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 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.     

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.     

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.     

The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism

2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway of Escherichia coli. The 2.1 A resolution X-ray structure of the native enzyme determined from orthorhombic crystals confirms that it is a member of the alpha/beta hydrolase fold family, comprising eight beta-strands interconnected by loops and helices. The 2.8 A resolution structure of the enzyme co-crystallised with the non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic acid (DKNDA) confirms the location of the active site in a buried channel including Ser110, His263 and Asp235, postulated contributors to a serine protease-like catalytic triad in homologous enzymes. It appears that the ligand binds in two separate orientations. In the first, the C6 keto group of the inhibitor forms a hemi-ketal adduct with the Ser110 side-chain, the C9 carboxylate group interacts, via the intermediacy of a water molecule, with Arg188 at one end of the active site, while the C1 carboxylate group of the inhibitor comes close to His114 at the other end. In the second orientation, the C1 carboxylate group binds at the Arg188 end of the active site and the C9 carboxylate group at the His114 end. These arrangements implicated His114 or His263 as plausible contributors to catalysis of the initial enol/keto tautomerisation of the substrate but lack of conservation of His114 amongst related enzymes and mutagenesis results suggest that His263 is the residue involved. Variability in the quality of the electron density for the inhibitor amongst the eight molecules of the crystal asymmetric unit appears to correlate with alternative positions for the side-chain of His114. This might arise from half-site occupation of the dimeric enzyme and reflect the apparent dissociation of approximately 50% of the keto intermediate from the enzyme during the catalytic cycle.     

Reassessing the type I dehydroquinate dehydratase catalytic triad: Kinetic and structural studies of Glu86 mutants

Dehydroquinate dehydratase (DHQD) catalyzes the third reaction in the biosynthetic shikimate pathway. Type I DHQDs are members of the greater aldolase superfamily, a group of enzymes that contain an active site lysine that forms a Schiff base intermediate. Three residues (Glu86, His143, and Lys170 in the Salmonella enterica DHQD) have previously been proposed to form a triad vital for catalysis. While the roles of Lys170 and His143 are well defined—Lys170 forms the Schiff base with the substrate and His143 shuttles protons in multiple steps in the reaction—the role of Glu86 remains poorly characterized. To probe Glu86′s role, Glu86 mutants were generated and subjected to biochemical and structural study. The studies presented here demonstrate that mutant enzymes retain catalytic proficiency, calling into question the previously attributed role of Glu86 in catalysis and suggesting that His143 and Lys170 function as a catalytic dyad. Structures of the Glu86Ala (E86A) mutant in complex with covalently bound reaction intermediate reveal a conformational change of the His143 side chain. This indicates a predominant steric role for Glu86, to maintain the His143 side chain in position consistent with catalysis. The structures also explain why the E86A mutant is optimally active at more acidic conditions than the wild‐type enzyme. In addition, a complex with the reaction product reveals a novel, likely nonproductive, binding mode that suggests a mechanism of competitive product inhibition and a potential strategy for the design of therapeutics.     

The dual histidine motif in the active site of Pin1 has a structural rather than catalytic role

The catalytic domain of the peptidyl-prolyl cis/ trans isomerase Pin1 is a member of the FKBP superfold family. Within its active site are two highly conserved histidine residues, H59 and H157. Despite their sequence conservation in parvulin PPIase domains, the role of these histidine residues remains unclear. Our previous work (Behrsin et al. (2007) J. Mol. Biol. 365, 1143- 1162.) was consistent with a model where one or both histidines had critical roles in a hydrogen bonding network in the active site. Here, we test this model by looking at the effect of mutations to H59 and H157 on Pin1 function, activity, and protein stability. Using a yeast complementation assay, we show that both H59 and H157 can be mutated to non-hydrogen bonding residues and still support viability. Surprisingly, a nonfunctional H59L mutation can be rescued by a mutation of H157, to leucine. This double mutation (H59L/H157L) also had about 5-fold greater isomerase activity than the H59L mutation with a phosphorylated substrate. Structural analyses suggest that rescue of function and activity results from partial rescue of protein stability. Our findings indicate that H59 and H157 are not required for hydrogen bonding within the active site, and in contrast to the active site C113, they do not participate directly in catalysis. Instead, we suggest these histidines play a key role in domain structure or stability.     

