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The evolution of new catalytic activities and specificities within an enzyme superfamily requires the exploration of sequence space for adaptation to a new substrate with retention of those elements required to stabilize key intermediates/transition states. Here, we propose that core residues in the large enzyme family, the haloalkanoic acid dehalogenase enzyme superfamily (HADSF) form a "mold" in which the trigonal bipyramidal transition states formed during phosphoryl transfer are stabilized by electrostatic forces. The vanadate complex of the hexose phosphate phosphatase BT4131 from Bacteroides thetaiotaomicron VPI-5482 (HPP) determined at 1.00 A resolution via X-ray crystallography assumes a trigonal bipyramidal coordination geometry with the nucleophilic Asp-8 and one oxygen ligand at the apical position. Remarkably, the tungstate in the complex determined to 1.03 A resolution assumes the same coordination geometry. The contribution of the general acid/base residue Asp-10 in the stabilization of the trigonal bipyramidal species via hydrogen-bond formation with the apical oxygen atom is evidenced by the 1.52 A structure of the D10A mutant bound to vanadate. This structure shows a collapse of the trigonal bipyramidal geometry with displacement of the water molecule formerly occupying the apical position. Furthermore, the 1.07 A resolution structure of the D10A mutant complexed with tungstate shows the tungstate to be in a typical "phosphate-like" tetrahedral configuration. The analysis of 12 liganded HADSF structures deposited in the protein data bank (PDB) identified stringently conserved elements that stabilize the trigonal bipyramidal transition states by engaging in favorable electrostatic interactions with the axial and equatorial atoms of the transferring phosphoryl group.

This article discusses three aspects of the Pseudomonas 4-hydroxybenzoyl-CoA thioesterase catalysis, namely, the binding of the CoA nucleotide to the active site, Asp17 contribution, and the polarization of the substrate thioester C=O using spectral, mutagenesis, and kinetic analyses.

Examines the chemical pathway for 4-chlorobenzoate:coenzyme A (4-CBA:CoA) ligase catalysis. HPLC analysis of product formation in multiple turnover reactions catalyzed by 4-CBA:CoA; Steady-state kinetic analysis; Isolation and structure determination.

Presents the results from pre-steady-state dead-end inhibition studies analyzing adenosine-5'-triphosphate/adenosine 5'-monophosphate and pyruvate/uni(pyruvate)uni binding sites. Linkage of two sites with catalytic histidine; Probing of the movement of the phosphorylhistidine residue between reaction sites.

4-Chlorobenzoate:CoA ligase (CBAL) is a member of a family of adenylate-forming enzymes that catalyze two-step adenylation and thioester-forming reactions. In previous studies, we have provided structural evidence that members of this enzyme family (exemplified by acetyl-CoA synthetase) use a large domain rotation to catalyze the respective partial reactions [A. M. Gulick, V. J. Starai, A. R. Horswill, K. M. Homick, and J. C. Escalante-Semerena, (2003) Biochemistry 42, 2866-28731. CBAL catalyzes the synthesis of 4-chlorobenzoyl-CoA, the first step in the 4-chlorobenzoate degredation pathway in PCB- degrading bacteria. We have solved the 2.0 Å crystal structure of the CBAL enzyme from Alcaligenes sp. AL3007 using multiwavelength anomalous dispersion. The results demonstrate that in the absence of any ligands, or bound to the aryl substrate 4-chlorobenzoate, the enzyme adopts the conformation poised for catalysis of the adenylate-forming half-reaction. We hypothesize that coenzyme A binding is required for stabilization of the alternate conformation, which catalyzes the 4-CBA-CoA thioester-forming reaction. We have also determined the structure of the enzyme bound to the aryl substrate 4-chlorobenzoate. The aryl binding pocket is composed of Phe184, His207, Val208, Val209, Phe249, Ala280, Ile303, Gly305, Met310, and Asn311. The structure of the 4-chlorobenzoate binding site is discussed in the context of the binding sites of other family members to gain insight into substrate specificity and evolution of new function. [ABSTRACT FROM AUTHOR]

