The molecular basis for inhibition of BphD, a C-C bond hydrolase involved in polychlorinated biphenyls degradation: large 3-substituents prevent tautomerization

The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H>F>Cl>Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A.3-Cl HOPDA and S112A.3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A.HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 A from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A.3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation.     

Kinetic and structural insight into the mechanism of BphD, a C-C bond hydrolase from the biphenyl degradation pathway

Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD(LB400)) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 degrees C showed that the enzyme rapidly (1/tau(1) approximately 500 s(-1)) transforms HOPDA (lambda(max) = 434 nm) into a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/tau(2) = 54 s(-1), 1/tau(3) = 6 s(-1) approximately k(cat)) with simultaneous biphasic appearance (48 and 8 s(-1)) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/tau(1) and 1/tau(2) but affected neither 1/tau(3) nor k(cat), suggesting that 1/tau(2) may reflect diffusive HPD dissociation, and 1/tau(3) represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and, therefore, postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (lambda(max) = 492 nm). The crystal structures of the wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 A resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogen-bonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.     

The tautomeric half-reaction of BphD, a C-C bond hydrolase. Kinetic and structural evidence supporting a key role for histidine 265 of the catalytic triad

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (lambda(max) is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (lambda(max) is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (lambda(max) is 506 nm) with a similar rate, 1/tau approximately 500 s(-1). The crystal structure of the S112A:HOPDA complex at 1.8-A resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7 A away.     

Identification of a serine hydrolase as a key determinant in the microbial degradation of polychlorinated biphenyls

The ability of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) of Burkholderia cepacia LB400 to hydrolyze polychlorinated biphenyl (PCB) metabolites was assessed by determining its specificity for monochlorinated HOPDAs. The relative specificities of BphD for HOPDAs bearing chlorine substituents on the phenyl moiety were 0.28, 0.38, and 1.1 for 8-Cl, 9-Cl, and 10-Cl HOPDA, respectively, versus HOPDA (100 mm phosphate, pH 7.5, 25 degrees C). In contrast, HOPDAs bearing chlorine substituents on the dienoate moiety were poor substrates for BphD, which hydrolyzed 3-Cl, 4-Cl, and 5-Cl HOPDA at relative maximal rates of 2.1 x 10(-3), 1.4 x 10(-4), and 0.36, respectively, versus HOPDA. The enzymatic transformation of 3-, 5-, 8-, 9-, and 10-Cl HOPDAs yielded stoichiometric quantities of the corresponding benzoate, indicating that BphD catalyzes the hydrolysis of these HOPDAs in the same manner as unchlorinated HOPDA. HOPDAs also underwent a nonenzymatic transformation to products that included acetophenone. In the case of 4-Cl HOPDA, this transformation proceeded via the formation of 4-OH HOPDA (t(12) = 2.8 h; 100 mm phosphate, pH 7.5, 25 degrees C). 3-Cl HOPDA (t(12) = 504 h) was almost 3 times more stable than 4-OH HOPDA. Finally, 3-Cl, 4-Cl and 4-OH HOPDAs competitively inhibited the BphD-catalyzed hydrolysis of HOPDA (K(ic) values of 0.57 +/- 0. 04, 3.6 +/- 0.2, and 0.95 +/- 0.04 microm, respectively). These results explain the accumulation of HOPDAs and chloroacetophenones in the microbial degradation of certain PCB congeners. More significantly, they indicate that in the degradation of PCB mixtures, BphD would be inhibited, thereby slowing the mineralization of all congeners. BphD is thus a key determinant in the aerobic microbial degradation of PCBs.     

