelate and 556-fold for (S)-mandelate. N197A shows only a 3.5-fold reduction in its affinity for the substrate analogue (R)-atrolactate but a 51- and 18-fold reduction in affinity for alpha-HBP and benzohydroxamate, respectively. Thus, interaction between Asn 197 and the substrate's alpha-hydroxyl function provides approximately 3.5 kcal/mol of transition-state stabilization free energy to differentially stabilize the transition state relative to the ground state.     

(S)-Mandelate dehydrogenase from Pseudomonas putida: mechanistic studies with alternate substrates and pH and kinetic isotope effects

(S)-Mandelate dehydrogenase from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, oxidizes (S)-mandelate to benzoylformate. The enzyme was purified with a carboxy-terminal histidine tag. Steady-state kinetic parameters indicate that it preferentially binds large substrates. A good correlation was obtained between the kcat, the substrate kinetic isotope effect (KIE), and the pKa of the substrate alpha-proton. The kcat decreased and the KIE increased for substrates whose alpha-protons have pKas higher than that of mandelate. These results support a mechanism involving a carbanion intermediate but are difficult to reconcile with one involving a direct hydride transfer. pH effects on steady-state parameters were determined with (S)-mandelate and a slow substrate, (R,S)-3-phenyllactate. The kcat/Km pH profile shows that two groups with apparent pKas of 5.5 and 8.9 in the free enzyme are important for activity. These pKas are shifted to 5.1 and 9.6 on binding (S)-mandelate, as shown in the kcat pH profile. The pH dependence of the KIEs suggests that the residues with these pKas are involved in the alpha-carbon-hydrogen bond-breaking step. pH dependencies of the inhibition constants for competitive inhibitors identified these residues as histidine 274 and arginine 277. We propose that histidine 274 is the base that abstracts the substrate alpha-proton and arginine 277 is important for substrate binding as well as stabilization of the carbanion/enolate intermediate.     

Role of arginine 277 in (S)-mandelate dehydrogenase from Pseudomonas putida in substrate binding and transition state stabilization

(S)-Mandelate dehydrogenase from Pseudomonas putida is an FMN-dependent alpha-hydroxy acid dehydrogenase. Structural studies of two homologous enzymes, glycolate oxidase and flavocytochrome b(2), indicated that a conserved arginine residue (R277 in MDH) interacts with the product carboxylate group [Lindqvist, Y., Branden, C.-I., Mathews, F. S., and Lederer, F. (1991) J. Biol. Chem. 266, 3198-3207]. The catalytic role of R277 was investigated by site-specific mutagenesis together with chemical rescue experiments. The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms. The k(cat) for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its K(m) value was 40-fold lower; in contrast, k(cat)s for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and K(m) values were 5-15-fold lower compared to R277K. The K(d)s for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. The k(cat) for R277G could be enhanced by the addition of exogenous guanidines or imidazoles; the maximum rescued k(cat) was approximately 70% of the wt-MDH value. Only reagents that were positively charged and could function as hydrogen bond donors were effective rescue agents. Our results indicate that R277 plays a major role in transition state stabilization through its positive charge-consistent with a mechanism involving a carbanion intermediate. The positive charge has a relatively small contribution toward substrate binding. R277 also forms a specific hydrogen bond with both the substrate and the transition state; this interaction contributes significantly to the low K(m) for (S)-mandelate.     

Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli

The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.     

Consequences of a modified putative substrate-activation site on catalysis by yeast pyruvate decarboxylase

Earlier, it had been proposed in the laboratories at Halle that a cysteine residue is responsible for the hysteretic substrate activation behavior of yeast pyruvate decarboxylase. More recently, this idea has received support in a series of studies from Rutgers with the identification of residue C221 as the site where substrate is bound to transmit the information to H92, to E91, to W412, and finally to the active center thiamin diphosphate. According to steady-state kinetic assays, the C221A/C222A variant is no longer subject to substrate activation yet is still a well-functioning enzyme. Several further experiments are reported on this variant: (1) The variant exhibits lag phases in the product formation progress curves, which can be attributed to a unimolecular step in the pre-steady-state stage of catalysis. (2) The rate of exchange with solvent deuterium of the thiamin diphosphate C2H atom is slowed by a factor of 2 compared to the wild-type enzyme, suggesting that the reduced activity that results from the substitutions some 20 A from the active center is also seen in the first key step of the reaction. (3) The solvent (deuterium oxide) kinetic isotope effect was found to be inverse on V(max)/K(m) (0.62), and small but normal on V(max) (1.26), virtually ruling out residue C221 as being responsible for the inverse effects reported for the wild-type enzyme at low substrate concentrations. The solvent kinetic isotope effects are compared to those on two related enzymes not subject to substrate activation, Zymomonas mobilis pyruvate decarboxylase and benzoylformate decarboxylase.     