Phosphonoacetaldehyde hydrolase (phosphonatase) from Bacillus cereus catalyzes hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) via a Schiff base intermediate formed with Lys53. A single turnover requires binding of Pald to the active site of the core domain, closure of the cap domain containing the Lys53 over the core domain, and dissociation of the products following catalysis. The ligand binding and dissociation steps occur from the "open conformer" (domains are separated and the active site is solvent-exposed), while catalysis occurs from the "closed conformer" (domains are bound together and the active site is sequestered from solvent). To test the hypothesis that bound substrate stabilizes the closed conformer, thus facilitating catalysis, the rates of chemical modification of Lys53 in the presence and absence of inert substrate and/or product analogues were compared. Acetylation of Lys53 with 2,4dinitrophenylacetate (DNPA) resulted in the loss of enzyme activity. The pseudo-first-order rate constant for inactivation varied with pH. The pH profile of inactivation is consistent with a pK[sub a] of 9.3 for Lys53. The inhibitors tungstate and vinyl sulfonate, which are known to bind to active site residues comprising the core domain, protected Lys53 from acetylation. These results are consistent with a dynamic equilibrium between the open and closed conformations of phosphonatase and the hypothesis that ligand binding stabilizes the closed conformation required for catalytic turnover. [ABSTRACT FROM AUTHOR]

Investigates the role of the domain linkers in separate site catalysis by clostridium symbosuim pyruvate phosphate dikinase. Mechanism of the adenosine triphosphate and pyruvate reaction; Movement of the central domain between active site during catalytic turnover; Analysis of linker mutants.

Herein, the structural determinants for substrate recognition and catalysis in two hotdog-fold thioesterase paralogs, YbdB and YdiI from Escherichia coli, are identified and analyzed to provide insight into the evolution of biological function in the hotdog-fold enzyme superfamily. The X-ray crystal structures of YbdB and YdiI, in complex with inert substrate analogs, determined in this study revealed the locations of the respective thioester substrate binding sites and the identity of the residues positioned for substrate binding and catalysis. The importance of each of these residues was assessed through amino acid replacements followed by steady-state kinetic analyses of the corresponding site-directed mutants. Transient kinetic and solvent 18O-labeling studies were then carried out to provide insight into the role of Glu63 posited to function as the nucleophile or general base in catalysis. Finally, the structure-function-mechanism profiles of the two paralogs, along with that of a more distant homolog, were compared to identify conserved elements of substrate recognition and catalysis, which define the core traits of the hotdog-fold thioesterase family, as well as structural features that are unique to each thioesterase. Founded on the insight gained from this analysis, we conclude that the promiscuity revealed by in vitro substrate activity determinations, and posited to facilitate the evolution of new biological function, is the product of intrinsic plasticity in substrate binding as well as in the catalytic mechanism. [ABSTRACT FROM AUTHOR]

The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp 17 in nucleophiic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 μM [14C]-4-HB-CoA catalyzed by 10 μM thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (Kis = 250 ± 70 μM; Kii = 900 ± 300 μM) and 4-HB (Kis = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D2O Solvent kinetic isotope effect (3.8) on the steady-state kcat value (18 s-1) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 μM 4-HB-CoA and 0.54 μM thioesterase), single-turnover reaction (5 μM [14C]-4-HB-CoA catalyzed by 50 μM thioesterase), steady-state reaction (5.2 μM 4-HB-CoA catalyzed by 0.003 μM thioesterase), and transient-state multiple-turnover reaction (50 μM [14C]-4-HB-CoA catalyzed by 10 μM thioesterase). Together with the results obtained from solvent 18O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl l7-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom. [ABSTRACT FROM AUTHOR]

4-Chlorobenzoate:CoA ligase (CBL) belongs to the adenylate-forming family of enzymes that catalyze a two-step reaction to first activate a carboxylate substrate as an adenylate and then transfer the carboxylate to the pantetheine group of either coenzyme A or an acyl-carrier protein. The active site is located at the interface of a large N-terminal domain and a smaller C-terminal domain. Crystallographic structures have been determined at multiple steps along the reaction pathway and form the basis for a proposal that the C-terminal domain rotates by ∼140° between the two states that catalyze the adenylation and thioester-forming half-reactions. The domain rotation is accompanied by a change in the main chain torsional angles of Asp402, a, conserved residue located at the interdomain hinge position. We have mutated the Asp402 residue to Pro in order to test the impact of reduced main chain flexibility at the putative hinge position. The crystal structure of the D402P mutant shows that the enzyme adopts the proposed adenylate-forming conformation with very little change to the overall structure. To examine the impact of this mutation on the ability of the enzyme to catalyze the complete reaction, single turnover kinetic experiments were performed. Whereas the ability of this mutant to catalyze the adenylate-forming half-reaction is reduced by ∼3-fold, catalysis of the second half-reaction is reduced by 4 orders of magnitude. The impact of the alanine replacement of Asp402 on the thioester-forming reaction is significant, although not as dramatic as the proline mutation, and provides evidence that the Asp402 carboxylate group, through ion pair formation with N-terminal domain residue Arg400, assists in the transition to the thioester-forming conformer. Together these results support the domain alternation hypothesis. [ABSTRACT FROM AUTHOR]