Evidence for a gem-diol reaction intermediate in bacterial C-C hydrolase enzymes BphD and MhpC from 13C NMR spectroscopy

C-C hydrolase enzymes MhpC and BphD catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid and Burkholderia xenovorans LB400 biphenyl degradation pathways and are both members of the alpha/beta-hydrolase family containing a Ser-His-Asp catalytic triad. The catalytic mechanism of this family of enzymes is thought to proceed via a gem-diol reaction intermediate, which has not been observed directly. Site-directed single mutants of BphD in which catalytic residues His-265 and Ser-112 were replaced with Ala were found to possess 10(4)-fold reduced k(cat) values, and in each case, the C-C cleavage step was shown by pre-steady-state kinetic analysis to be rate-limiting. The processing of a 6-(13)C-labeled aryl-containing substrate by these H265A or S112A mutant BphD enzymes was monitored directly by (13)C NMR spectroscopy. A new line-broadened signal was observed at 128 ppm for each enzyme, corresponding to the proposed gem-diol reaction intermediate, over a time scale of 1-24 h. A similar signal was observed upon incubation of the (13)C-labeled substrate with an H114A MhpC mutant, which is able to accept the 6-phenyl-containing substrate, on a shorter time scale. The direct observation of a gem-diol intermediate provides further evidence that supports a general base mechanism for this family of enzymes.     

Comparative specificities of two evolutionarily divergent hydrolases involved in microbial degradation of polychlorinated biphenyls

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs) by Burkholderia sp. strain LB400 (S. Y. K. Seah, G. Labbé, S. Nerdinger, M. Johnson, V. Snieckus, and L. D. Eltis, J. Biol. Chem. 275:15701-15708, 2000). To determine whether this is also true in divergent biphenyl degraders, the homologous hydrolase of Rhodococcus globerulus P6, BphD(P6), was hyperexpressed, purified to apparent homogeneity, and studied by steady-state kinetics. BphD(P6) hydrolyzed HOPDA with a k(cat)/K(m) of 1.62 (+/- 0.03) x 10(7) M(-1) s(-1) (100 mM phosphate [pH 7.5], 25 degrees C), which is within 70% of that of BphD(LB400). BphD(P6) was also similar to BphD(LB400) in that it catalyzed the hydrolysis of HOPDAs bearing chloro substituents on the phenyl moiety at least 25 times more specifically than those bearing chloro substituents on the dienoate moiety. However, the rhodococcal enzyme was significantly more specific for 9-Cl and 10-Cl HOPDAs, catalyzing the hydrolysis of 9-Cl, 10-Cl, and 9,10-diCl HOPDAs two- to threefold respectively, more specifically than HOPDA. Moreover, 4-Cl HOPDA competitively inhibited BphD(P6) more effectively than 3-Cl HOPDA, which is the inverse of what was observed in BphD(LB400). These results demonstrate that BphD is a key determinant in the aerobic transformation of PCBs by divergent biphenyl degraders, but that there exists significant diversity in the specificity of these biphenyl hydrolases.     

Characterization of a C-C bond hydrolase from Sphingomonas wittichii RW1 with novel specificities towards polychlorinated biphenyl metabolites

Sphingomonas wittichii RW1 degrades chlorinated dibenzofurans and dibenzo-p-dioxins via meta cleavage. We used inverse PCR to amplify dxnB2, a gene encoding one of three meta-cleavage product (MCP) hydrolases identified in the organism that are homologues of BphD involved in biphenyl catabolism. Purified DxnB2 catalyzed the hydrolysis of 8-OH 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) approximately six times faster than for HOPDA at saturating substrate concentrations. Moreover, the specificity of DxnB2 for HOPDA (k(cat)/K(m) = 1.2 x 10(7) M(-1) s(-1)) was about half that of the BphDs of Burkholderia xenovorans LB400 and Rhodococcus globerulus P6, two potent polychlorinated biphenyl (PCB)-degrading strains. Interestingly, DxnB2 transformed 3-Cl and 4-OH HOPDAs, compounds that inhibit the BphDs and limit PCB degradation. DxnB2 had a higher specificity for 9-Cl HOPDA than for HOPDA but a lower specificity for 8-Cl HOPDA (k(cat)/K(m) = 1.7 x 10(6) M(-1) s(-1)), the chlorinated analog of 8-OH HOPDA produced during dibenzofuran catabolism. Phylogenetic analyses based on structure-guided sequence alignment revealed that DxnB2 belongs to a previously unrecognized class of MCP hydrolases, evolutionarily divergent from the BphDs although the physiological substrates of both enzyme types are HOPDAs. However, both classes of enzymes have mainly small hydrophobic residues lining the subsite that binds the C-6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phe106, Phe135, Trp150, and Phe197) found in the class II enzymes that prefer substrates possessing a C-6 alkyl. Thr196 and/or Asn203 appears to be an important determinant of specificity for DxnB2, potentially forming hydrogen bonds with the 8-OH substituent. This study demonstrates that the substrate specificities of evolutionarily divergent hydrolases may be useful for degrading mixtures of pollutants, such as PCBs.     

Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD

The alpha/beta-hydrolase superfamily, comprised mainly of esterase and lipase enzymes, contains a family of bacterial C-C hydrolases, including MhpC and BphD which catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid pathway and Burkholderia xenovorans LB400 biphenyl degradation pathway, respectively. Five active site amino acid residues (Arg-188, Asn-109, Phe-173, Cys-261, and Trp-264) were identified from sequence alignments that are conserved in C-C hydrolases, but not in enzymes of different function. Replacement of Arg-188 in MhpC with Gln and Lys led to 200- and 40-fold decreases, respectively, in k(cat); the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in k(cat). Pre-steady-state kinetic analysis of the R188Q MhpC mutant revealed that the first step of the reaction, keto-enol tautomerization, had become rate-limiting, indicating that Arg-188 has a catalytic role in ketonization of the dienol substrate, which we propose is via substrate destabilization. Mutation of nearby residues Phe-173 and Trp-264 to Gly gave 4-10-fold reductions in k(cat) but 10-20-fold increases in K(m), indicating that these residues are primarily involved in substrate binding. The X-ray structure of a succinate-H263A MhpC complex shows concerted movements in the positions of both Phe-173 and Trp-264 that line the approach to Arg-188. Mutation of Asn-109 to Ala and His yielded 200- and 350-fold reductions, respectively, in k(cat) and pre-steady-state kinetic behavior similar to that of a previous S110A mutant, indicating a role for Asn-109 is positioning the active site loop containing Ser-110. The catalytic role of Arg-188 is rationalized by a hydrogen bond network close to the C-1 carboxylate of the substrate, which positions the substrate and promotes substrate ketonization, probably via destabilization of the bound substrate.     

Kinetic analysis of the action of tissue transglutaminase on peptide and protein substrates

Tissue transglutaminase (TGase) catalyzes transfer of gamma-acyl moieties of Gln residues in peptides or protein substrates to either water or amine nucleophiles through an acyl-enzyme intermediate formed from initial acyl-transfer to an active site Cys residue. Natural substrates for this enzyme include proteins (e.g., tau, alpha-synuclein, and huntingtin) whose TGase-promoted polymerization may be causative in neurodegenerative diseases. As part of a program to find inhibitors of TGase, we have undertaken kinetic and mechanistic studies of the enzyme from guinea pig (gpTGase) and humans (hTGase). Key findings of this study include: (i) gpTGase-catalyzed transamidation of Z-Gln-Gly by Gly-OMe proceeds essentially as described above but with the involvement of substrate inhibition by Gly-OMe. This phenomena, resulting from the binding of nucleophile to free enzyme, appears to be a common feature of TGase-catalyzed reactions. (ii) Solvent deuterium isotope effects for hydrolysis of Z-Gln-Gly by gpTGase are (D)(k(c)/K(m)) = 0.45 and (D)k(c) = 3.6. While the latter results from general catalysis of deacylation, the former originates purely from the reactant state, hydrogen fractionation factor of the active site thiol with no involvement of general catalysis of acylation. (iii) Studies of the transamidation of N,N-dimethylated casein by Gly-OMe and dansyl-cadaverine suggest a complex kinetic mechanism for both enzymes that reflects contributions from four reactions: Gln hydrolysis, intramolecular transpeptidation, intermolecular transpeptidation, and transamidation by added nucleophile.     