Construction of an electro-enzymatic bioreactor for the production of (R)-mandelate from benzoylformate

Coupling both the electrocatalytic recycling of NADH and the enzymatic reduction of the substrate was used to produce (R)-mandelate from benzoylformate using benzoylformate reductase (BFR). The reduction of benzoylformate by BFR in combination with FAD-mediated electrolysis (at -0.5 V vs. Ag/AgCl) was complete in about 18 h and gave 47.5 mM (R)-mandelate from 50 mM substrate, while the process involving MV2+-mediated procedure (at -0.7 V vs. Ag/AgCl) produced 40 mM (R)-mandelate after 30 h. The overpotential for the NAD+ reduction could be decreased by about 0.2 V by substituting a toxic viologen derivative, MV2+, with a natural electron carrier, FAD. MV2+, however, decreased the productivity as BFR lost about 50% of its initial activity after 6 d in its presence.     

Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate: analysis of volume changes upon reaction and hysteretic behavior

Hydrolysis of the neutral substrate N-methylindoxyl acetate (NMIA) by wild-type human butyrylcholinesterase (BuChE) and peripheral site mutants (D70G, Y332A, D70G/Y332A) was found to follow the Michaelis-Menten kinetics. K(m) was 0.14 mM for wild-type, and 0.07-0.16 mM for D70G, Y332A and D70G/Y332A, indicating that the peripheral site is not involved in NMIA binding. The values of k(cat) were of the same order for all enzymes: 12,000-18,000 min(-1). Volume changes upon substrate binding (-DeltaV(K(m))) and the activation volumes (DeltaV++(k(cat)) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of -DeltaV(K(m)) indicate that NMIA binds to an aromatic residue, presumed to be W82, the active site binding locus. Binding is accompanied by a release of water molecules from the gorge. Residue 70 controls the number of water molecules that are released upon substrate binding. The values of DeltaV++(k(cat)), which are positive for wild-type and faintly positive for D70G, clearly indicate that the catalytic steps are accompanied by re-entry of water into the gorge. Results support the premise that residue D70 is involved in the conformational stabilization of the active site gorge and in control of its hydration. A slow transient, preceding the steady state, was seen on a time scale of several minutes. The induction time rapidly increased with NMIA concentration to reach a limit at substrate saturation. Much shorter induction times (<1 min) were seen for hydrolysis of benzoylcholine (BzCh) by wild-type BuChE and for hydrolysis of butyrylthiocholine (BuSCh) by the active site mutants E197Q and E197Q/G117H. This slow transient was interpreted in terms of hysteresis without kinetic cooperativity. The hysteretic behavior of BuChE results from a slow conformational equilibrium between two enzyme states E and E'. NMIA binds only to the primed form E'. Kosmotropic salts and hydrostatic pressure were found to shift the equilibrium toward E'. The E-->E' transition is accompanied by a negative activation volume (DeltaV++(0)= -45+/-10 ml/mol), and the E' form is more compact than E. Hydration water in the gorge of E' appears to be more structured than in the unprimed form.     

Neutron and X-ray crystallographic analysis of Achromobacter protease I at pD 8.0: Protonation states and hydration structure in the free-form

The structure of the free-form of Achromobacter protease I (API) at pD 8.0 was refined by simultaneous use of single crystal X-ray and neutron diffraction data sets to investigate the protonation states of key catalytic residues of the serine protease. Occupancy refinement of the catalytic triad in the active site of API free-form showed that ca. 30% of the imidazole ring of H57 and ca. 70% of the hydroxyl group of S194 were deuterated. This observation indicates that a major fraction of S194 is protonated in the absence of a substrate. The protonation state of the catalytic triad in API was compared with the bovine β-trypsin–BPTI complex. The comparison led to the hypothesis that close contact of a substrate with S194 could lower the acidity of its hydroxyl group, thereby allowing H57 to extract the hydrogen from the hydroxyl group of S194. H210, which is a residue specific to API, does not form a hydrogen bond with the catalytic triad residue D113. Instead, H210 forms a hydrogen bond network with S176, H177 and a water molecule. The close proximity of the bulky, hydrophobic residue W169 may protect this hydrogen bond network, and this protection may stabilize the function of API over a wide pH range.     