Catalytic mechanism of a C-C hydrolase enzyme: evidence for a gem-diol intermediate, not an acyl enzyme

2-Hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli catalyses the hydrolytic cleavage of the extradiol ring fission product on the phenylpropionate catabolic pathway and is a member of the alpha/beta hydrolase family. The catalytic mechanism of this enzyme has previously been shown to proceed via initial ketonization of the dienol substrate (Henderson, I. M. J., and Bugg, T. D. H. (1997) Biochemistry 36, 12252-12258), followed by stereospecific fragmentation. Despite the implication of an active site serine residue in the alpha/beta hydrolase family, attempts to verify a putative acyl enzyme intermediate by radiochemical trapping methods using a (14)C-labeled substrate yielded a stoichiometry of <1% covalent intermediate, which could be accounted for by nonenzymatic processes. In contrast, incorporation of 5-6% of two atoms of (18)O from H(2)(18)O into succinic acid was observed using the natural substrate, consistent with the reversible formation of a gem-diol intermediate. Furthermore, time-dependent incorporation of (18)O from H(2)(18)O into the carbonyl group of a nonhydrolysable analogue 4-keto-nona-1,9-dioic acid was observed in the presence of MhpC, consistent with enzyme-catalyzed attack of water at the ketone carbonyl. These results favor a catalytic mechanism involving base-catalyzed attack of water, rather than nucleophilic attack of an active site serine. The implication of this work is that the putative active site serine in this enzyme may have an alternative function, for example, as a base.     

Mutational, structural, and kinetic evidence for a dissociative mechanism in the GDP-mannose mannosyl hydrolase reaction

GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-alpha-d-sugars by nucleophilic substitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124. Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP.Tris(+) complex [Gabelli, S. B., et al. (2004) Structure 12, 927-935], the GDP leaving group interacts with five catalytic components: R37, Y103, R52, R65, and the essential Mg(2+). As determined by the effects of site-specific mutants on k(cat), these components contribute factors of 24-, 100-, 309-, 24-, and >/=10(5)-fold, respectively, to catalysis. Both R37 and Y103 bind the beta-phosphate of GDP and are only 5.0 A apart. Accordingly, the R37Q/Y103F double mutant exhibits partially additive effects of the two single mutants on k(cat), indicating cooperativity of R37 and Y103 in promoting catalysis, and antagonistic effects on K(m). Second, the conserved residue, D22, is positioned to accept a hydrogen bond from the C2-OH group of the sugar undergoing substitution at C1, as was shown by modeling an alpha-d-mannosyl group into the sugar binding site. The D22A and D22N mutations decreased k(cat) by factors of 10(2.1) and 10(2.6), respectively, for the hydrolysis of GDP-alpha-d-mannose, and showed smaller effects on K(m), suggesting that the D22 anion stabilizes a cationic oxocarbenium transition state. Third, the fluorinated substrate, GDP-2F-alpha-d-mannose, for which a cationic oxocarbenium transition state would be destabilized by electron withdrawal, exhibited a 16-fold decrease in k(cat) and a smaller, 2.5-fold increase in K(m). The D22A and D22N mutations further decreased the k(cat) with GDP-2F-alpha-d-mannose to values similar to those found with GDP-alpha-d-mannose, and decreased the K(m) of the fluorinated substrate. The choice of histidine as the general base over glutamate, the preferred base in other Nudix enzymes, is not due to the greater basicity of histidine, since the pK(a) of E124 in the active complex (7.7) exceeded that of H124 (6.7), and the H124E mutation showed a 10(2.2)-fold decrease in k(cat) and a 4.0-fold increase in K(m) at pH 9.3. Similarly, the catalytic triad detected in the X-ray structure (H124- - -Y127- - -P120) is unnecessary for orienting H124, since the Y127F mutation had only 2-fold effects on k(cat) and K(m) with either H124 or E124 as the general base. Hence, a neutral histidine rather than an anionic glutamate may be necessary to preserve electroneutrality in the active complex.     