Mechanistic origins of the substrate selectivity of serine proteases

Serine proteases catalyze the hydrolysis of amide bonds of their protein and peptide substrates through a mechanism involving the intermediacy of an acyl-enzyme. While the rate constant for formation of this intermediate, k(2), shows a dramatic dependence on peptide chain length, the rate constant for the intermediate's hydrolysis is relatively insensitive to chain length. To probe the mechanistic origins of this phenomenon, we determined temperature dependencies and solvent isotope effects for the alpha-chymotrypsin-catalyzed hydrolysis of Suc-Phe-pNA (K(s) = 1 mM, k(2) = 0.04 s(-)(1), and k(3) = 11 s(-)(1)), Suc-Ala-Phe-pNA (K(s) = 4 mM, k(2) = 0.9 s(-)(1), and k(3) = 42 s(-)(1)), and Suc-Ala-Ala-Pro-Phe-pNA (K(s) = 0.1 mM, k(2) = 98 s(-)(1), and k(3) = 71 s(-)(1)). We found that while the van't Hoff plots for K(s) and the Eyring plots for k(3) are linear for all three reactions, the Eyring plots for k(2) are convex, indicating that the process governed by k(2) is complex, possibly involving a coupling between active site chemistry and protein conformational isomerization. This interpretation is strengthened by solvent isotope effects on k(2) that are largely temperature-independent. Furthermore, the dependence of k(2) on peptide length is manifested entirely in the enthalpy of activation, suggesting a mechanism of catalysis by distortion. Taken together, this analysis of acylation suggests that extended substrates which can engage in subsite interactions are able to efficiently trigger the coupling mechanism between chemistry and a conformational isomerization that distorts the substrate and thereby promotes nucleophilic attack.     

Effect of an E461G mutation of beta-galactosidase (Escherichia coli, lac Z) on pL rate profiles and solvent deuterium isotope effects.

An E461G mutation of beta-galactosidase results in the disappearance of the high pL (L = H, D) downward break in the rate profiles for k(cat)/K(m) for wild-type enzyme-catalyzed hydrolysis of 4-nitrophenyl beta-D-galactopyranoside (Gal-OPNP) and a decrease from (k(cat))(HOH)/(k(cat))(DOD) = 1.7 to (k(cat))(HOH)/(k(cat))(DOD) = 1.2 in the solvent deuterium isotope effect. These observations provide evidence that the propionic acid side chain of Glu 461 is protonated at catalytically active free beta-galactosidase and they are consistent with a role for this residue in Br?nsted acid catalysis at the leaving group. The earlier observation that this same E461G mutation results in the loss of a downward break at high pH in the rate profile for k(s) for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to water cannot be simply explained by a mechanism in which the single side chain of Glu 461 functions to provide general acid catalysis in the rate limiting step for formation of the beta-D-galactopyranosyl intermediate and general base catalysis of breakdown of this intermediate. Evidence is presented that there may be different catalytic mechanisms, with different roles for the side chain for Glu-461, for nucleophilic addition of water and of small alkyl alcohols to the beta-D-galactopyranosyl reaction intermediate.     

Investigation of the role of a second conserved serine in carboxylesterases via site-directed mutagenesis

Carboxylesterases are enzymes that catalyze the hydrolysis of ester and amide moieties. These enzymes have an active site that is composed of a nucleophile (Ser), a base (His), and an acid (Glu) that is commonly known as a catalytic triad. It has previously been observed that the majority of carboxylesterases and lipases contain a second conserved serine in their active site [Proteins, 34 (1999) 184]. To investigate whether this second serine is also involved in the catalytic mechanism, it was mutated to an alanine, a glycine or a cysteine. Site-directed mutagenesis of this conserved serine resulted in a loss of specific activity, in both the S247G and S247A mutants (5- to 15-fold), which was due to a decrease in the rate of catalysis (kcat). Due to the instability of the S247C mutant no reliable data could be attained. A carbamate inhibitor, carbaryl, was then employed to investigate whether this decrease in the kcat was due to the rate of formation of the acyl-enzyme intermediate (k2) or the rate of deacylation (k3). The S247A mutant was found only to alter k2 (2.5-fold decrease), with no effect on k3. Together with information inferred from a human carboxylesterase crystal structure, it was concluded that this serine provides an important structural support for the spatial orientation of the glutamic acid, stabilizing the catalytic triad so that it can perform the hydrolysis.     