Biochemical characterization and structural analysis of a highly proficient cocaine esterase

The bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaine esterase. Hydrolysis of the cocaine benzoyl ester follows Michaelis-Menten kinetics with k(cat) = 7.8 s(-1) and K(M) = 640 nM. A similar rate is observed for hydrolysis of cocaethylene, a more potent cocaine metabolite that has been observed in patients who concurrently abuse cocaine and alcohol. The high catalytic proficiency, lack of observable product inhibition, and ability to hydrolyze both cocaine and cocaethylene make cocE an attractive candidate for rapid cocaine detoxification in an emergency setting. Recently, we determined the crystal structure of this enzyme, and showed that it is a serine carboxylesterase, with a catalytic triad formed by S117, H287, and D259 within a hydrophobic active site, and an oxyanion hole formed by the backbone amide of Y118 and the Y44 hydroxyl. The only enzyme previously known to use a Tyr side chain to form the oxyanion hole is prolyl oligopeptidase, but the Y44F mutation of cocE has a more deleterious effect on the specificity rate constant (k(cat)/K(M)) than the analogous Y473F mutation of prolyl oligopeptidase. Kinetic studies on a series of cocE mutants both validate the proposed mechanism, and reveal the relative contributions of active site residues toward substrate recognition and catalysis. Inspired by the anionic binding pocket of the cocaine binding antibody GNC92H2, we found that a Q55E mutation within the active site of cocE results in a modest (2-fold) improvement in K(M), but a 14-fold loss of k(cat). The pH rate profile of cocE was fit to the ionization of two groups (pK(a1) = 7.7; pK(a2) = 10.4) that likely represent titration of H287 and Y44, respectively. We also describe the crystal structures of both S117A and Y44F mutants of cocE. Finally, urea denaturation studies of cocE by fluorescence and circular dichroism show two unfolding transitions (0.5-0.6 M and 3.2-3.7 M urea), with the first transition likely representing pertubation of the active site.     

Intermediates and transition states in thiamin diphosphate-dependent decarboxylases. A kinetic and NMR study on wild-type indolepyruvate decarboxylase and variants using indolepyruvate, benzoylformate, and pyruvate as substrates

The thiamin diphosphate (ThDP)-dependent enzyme indolepyruvate decarboxylase (IPDC) is involved in the biosynthetic pathway of the phytohormone 3-indoleacetic acid and catalyzes the nonoxidative decarboxylation of 3-indolepyruvate to 3-indoleacetaldehyde and carbon dioxide. The steady-state distribution of covalent ThDP intermediates of IPDC reacting with 3-indolepyruvate and the alternative substrates benzoylformate and pyruvate has been analyzed by (1)H NMR spectroscopy. For the first time, we are able to isolate and directly assign covalent intermediates of ThDP with aromatic substrates. The intermediate analysis of IPDC variants is used to infer the involvement of active site side chains and functional groups of the cofactor in distinct catalytic steps during turnover of the different substrates. As a result, three residues (glutamate 468, aspartate 29, and histidine 115) positioned perpendicular to the thiazolium moiety of ThDP are involved in binding of all substrates and decarboxylation of the respective tetrahedral ThDP-substrate adducts. Most likely, interactions of these side chains with the substrate-derived carboxylate account for an optimal orientation of the substrate and/or intermediate in the course of carbon-carbon ligation and decarboxylation supporting the suggested least-motion, maximum overlap mechanism. The active site residue glutamine 383, which is located at the opposite site of the thiazolium nucleus as the "carboxylate pocket" (formed by the Glu-Asp-His triad), is central to the substrate specificity of IPDC, probably through orbital alignment. The Glu51-cofactor proton shuttle is, conjointly with the Glu-Asp-His triad, involved in multiple proton transfer steps, including ylide generation, substrate binding, and product release. Studies with para-substituted benzoylformate substrates demonstrate that the electronic properties of the substrate affect the stabilization or destabilization of the carbanion intermediate or carbanion-like transition state and in that way alter the rate dependence on decarboxylation. In conclusion, general mechanistic principles of catalysis of ThDP-dependent enzymes are discussed.     

Improved Method for Rapid Microbioassay of Amino Acids

Single and double asymmetric inductions were achieved by the sodium borohydride reduction of α-ketoesters in the presence of chirai phase transfer catalysts. The quaternary ammonium salts with hydroxyl group in the substituent moiety showed the effective catalytic activity. The reduction of (–)-(3R)-menthyl benzoylformate gave (3R)-menthyl (S)-mandelate in contrast to the prediction by the Prelog rule.     