Investigation of the substrate binding and catalytic groups of the P-C bond cleaving enzyme, phosphonoacetaldehyde hydrolase.

Kinetic studies with substrate analogs and group-directed chemical modification agents were carried out for the purpose of identifying the enzyme-substrate interactions required for phosphonoacetaldehyde (P-Ald) binding and catalyzed hydrolysis by P-Ald hydrolase (phosphonatase). Malonic semialdehyde (Ki = 1.6 mM), phosphonoacetate (Ki = 10 mM), phosphonoethanol (Ki = 10 mM), and fluorophosphate (Ki = 20 mM) were found to be competitive inhibitors of the enzyme but not substrates. Thiophosphonoacetaldehyde and acetonyl phosphonate underwent phosphonatase-catalyzed hydrolysis but at 20-fold and 140-fold slower rates, respectively, than did P-Ald. In the presence of NaBH4, acetonyl-phosphonate inactivated phosphonatase at a rate exceeding that of its turnover. Sequence analysis of the radiolabeled tryptic peptide generated from [3-3H]acetonylphosphonate/NaBH4-treated phosphonatase revealed that Schiff base formation had occurred with the catalytic lysine. From the Vm/Km and Vm pH profiles for phosphonatase-catalyzed P-Ald hydrolysis, an optimal pH range of 6-8 was defined for substrate binding and catalysis. The pH dependence of inactivation by acetylation of the active site lysine with acetic anhydride and 2,4-dinitrophenyl acetate evidenced protonation of the active site lysine residue as the cause for activity loss below pH 6. The pH dependence of inactivation of an active site cysteine residue with methyl methanethiol-sulfonate indicated that deprotonation of this residue may be the cause for the loss of enzyme activity above pH 8.     

Kinetic and mutagenic evidence for the role of histidine residues in the Lycopersicon esculentum 1-aminocyclopropane-1-carboxylic acid oxidase

The ACCO gene from Lycopersicon esculentum (tomato) has been cloned into the expression vector PT7-7. The highly expressed protein was recovered in the form of inclusion bodies. ACCO is inactivated by diethyl pyrocarbonate (DEPC) with a second-order rate constant of 170 M^–1 min^–1. The pH–inactivation rate data imply the involvement of an amino acid residue with a pK value of 6.05. The difference UV spectrum of the the DEPC-inactivated versus native ACCO showed a single peak at 242 nm indicating the modification of histidine residues. The inactivation was reversed by the addition of hydroxylamine to the DEPC-inactivated ACCO. Substrate/cofactor protection studies indicate that both iron and ACC bind near the active site, which contains histidine residues. Four histidines of ACCO were individually mutated to alanine and glycine. H39A is catalytically active, while H177A, H177G, H211A, H211G, H234A, and H234G are basically inactive. The results indicate that histidine residues 177, 211, and 234 may serve as ligands for the active-site iron of ACCO and/or may play some important structural or catalytic role.     

The role of histidine 63 in the catalytic mechanism of Bordetella pertussis adenylate cyclase.

Of the 9 histidines located in the catalytic domain of Bordetella pertussis adenylate cyclase, three (His63, His106, and His298) were found to be conserved in the adenylate cyclase of Bacillus anthracis, another calmodulin-dependent enzyme. Substitution of His63 with Arg, Glu, Gln, or Val decreased the catalytic efficiency of adenylate cyclase between 2 and 3 orders of magnitude and altered the kinetic properties of the enzyme. These effects varied in relation to the nature of the substituting residue, pH, and direction of the reaction, i.e. ATP cyclization (forward) or ATP synthesis (reverse). Arg was the best substituent for His63 as catalyst in the forward reaction, with shift of the optimum pH to the alkaline side, whereas Glu was the best substituent for His63 in the reverse reaction, with shift of the optimum pH to the acidic side. Diethyl pyrocarbonate, which had a deleterious effect on wild-type adenylate cyclase was ineffective on His63 mutants. From these results we conclude that His63 is involved in the reaction mechanism of adenylate cyclase, which requires a general acid/base catalyst, most probably as an intermediate in a charge-relay system.     