The key role of a non-active-site residue Met148 on the catalytic efficiency of meta-cleavage product hydrolase BphD

meta-Cleavage product (MCP) hydrolases (EC 3.7.1.9) can catalyze a specific C–C bond fission during the microbial aerobic degradation of aromatics. The previous studies on structure–function relationship of MCP hydrolases mainly focus on the active site residues by site-directed mutagenesis. However, the information about the role of the non-active-site residues is still unclear. In this study, a non-active-site residue Met148 of MCP hydrolase BphD was selected as the mutagenesis site according to the sequence alignments, structure superimpose and the tunnel analysis, which underwent the saturation mutagenesis resulting 19 mutants. The catalytic efficiencies of the mutants on 6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) were all decreased compared with the wild-type one except for the M148D mutant. Especially, the M148P mutant exhibited 290-fold lower k _cat/K _m than that of the wild-type BphD. Transient kinetic analyses of M148P showed the reciprocal relaxation time corresponded to C–C bond cleavage and product release steps (9.6 s^?1) was 4.08-fold lower than BphD WT (39.2 s^?1). Tunnel cluster analysis of BphD WT, M148P and M148W demonstrated that only the bulky Trp148 could block tunnel T2 in the BphD WT, but it exhibited slight effects on the catalytic efficiency (0.94-fold of BphD WT). Therefore, product release was not the main reason for the efficiency decrease of M148P. On the other hand, molecular dynamics simulations on the BphD WT and BphD M148P in complex with HOPDA indicated that the dramatic decrease of the catalytic efficiencies of BphD M148P should be due to the unproductive binding of HOPDA. The study demonstrated the catalytic efficiency of MCP hydrolase can be engineered by modification of non-active site residue.     

Enzyme:substrate hydrogen bond shortening during the acylation phase of serine protease catalysis

Atomic resolution (相似文献   

The QM/MM molecular dynamics and free energy simulations of the acylation reaction catalyzed by the serine-carboxyl peptidase kumamolisin-As

Quantum mechanical/molecular mechanical molecular dynamics and free energy simulations are performed to study the acylation reaction catalyzed by kumamolisin-As, a serine-carboxyl peptidase, and to elucidate the catalytic mechanism and the origin of substrate specificity. It is demonstrated that the nucleophilic attack by the serine residue on the substrate may not be the rate-limiting step for the acylation of the GPH*FF substrate. The present study also confirms the earlier suggestions that Asp164 acts as a general acid during the catalysis and that the electrostatic oxyanion hole interactions may not be sufficient to lead a stable tetrahedral intermediate along the reaction pathway. Moreover, Asp164 is found to act as a general base during the formation of the acyl-enzyme from the tetrahedral intermediate. The role of dynamic substrate assisted catalysis (DSAC) involving His at the P1 site of the substrate is examined for the acylation reaction. It is demonstrated that the bond-breaking and -making events at each stage of the reaction trigger a change of the position for the His side chain and lead to the formation of the alternative hydrogen bonds. The back and forth movements of the His side chain between the C=O group of Pro at P2 and Odelta2 of Asp164 in a ping-pong-like mechanism and the formation of the alternative hydrogen bonds effectively lower the free energy barriers for both the nucleophilic attack and the acyl-enzyme formation and may therefore contribute to the relatively high activity of kumamolisin-As toward the substrates with His at the P1 site.     

Stereoelectronic control of bond formation in Escherichia coli tryptophan synthase: substrate specificity and enzymatic synthesis of the novel amino acid dihydroisotryptophan