Energetic and mechanistic studies of glucoamylase using molecular recognition of maltose OH groups coupled with site-directed mutagenesis

Molecular recognition using a series of deoxygenated maltose analogues was used to determine the substrate transition-state binding energy profiles of 10 single-residue mutants at the active site of glucoamylase from Aspergillus niger. The individual contribution of each substrate hydroxyl group to transition-state stabilization with the wild type and each mutant GA was determined from the relation Delta(DeltaG()) = -RT ln[(k(cat)/K(M))(x)/(k(cat)/K(M))(y)], where x represents either a mutant enzyme or substrate analogue and y the wild-type enzyme or parent substrate. The resulting binding energy profiles indicate that disrupting an active site hydrogen bond between enzyme and substrate, as identified in crystal structures, not only sharply reduces or eliminates the energy contributed from that particular hydrogen bond but also perturbs binding contributions from other substrate hydroxyl groups. Replacing the active site acidic groups, Asp55, Glu180, or Asp309, with the corresponding amides, and the neutral Trp178 with the basic Arg, all substantially reduced the binding energy contribution of the 4'- and 6'-OH groups of maltose at subsite -1, even though both Glu180 and Asp309 are localized at subsite 1. In contrast, the substitution, Asp176 --> Asn, located near subsites -1 and 1, did not substantially perturb any of the individual hydroxyl group binding energies. Similarly, the substitutions Tyr116 --> Ala, Ser119 --> Tyr, or Trp120 --> Phe also did not substantially alter the energy profiles even though Trp120 has a critical role in directing conformational changes necessary for activity. Since the mutations at Trp120 and Asp176 reduced k(cat) values by 50- and 12-fold, respectively, a large effect on k(cat) is not necessarily accompanied by changes in hydroxyl group binding energy contributions. Two substitutions, Asn182 --> Ala and Tyr306 --> Phe, had significant though small effects on interactions with 3- and 4'-OH, respectively. Binding interactions between the enzyme and the glucosyl group in subsite -1, particularly with the 4'- and 6'-OH groups, play an important role in substrate binding, while subsite 1 interactions may play a more important role in product release.     

Investigation of the functional contributions of invariant serine residues in yeast mevalonate diphosphate decarboxylase

Alignment of more than 20 deduced sequences for mevalonate diphosphate decarboxylase (MDD) indicates that serines 34, 36, 120,121, 153, and 155 are invariant residues that map within a proposed interdomain active site cleft. To test possible active site roles for these invariant serines, each has been mutated to alanine. S34A exhibits limited solubility and impaired binding of the fluorescent ATP analogue, trinitrophenyl-ATP (TNP-ATP), suggesting that Ser-34 substitution destabilizes proper enzyme folding. All other serine mutants retain structural integrity, as indicated by their ability to bind TNP-ATP at levels comparable to wild-type enzyme. S153A exhibits a 18-fold inflation in K(d) for Mg-ATP, as indicated by competitive displacement of TNP-ATP; the enzyme also is characterized by a 35-fold inflation in K(m) for Mg-ATP. S155A exhibits a 26-fold inflation in K(m) for Mg-ATP, but competitive displacement of TNP-ATP indicates only a 2-fold inflation in K(d) for this substrate. S155A exhibits both a 16-fold inflation in K(m) for mevalonate diphosphate and a 14-fold inflation in K(i(slope)) for the substrate analogue, diphosphoglycolylproline. These observations suggest roles for Ser-153 and Ser-155 in substrate binding. Catalytic consequences of mutating invariant serines 36, 120, 153, and 155 are modest (<8-fold diminution in k(cat)). In contrast, S121A, which exhibits only modest changes in K(d) for Mg-ATP and K(m) for mevalonate diphosphate, is characterized by a >42,000-fold diminution in k(cat), indicating the critical involvement of Ser-121 in reaction catalysis. The selective involvement of the latter of two tandem serine residues (Ser-120, Ser-121) in a conserved sequence motif suggests mechanistic similarities within the GHMP kinase superfamily of proteins.     

Involvement of the protocatechuate pathway in the metabolism of mandelic acid by Aspergillus niger

Cell-free extracts of Aspergillus niger UBC 814 grown in the presence of dl-mandelate oxidized both d(-)- and l(+)-mandelate via benzoylformate and benzaldehyde to benzoate. dl-p-Hydroxymandelate was oxidized, presumably through a parallel pathway, to p-hydroxybenzoate. A particulate d(-)-mandelate dehydrogenase and a supernatant fraction l(+)-mandelate dehydrogenase converted their respective substrates to benzoylformate. Both flavine adenine dinucleotide and flavine mononucleotide showed a stimulatory effect on the activity of the l(+)-mandelate dehydrogenase. Benzoylformate was decarboxylated to benzaldehyde by an enzyme requiring thiamine pyrophosphate for maximal activity. Two benzaldehyde dehydrogenases dependent on nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), respectively, for their activity dehydrogenated benzaldehyde to benzoate. In the presence of reduced NADP (NADPH), benzoate was oxidized via p-hydroxybenzoate and protocatechuate. Reduced NAD could not replace NADPH. Sensitive methods of assay for d(-)-mandelate dehydrogenase and benzoylformate decarboxylase are described. The fungal pathway is compared with these systems in bacteria.     

Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase.

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.