Structure-function analysis of human triacylglycerol hydrolase by site-directed mutagenesis: identification of the catalytic triad and a glycosylation site

Triacylglycerol hydrolase is a microsomal enzyme that hydrolyzes stored cytoplasmic triacylglycerol in the liver and participates in the lipolysis/re-esterification cycle during the assembly of very-low-density lipoproteins. The structure-activity relationship of the enzyme was investigated by site-directed mutagenesis and heterologous expression. Expression of human TGH in Escherichia coli yields a protein without enzymatic activity, which suggests that posttranslational processing is necessary for the catalytic activity. Expression in baculovirus-infected Sf-9 cells resulted in correct processing of the N-terminal signal sequence and yielded a catalytically active enzyme. A putative catalytic triad consisting of a nucleophilic serine (S221), glutamic acid (E354), and histidine (H468) was identified. Site-directed mutagenesis of the residues (S221A, E354A, and H468A) yielded a catalytically inactive enzyme. CD spectra of purified mutant proteins were very similar to that of the wild-type enzyme, which suggests that the mutations did not affect folding. Human TGH was glycosylated in the insect cells. Mutagenesis of the putative N-glycosylation site (N79A) yielded an active nonglycosylated enzyme. Deletion of the putative C-terminal endoplasmic reticulum retrieval signal (HIEL) did not result in secretion of the mutant protein. A model of human TGH structure suggested a lipase alpha/beta hydrolase fold with a buried active site and two disulfide bridges (C87-C116 and C274-C285).     

The crystal structure of three site-directed mutants of Escherichia coli dihydrodipicolinate synthase: further evidence for a catalytic triad

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes: the condensation of (S)-aspartate-beta-semialdehyde and pyruvate. The crystal structure of wild-type DHDPS has been published to 2.5A, revealing a tetrameric molecule comprised of four identical (beta/alpha)(8)-barrels, each containing one active site. Previous workers have hypothesised that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Tyr133, Thr44 and Tyr107, which provide a proton shuttle to transport protons from the active site to solvent. We have tested this hypothesis using site-directed mutagenesis to produce three mutant enzymes: DHDPS-Y133F, DHDPS-T44V and DHDPS-Y107F. Each of these mutants has substantially reduced activity, consistent with the catalytic triad hypothesis. We have determined each mutant crystal structure to at least 2.35A resolution and compared the structures to the wild-type enzyme. All mutant enzymes crystallised in the same space group as the wild-type form and only minor differences in structure are observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS.     

A critical role for the histidine residues in the catalytic function of acyl-CoA:cholesterol acyltransferase catalysis: evidence for catalytic difference between ACAT1 and ACAT2

To investigate a role for histidine residues in the expression of normal acyl-CoA:cholesterol acyltransferase (ACAT) activity, the histidine residues located at five different positions in two isoenzymes were substituted by alanine, based on the sequence homology between ACAT1 and ACAT2. Among the 10 mutants generated by baculovirus expression technology, H386A-ACAT1, H460A-ACAT1, H360A-ACAT2, and H399A-ACAT2 lost their enzymatic activity completely. A reduction in catalytic activity is unlikely to result from structural changes in the substrate-binding pocket, because their substrate-binding affinities were normal. However, the enzymatic activity of H386A-ACAT1 was restored to <37% of the level of the wild-type activity when cholesterol was replaced by 25-hydroxycholesterol as substrate. H527A-ACAT1 and H501A-ACAT2, termed carboxyl end mutants, exhibit activities of ∼96% and ∼75% of that of the wild-type. Interestingly, H425A-ACAT1 showed 59% of the wild-type activity, in contrast to its equivalent mutant, H399A-ACAT2. These results demonstrate that the histidine residues located at the active site are very crucial both for the catalytic activity of the enzyme and for distinguishing ACAT1 from ACAT2 with respect to enzyme catalysis and substrate specificity.     