ABSTRACT: Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA) is a member of the class II zinc-dependent aldolase family that catalyzes the cleavage of D-fructose 1,6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (G3P). In addition to the active site zinc, the catalytic apparatus of FBPA employs an aspartic acid, Asp83 in the G. lamblia enzyme, which when replaced with an alanine residue renders the enzyme inactive. A comparison of the crystal structures of D83A FBPA in complex with FBP and of wild-type FBPA in the unbound state revealed a substrate-induced conformational transition of loops in the vicinity of the active site and a shift in the location of Zn[sup2+] When FBP binds, the Zn[sup2+] shifts up to 4.6 A toward the catalytic Asp83, which brings the metal within coordination distance of the Asp83 carboxylate group. In addition, the structure of wild- type FBPA was determined in complex with the competitive inhibitor D-tagatose 1,6-bisphosphate (TBP), a FBP stereoisomer. In this structure, the zinc binds in a site close to that previously seen in the structure of FBPA in complex with phosphoglycolohydroxamate, an analogue of the postulated DHAP ene-diolate intermediate. Together, the ensemble of structures suggests that the zinc mobility is necessary to orient the Asp83 side chain and to polarize the substrate for proton transfer from the FBP C(4) hydroxyl group to the Asp83 carboxyl group. In the absence of FBP, the alternative zinc position is too remote for coordinating the Asp83. We propose a modification of the catalytic mechanism that incorporates the novel features observed in the FBPA-FBP structure. The mechanism invokes coordination and coplanarity of the Zn[sup2+] with the FBP's O-C(3)-C(4)-O group concomitant with coordination of the Asp83 carboxylic group. Catalysis is accompanied by movement of Zn[sup2+] to a site coplanar with the O-C(2)-C(3)-O group of the DHAP. g/FBPA exhibits strict substrate specificity toward FBP and does not cleave TBP. The active sites of FBPAs contain an aspartate residue equivalent to Asp255 of g/FBPA, whereas tagatose-1,6-bisphosphate aldolase contains an alanine in this position. We and others hypothesized that this aspartic acid is a likely determinant of FBP versus TBP specificity. Replacement of Asp255 with an alanine resulted in an enzyme that possesses double specificity, now cleaving TBP (albeit with low efficacy; k[subcat]/K[subm] = 80 M[sup-1] s[sup-1]) while maintaining activity toward FBP at a 50-fold lower catalytic efficacy compared with that of wild-type FBPA. The collection of structures and sequence analyses highlighted additional residues that may be involved in substrate discrimination. [ABSTRACT FROM AUTHOR]

The β-phosphoglucomutase (β-PGM) of the haloacid dehalogenase enzyme superfamily (HADSF) catalyzes the conversion of β-glucose 1-phosphate (βG1P) to glucose 6-phosphate (G6P) using Asp8 of the core domain active site to mediate phosphoryl transrer from β-glucose 1 ,6-(bis)phosphate (βG1 ,6bisP) to βG1P. Herein, we explore the mechanism by which hydrolysis of the β-PGM phospho-Asp8 is avoided during the time that the active site must remain open to solvent to allow the exchange of the bound product G6P with the substrate βG1P. On the basis of structural information, a model of catalysis is proposed in which the general acid/base (Asp10) side chain moves from a position where it forms a hydrogen bond to the Thr16-A1a17 portion of the domain--domain linker to a functional position where it forms a hydrogen bond to the substrate leaving group p and a His20-Lys76 pair of the cap domain. This repositioning of the general acid/base within the core domain active site is coordinated with substrate-induced closure of the cap domain over the core domain. The model predicts that Asp10 is required for general acid/base catalysis and for stabilization of the enzyme in the cap-closed conformation. It also predicts that hinge residue Thr16 plays a key role in productive domain--domain association, that hydrogen bond interaction with the Thr16 backbone amide NH group is required to prevent phospho-Asp8 hydrolysis in the cap-open conformation, and that the His20-Lys76 pair plays an important role in substrate-induced cap closure. The model is examined via kinetic analyses of Asp10, Thr16, His20, and Lys76 site-directed mutants. Replacement of Asp10 with Ala, Ser, Cys, Asn, or Glu resulted in no observable activity. The kinetic consequences of the replacement or linker residue Thr16 with Pro include a reduced rate of Asp8 phosphorylation by βG1 ,6bisP, a reduced rate of cycling of the phosphorylated enzyme to convert βG1P to G6P, and an enhanced rate of phosphoryl transfer from phospho-Asp8 to water. The X-ray crystal structure of the T16P mutant at 2.7 Å resolution provides a snapshot of the enzyme in an unnatural cap-open conformation where the Asp10 side chain is located in the core domain active site. The His20 and Lys76 site-directed mutants exhibit reduced activity in catalysis of the Asp8-mediated phosphoryl transfer between βG1,6bisP and βG1P but no reduction in the rate of phospho-Asp8 hydrolysis. Taken together, the results support a substrate induced-fit model of catalysis in which βG1P binding to the core domain facilitates recruitment of the general acid/base Asp10 to the catalytic site and induces cap closure. [ABSTRACT FROM AUTHOR]