The reactions of the indole analogues indoline and aniline with the Escherichia coli tryptophan synthase alpha-aminoacrylate Schiff base intermediate have been characterized by UV-visible and 1H NMR absorption spectroscopy and compared with the interactions of indole and the potent inhibitor benzimidazole. Indole, via the enamine functionality of the pyrrole ring, reacts with the alpha-aminoacrylate intermediate, forming a transient quinonoid species with lambda max 476 nm as the new C-C bond is synthesized. Conversion of this quinonoid to L-tryptophan is the rate-limiting step in catalysis [Lane, A., & Kirschner, K. (1981) Eur. J. Biochem. 120, 379-398]. Both aniline and indoline undergo rapid N-C bond formation with the alpha-aminoacrylate to form quinonoid intermediates; benzimidazole binds rapidly and tightly to the alpha-aminoacrylate but does not undergo covalent bond formation. The indoline and aniline quinonoids (lambda max 464 and 466 nm, respectively) are formed via nucleophilic attack on the electrophilic C-beta of the alpha-aminoacrylate. The indoline quinonoid decays slowly, yielding a novel, new amino acid, dihydroisotryptophan. The aniline quinonoid is quasi-stable, and no new amino acid product was detected. We conclude that nucleophilic attack requires the precise alignment of bonding orbitals between nucleophile and the alpha-aminoacrylate intermediate. The constraints imposed by the geometry of the indole subsite force the aromatic rings of indoline, aniline, and benzimidazole to bind in the same plane as indole; thus nucleophilic attack occurs with the N-1 atoms of indoline and aniline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and mechanism of the serine beta-lactamase catalyzed hydrolysis of depsipeptides

Steady-state kinetic parameters have been determined for the hydrolysis of a series of acyclic depsipeptides (ester analogues of acyl-D-alanyl-D-alanine peptides) catalyzed by representative class C (Enterobacter cloacae P99) and class A (Bacillus cereus I, TEM-2, and Staphylococcus aureus PC1) beta-lactamases. The best of these substrates, and the one most used in this work, was m-[[(phenylacetyl)-glycyl]oxy]benzoic acid, whose rates of cleavage could be followed spectrophotometrically. The P99 enzyme also catalyzed the methanolysis of these substrates in aqueous methanol solutions. Quantitative evaluation of the effects of methanol on the kinetics of the competing hydrolysis and methanolysis reactions, and on the product distribution, supports a reaction mechanism involving an acyl-enzyme intermediate whose formation is rate-determining under conditions of substrate saturation. Consideration of the variation of these kinetic parameters with the structure of the depsipeptides and comparison with the analogous parameters for bicyclic beta-lactam substrates suggest that a variety of substrate binding modes exist on this enzyme. The class A enzymes, B. cereus beta-lactamase I and the TEM-2 beta-lactamase, catalyze depsipeptide and benzylpenicillin hydrolyses but not methanolysis. The acyl-enzyme derived from both types of substrate is thus shielded from external nucleophiles; the shielding is therefore not an effect, direct or indirect, of the thiazolidinyl group in the penicilloyl-enzyme. The class A beta-lactamase of the PC1 plasmid of S. aureus is distinctly different from the above two representatives of that class, in that it does catalyze methanolysis of depsipeptides (but not of benzylpenicillin). The methanolysis kinetics suggest that deacylation is rate-determining at saturation, a conclusion supported by the demonstration of an intermediate during the hydrolysis of m-[[(phenylacetyl)glycyl]oxy]benzoate, subsequent to leaving-group departure. The beta-lactamases have thus been shown to catalyze the hydrolysis of specific depsipeptides with comparable facility to that demonstrated by D-alanyl-D-alanine carboxypeptidase/transpeptidases. The former enzymes, however, differ in being unable to cleave the analogous peptides.     

pH-dependence and structure–activity relationships in the papain-catalysed hydrolysis of anilides

The pH-dependence of the Michaelis-Menten parameters for the papain-catalysed hydrolysis of N-acetyl-l-phenylalanylglycine p-nitroanilide was determined. The equilibrium binding constant, K(s), is independent of pH between 3.7 and 9.3, whereas the acylation constant, k(+2), shows bell-shaped pH-dependence with apparent pK(a) values of 4.2 and 8.2. The effect of substituents in the leaving group on the acylation constant of the papain-catalysed hydrolysis of hippuryl anilides and N-acetyl-l-phenylalanylglycine anilides gives rise in both series to a Hammett rho value of -1.04. This indicates that the enzyme provides electrophilic, probably general-acid, catalysis, as well as the nucleophilic or general-base catalysis previously found. A mechanism involving a tetrahedral intermediate whose formation is general-base-catalysed and whose breakdown is general-acid-catalysed seems most likely. The similarity of the Hammett rho values appears to exclude facilitated proton transfer as a means through which the specificity of papain is expressed.     