Low-barrier hydrogen bond hypothesis in the catalytic triad residue of serine proteases: correlation between structural rearrangement and chemical shifts in the acylation process

To elucidate the catalytic advantage of the low-barrier hydrogen bond (LBHB), we analyze the hydrogen bonding network of the catalytic triad (His57-Asp102-Ser195) of serine protease trypsin, one of the best examples of the LBHB reaction mechanism. Especially, we focus on the correlation between the change of the chemical shifts and the structural rearrangement of the active site in the acylation process. To clarify LBHB, we evaluate the two complementary properties. First, we calculate the NMR chemical shifts of the imidazole ring of His57 by the gauge-including atomic orbital (GIAO) approach within the ab initio QM/MM framework. Second, the free energy profile of the proton transfer from His57 to Asp102 in the tetrahedral intermediate is obtained by ab initio QM/MM calculations combined with molecular dynamics free energy perturbation (MD-FEP) simulations. The present analyses reveal that the calculated shifts reasonably reproduce the observed values for (1)H chemical shift of H(epsilon)(1) and H(delta)(1) in His57. The (15)N and (13)C chemical shifts are also consistent with the experiments. It is also shown that the proton between His57 and Asp102 is localized at the His57 side. This largely downfield chemical shift is originated from the strong electrostatic interaction, not a covalent-like bonding character between His57 and Asp102. Also, it is proved that a slight downfield character of H(epsilon)(1) is originated from a electrostatic interaction between His57 and the backbone carbonyl group of Val213 and Ser214. These downfield chemical shifts are observed only when the tetrahedral intermediate is formed in the acylation process.     

Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad

Escherichia coli Spr is a membrane-anchored cell wall hydrolase. The solution NMR structure of the C-terminal NlpC/P60 domain of E. coli Spr described here reveals that the protein adopts a papain-like alpha+beta fold and identifies a substrate-binding cleft featuring several highly conserved residues. The active site features a novel Cys-His-His catalytic triad that appears to be a unique structural signature of this cysteine peptidase family. Moreover, the relative orientation of these catalytic residues is similar to that observed in the analogous Ser-His-His triad, a variant of the classic Ser-His-Asp charge relay system, suggesting the convergent evolution of a catalytic mechanism in quite distinct peptidase families.     

3 beta, 17 beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni. Kinetic evidence for the bifunctional activity at a common catalytic site

3 beta, 17 beta-Hydroxysteroid dehydrogenase (3 beta 17 beta HSDH) is an NAD-dependent dehydrogenase which has a double specificity for the 3- and 17-positions on the steroid skeleton. When dehydroepiandrosterone (DHEA) is used as steroid substrate, and the assay coupled with ketosteroid-isomerase, the two reactions occur alternately and each reaction on the 3-position produces a chromophoric molecule. These two reactions can follow one another without dissociation of the coenzyme from the enzyme binding site. This is confirmed by competition experiments with another dehydrogenase.     

The role of electrostatic interactions in the mechanism of peptide bond hydrolysis by a Ser-Lys catalytic dyad.

General-base catalysis in the active site of serine proteases is carried out by the imidazole side chain of a histidine. During formation of the transition state, an adjacent carboxylic acid group stabilizes the positive charge that forms on the general-base catalyst and as a result contributes several orders of magnitude to the catalytic efficiency of these enzymes. In the recently discovered family of self-cleaving proteins exemplified by the LexA repressor of Escherichia coli, instead of the imidazole of a histidine, the active-site general-base catalyst was found to be the epsilon-amino of a lysine. The considerably higher capacity of the lysine side chain for proton acceptance raises interesting questions concerning the role of electrostatic interactions in the mechanism of proton transfer by this highly basic group. The negative charge elimination studies described here and their effects on the kmax and pK of LexA self-cleavage are consistent with a model in which electrostatic interactions between an acidic side chain and the general-base catalyst form a barrier to proton transfer. The implications are that the epsilon-amino group, unlike the imidazole group, is capable of effecting proton transfer without the intervention of a countercharge.