Abstract: The Aspergillus niger genome contains four genes that encode proteins exhibiting greater than 30% amino acid sequence identity to the confirmed oxaloacetate acetyl hydrolase (OAH), an enzyme that belongs to the phosphoenolpyruvate mutase/isocitrate lyase superfamily. Previous studies have shown that a mutant A. niger strain lacking the OAH gene does not produce oxalate. To identify the function of the protein sharing the highest amino acid sequence identity with the OAH (An07g08390, Swiss-Prot entry Q2L887, 57% identity), we produced the protein in Escherichia coli and purified it for structural and functional studies. A focused substrate screen was used to determine the catalytic function of An07g08390 as (2R,3S)-dimethylmalate lyase (DMML): k cat =19.2 s−1 and K m =220 μM. DMML also possesses significant OAH activity (k cat =0.5 s−1 and K m =220 μM). DNA array analysis showed that unlike the A. niger oah gene, the DMML encoding gene is subject to catabolite repression. DMML is a key enzyme in bacterial nicotinate catabolism, catalyzing the last of nine enzymatic steps. This pathway does not have a known fungal counterpart. BLAST analysis of the A. niger genome for the presence of a similar pathway revealed the presence of homologs to only some of the pathway enzymes. This and the finding that A. niger does not thrive on nicotinamide as a sole carbon source suggest that the fungal DMML functions in a presently unknown metabolic pathway. The crystal structure of A. niger DMML (in complex with Mg2+ and in complex with Mg2+ and a substrate analog: the gem-diol of 3,3-difluoro-oxaloacetate) was determined for the purpose of identifying structural determinants of substrate recognition and catalysis. Structure-guided site-directed mutants were prepared and evaluated to test the contributions made by key active-site residues. In this article, we report the results in the broader context of the lyase branch of the phosphoenolpyruvate mutase/isocitrate lyase superfamily to provide insight into the evolution of functional diversity. [Copyright &y& Elsevier]

L-Argtntne deiminase (AD!) catalyzes the hydrolysis of L-argtntne to form L-citrulliue 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 Hit. Accordingly, in the first partial reaction, the Cys attacks the substrate guanidino C~ 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~ atom to form the second tetrahedral intermediate, which eliminates the Cys in formation of the L-citrutline 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 Cs Iltiolate anion. The studies described in this paper explore the mechanism of stabilization of the Cys thiolate. First, the Iog(k~/K~) and log k~ pH rate profiles were measured for several structurally divergent Al)k to establish the pH range for ADI catalysis. All ADIs were optimally active at pH 5, which suggested that the Cys pK, is strongly perturbed by the prevailing electrostatics of the ADI active site. The pK~ of the Bad/u.s cereu,s ADI (BcADI) was determined by UV-pH titration to be 9.6. In contrast, the pK~ determined by iodoacetamide Cys alkyltttion is 6.9. These results suggest that the negative electrostatic field from the Iwo opposing Asp carboxylates perturbs thc Cys pK~ upward in the apoenzyme and that the binding of the iodoacetamide (a truncated analogue of the citrulline product) between the Cys thiol aitd the two Asp carboxylates shields the Cys thiol. thereby reducing its pK~. It is hypothesized that the hound positively charged gitanidinium group of the t.-arginine substrate further stabilizes the Cys thiolale. The so-called "substrate-assisted" Cys ionization. first reported by Fast and co-workers to operate in the related enzyme diinethylarginine dinsethylaminohydrolase Stone, E. M., Costello, A. L., Tierney, D. L., and Fast. W. (2006) Biochemistry 45, 5618-563)11. was further explored computationally in ADI by using an ab initio quantum mechanics/niolecular 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 coitsists of the (`ys thiolate (nucleophilel and His imidazolium ion (general acid) and that the energy harriers for the nucleophilic addition of Cys thiolate to the thiouronium Cr atom and 1-1k imidazolium ion-assisted elimination from the tetrahedral intermediate are small, [ABSTRACT FROM AUTHOR]