The electrostatic driving force for nucleophilic catalysis in L-arginine deiminase: a combined experimental and theoretical study

L-arginine deiminase (ADI) catalyzes the hydrolysis of L-arginine to form L-citrulline and ammonia via two partial reactions. A working model of the ADI catalytic mechanism assumes nucleophilic catalysis by a stringently conserved active site Cys and general acid-general base catalysis by a stringently conserved active site His. Accordingly, in the first partial reaction, the Cys attacks the substrate guanidino C zeta atom to form a tetrahedral covalent adduct, which is protonated by the His at the departing ammonia group to facilitate the formation of the Cys- S-alkylthiouronium intermediate. In the second partial reaction, the His activates a water molecule for nucleophilic addition at the thiouronium C zeta atom to form the second tetrahedral intermediate, which eliminates the Cys in formation of the L-citrulline product. The absence of a basic residue near the Cys thiol suggested that the electrostatic environment of the Cys thiol, in the enzyme-substrate complex, stabilizes the Cys thiolate anion. The studies described in this paper explore the mechanism of stabilization of the Cys thiolate. First, the log(k(cat)/K(m)) and log k(cat) pH rate profiles were measured for several structurally divergent ADIs to establish the pH range for ADI catalysis. All ADIs were optimally active at pH 5, which suggested that the Cys pKa is strongly perturbed by the prevailing electrostatics of the ADI active site. The p K a of the Bacillus cereus ADI (BcADI) was determined by UV-pH titration to be 9.6. In contrast, the pKa determined by iodoacetamide Cys alkylation is 6.9. These results suggest that the negative electrostatic field from the two opposing Asp carboxylates perturbs the Cys pKa upward in the apoenzyme and that the binding of the iodoacetamide (a truncated analogue of the citrulline product) between the Cys thiol and the two Asp carboxylates shields the Cys thiol, thereby reducing its pKa. It is hypothesized that the bound positively charged guanidinium group of the L-arginine substrate further stabilizes the Cys thiolate. The so-called "substrate-assisted" Cys ionization, first reported by Fast and co-workers to operate in the related enzyme dimethylarginine dimethylaminohydrolase [Stone, E. M., Costello, A. L., Tierney, D. L., and Fast, W. (2006) Biochemistry 45, 5618-5630], was further explored computationally in ADI by using an ab initio quantum mechanics/molecular mechanics method. The energy profiles for formation of the tetrahedral intermediate in the first partial reaction were calculated for three different reaction scenarios. From these results, we conclude that catalytic turnover commences from the active configuration of the ADI(L-arginine) complex which consists of the Cys thiolate (nucleophile) and His imidazolium ion (general acid) and that the energy barriers for the nucleophilic addition of Cys thiolate to the thiouronium C zeta atom and His imidazolium ion-assisted elimination from the tetrahedral intermediate are small.     

Pseudo-enzymatic hydrolysis of 4-nitrophenyl myristate by human serum albumin

Most of the esterase properties of human serum albumin (HSA) are the result of multiple irreversible chemical modifications rather than turnover. The HSA-catalyzed hydrolysis of 4-nitrophenyl myristate (NphOMy) is consistent with the minimum three-step mechanism involving the acyl-enzyme intermediate HSA-OMy: Under all the experimental conditions, values of K(s) (= k(-1)/k(+1)), k(+2), and k(+2)/K(s) determined under conditions where [HSA] ≥ 5 × [NphOMy] and [NphOMy] ≥ 5 × [HSA] match very well each other. The deacylation process is rate limiting in catalysis (i.e., k(+3) < k(+2)) and k(-2)~k(-3)~0 s(-1). The pH dependence of k(+2)/K(s), k(+2), and K(s) reflects the acidic pK(a)-shift of one ionizing group from 8.9 ± 0.2 in NphOMy-free HSA to 6.8 ± 0.3 in the HSA:NphOMy adduct. The HSA-catalyzed hydrolysis of NphOMy is inhibited competitively by diazepam, indicating that Tyr411 is the active-site nucleophile.