Arginine deiminase (ADI) catalyzes the hydrolytic conversion of L-arginine to ammonia and L-citrulline as part of the energy-producing L-arginine degradation pathway. The chemical mechanism for ADI catalysis involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate. The structure of the Pseudomorias aeruginosa ADI-(L-arginine) complex guided the design of arginine analogs that might react with the ADIs to form inactive covalent adducts during catalytic turnover. One such candidate is L-canavanine, in which an N-methylene of L-arginine is replaced by an N-O. This substance was shown to be a slow substrate-producing O-ureido-L-homoserine. An in depth kinetic and mass spectrometric analysis of P. aeruginosa ADI inhibition by L-canavanine showed that two competing pathways are followed that branch at the Cys-alkylthiouronium ion intermediate. One pathway leads to direct formation of O-ureido-L-homoserine via a reactive thiouronium intermediate. The other pathway leads to an inactive form of the enzyme, which was shown by chemical model and mass spectrometric studies to be a Cys-alkylisothiourea adduct. This adduct undergoes slow hydrolysis to form O-ureido-L-homoserine and regenerated enzyme. In contrast, kinetic and mass spectrometric investigations demonstrate that the Cys-alkylthiouronium ion intermediate formed in the reaction of L-canavanine with Bacillus cereus ADI partitions between the product forming pathway (O-ureido-L-homoserine and free enzyme) and an inactivation pathway that leads to a stable Cys-alkylthiocarbamate adduct. The ADIs from Escherichia co/i, Burkholderia mallei, and Giardia intestinalis were examined in order to demonstrate the generality of the L-canavanine slow substrate inhibition and to distinguish the kinetic behavior that defines the irreversible inhibition observed with the B. cereus ADI from the time controlled inhibition observed with the P. aeruginosa, E. co/i, B. mallei, and G. intestinalis ADI5. [ABSTRACT FROM AUTHOR]

The structure and biochemical function of the hot dog-fold thioesterase PaaI operative in the aerobic phenylacetate degradation pathway are examined. PaaI showed modest activity with phenylacetyl-coenzyme A, suggestive of a role in coenzyme A release from this pathway intermediate in the event of limiting downstream pathway enzymes. Minimal activity was observed with aliphatic acyl-coenzyme A thioesters, which ruled out PaaI function in the lower phenylacetate pathway. PaaI was most active with ring-hydroxylated phenylacetyl-coenzyme A thioesters. The x-ray crystal structure of the Escherichia coli thioesterase is reported and analyzed to define the structural basis of substrate recognition and catalysis. The contributions of catalytic and substrate binding residues, thus, identified were examined through steady-state kinetic analysis of site-directed mutant proteins. [ABSTRACT FROM AUTHOR]

Lactococcus lactis β-phosphoglucomutase (β-PGM) catalyzes the interconversion of β-D-glucose 1-phosphate (β-G1P) and β-D-glucose 6-phosphate (G6P), forming β-D-glucose 1,6-(bis)phosphate (β- G16P) as an intermediate. β-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine β-PGM activation by the Mg2+ cofactor, β-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 Å resolution X-ray crystal structure of the Mg2+-β-PGM complex is examined in the context of previously reported structures of the Mg2+-α-D-galactose-1-phosphate-β-PGM, Mg2+-phospho-β-PGM, and Mg2+-β-glucose-6-phosphate-1-phosphorane-β-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg2+ in the different complexes shows that a single Mg2+ coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg2+ anchoring in β-PGM is consistent with the relatively loose binding indicated by the large Km for Mg2+ activation (270 ± 20 μM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp 10 for acid—base catalysis. Kinetic analyses of the specificity of β-PGM toward phosphoryl group donors and the specificity of phospho-β-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of β-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of β-PGM by the substrate β-G1P accounts for the origin of phospho-β-PGM in the cell. [ABSTRACT FROM AUTHOR]

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the α/β core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight β-turn in the helix-loop--helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and β-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members. [ABSTRACT FROM AUTHOR]

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Paid) to form orthophosphate and acetaldehyde. The reaction proceeds via a Schiff-base intermediate formed between Lys-53 and the Paid carbonyl. The x-ray crystal structures of the wild.type phosphonatase complexed with Mg(II) alone or with Mg(II) plus vinylsulfonate (a phosphonoethylenamine analog) were determined to 2.8 and 2.4 Å, respectively. These structures were used to determine the identity and positions of active site residues surrounding the Lys-53 ammonium group and the Paid carbonyl. These include Cys-22, His-56, Tyr-128, and Met-49. Site-directed mutagenesis was then employed to determine whether or not these groups participate in catalysis. Based on rate contributions, Tyr-128 and Cys-22 were eliminated as potential catalytic groups. The Lys-53 ε-amino group, positioned for reaction with the Paid carbonyl, forms a hydrogen bond with water 120. Water 120 is also within hydrogen bond distance of an imidazole nitrogen of His-56 and the sulfur atom of Met-49. Kinetic constants for mutants indicated that His-56 (1000-fold reduction in kcat/Km upon Ala substitution) and Met-49 (17,000-fold reduction in kcat/Km upon Leu substitution) function in catalysis of Schiff-base formation. Based on these resuits, it is proposed that a network of hydrogen bonds among Lys-53, water 120, His-56, and Met49 facilitate proton transfer from Lys-53 to the carbinolamine intermediate. Comparison of the vinylsulfonate complex versus unliganded structures indicated that association of the cap and core domains is essential for the positioning of the Lys-53 for attack at the Paid carbonyl and that substrate binding at the core domain stabilizes cap domain binding. [ABSTRACT FROM AUTHOR]

In this study, we have shown that recombinant BH1999 from Bacillus halodurans catalyzes the hydrolysis of gentisyl coenzyme A (CoA) (2,5-dihydroxybenzoyl-coenzyme A) at physiological pH with a k[sub cat]/K[sub m] of 1.6 × 10[sup 6] M[sup -1] s[sup -1] and the hydrolysis of 3-hydroxybenzoyl-CoA with a k[sub cat]/K[sub m] of 3.0 × 10[sup 5] M[sup -1] s[sup -1]. All other acyl-CoA thioesters tested had low or no substrate activity. The BH1999 gene is juxtaposed with a gene cluster that contains genes believed to function in gentisate oxidative degradation. It is hypothesized that BH1999 functions as a gentisyl-CoA thioesterase. Gentisyl-CoA thioesterase shares the backbone fold and the use of an active site aspartate residue to mediate catalysis with the 4-hydroxybenzoyl-CoA thioesterase of the hotdog fold enzyme superfamily. A comparative study of these two enzymes showed that they differ greatly in the rate contribution made by the catalytic aspartate, in the pH dependence of catalysis, and in substrate specificity. [ABSTRACT FROM AUTHOR]

Examines the mechanisms of substrate recognition and catalysis on thioesterase family. Structures of the liganded thioesterase; Interactions between the positively charged residues and the negatively charged phosphates; Typology of protein surfaces.

Examines the contribution of the active site Histidine 90 (His90) to the 4-chlorobenzoyl-coenzyme A dehalogenase catalysis. Replacement of His90 by glutamine by site-directed mutagenesis; Use of X-ray crystallographic analysis; Evaluation of the kinetic binding and catalytic turnover rate.

Presents a Raman study of the polarizing forces promoting catalysis in 4-chlorobenzoate-CoA dehalogenase enzymes. Carbonyl group's participation in normal modes occurring in the Raman spectroscopy; Estimation of the degree of electron polarization; Factors affecting the polarization of electrons; Description of the hydrogen strength value.

Arginine deiminase, catalyzes the hydrolysis of L-arginine to citrulline and ammonium ion; the first step of the L-argnine degradation pathway used by specialized microorganisms as an essential pathway for energy production. Arginine deiminase shares structural homology with peptidyl-arginine deiminase, amidinotransferase, and N(G),N(G)-dimethyl-arginine deiminase. The conserved cysteine (Cys) residue may mediate group transfer from the guanidinium centers of the respective substrates. The possibility of Cys nucleophilic catalysis has important implications for the development of arginine deiminase inhibitors, where design strategies may center on the use of mechanism-based, irreversible inhibitors.