Reaction mechanism of the binuclear zinc enzyme glyoxalase II - A theoretical study

The glyoxalase system catalyzes the conversion of toxic methylglyoxal to nontoxic d-lactic acid using glutathione (GSH) as a coenzyme. Glyoxalase II (GlxII) is a binuclear Zn enzyme that catalyzes the second step of this conversion, namely the hydrolysis of S-d-lactoylglutathione, which is the product of the Glyoxalase I (GlxI) reaction. In this paper we use density functional theory method to investigate the reaction mechanism of GlxII. A model of the active site is constructed on the basis of the X-ray crystal structure of the native enzyme. Stationary points along the reaction pathway are optimized and the potential energy surface for the reaction is calculated. The calculations give strong support to the previously proposed mechanism. It is found that the bridging hydroxide is capable of performing nucleophilic attack at the substrate carbonyl to form a tetrahedral intermediate. This step is followed by a proton transfer from the bridging oxygen to Asp58 and finally C-S bond cleavage. The roles of the two zinc ions in the reaction mechanism are analyzed. Zn2 is found to stabilize the charge of tetrahedral intermediate thereby lowering the barrier for the nucleophilic attack, while Zn1 stabilizes the charge of the thiolate product, thereby facilitating the C-S bond cleavage. Finally, the energies involved in the product release and active-site regeneration are estimated and a new possible mechanism is suggested.     

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.     

Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center

Dihydroorotase plays a key role in pyrimidine biosynthesis by catalyzing the reversible interconversion of carbamoyl aspartate to dihydroorotate. Here we describe the three-dimensional structure of dihydroorotase from Escherichia coli determined and refined to 1.7 A resolution. Each subunit of the homodimeric enzyme folds into a "TIM" barrel motif with eight strands of parallel beta-sheet flanked on the outer surface by alpha-helices. Unexpectedly, each subunit contains a binuclear zinc center with the metal ions separated by approximately 3.6 A. Lys 102, which is carboxylated, serves as a bridging ligand between the two cations. The more buried or alpha-metal ion in subunit I is surrounded by His 16, His 18, Lys 102, Asp 250, and a solvent molecule (most likely a hydroxide ion) in a trigonal bipyramidal arrangement. The beta-metal ion, which is closer to the solvent, is tetrahedrally ligated by Lys 102, His 139, His 177, and the bridging hydroxide. L-Dihydroorotate is observed bound to subunit I, with its carbonyl oxygen, O4, lying 2.9 A from the beta-metal ion. Important interactions for positioning dihydroorotate into the active site include a salt bridge with the guanidinium group of Arg 20 and various additional electrostatic interactions with both protein backbone and side chain atoms. Strikingly, in subunit II, carbamoyl L-aspartate is observed binding near the binuclear metal center with its carboxylate side chain ligating the two metals and thus displacing the bridging hydroxide ion. From the three-dimensional structures of the enzyme-bound substrate and product, it has been possible to propose a unique catalytic mechanism for dihydroorotase. In the direction of dihydroorotate hydrolysis, the bridging hydroxide attacks the re-face of dihydroorotate with general base assistance by Asp 250. The carbonyl group is polarized for nucleophilic attack by the bridging hydroxide through a direct interaction with the beta-metal ion. During the cyclization of carbamoyl aspartate, Asp 250 initiates the reaction by abstracting a proton from N3 of the substrate. The side chain carboxylate of carbamoyl aspartate is polarized through a direct electrostatic interaction with the binuclear metal center. The ensuing tetrahedral intermediate collapses with C-O bond cleavage and expulsion of the hydroxide which then bridges the binuclear metal center.     

Structural consequences of the active site substitution Cys181 ==> Ser in metallo-beta-lactamase from Bacteroides fragilis

The metallo-beta-lactamases require divalent cations such as zinc or cadmium for hydrolyzing the amide bond of beta-lactam antibiotics. The crystal structure of the Zn2+ -bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam bond of the substrate. It was previously reported that the replacement of the active site Cys181 by a serine residue severely impaired catalysis while atomic absorption measurements indicated that binding of the two zinc ions remained intact. Contradicting data emerge from recent mass spectrometry results, which show that only a single zinc ion binds to the C181S metallo-beta-lactamase. In the current study, the C181S mutant enzyme was examined at the atomic level by determining the crystal structure at 2.6 A resolution. The overall structure of the mutant enzyme is the same as that of the wild-type enzyme. At the mutation site, the side chain of Ser181 occupies the same position as that of the side chain of Cys181 in the wild-type protein. One zinc ion, Zn1, is present in the crystal structure; however, the site of the second zinc ion, Zn2 is unoccupied. A water molecule is associated with Zn1, reminiscent of the hydroxide seen in the structure of the wild-type enzyme but farther from the metal. The position of the water molecule is off the plane of the carboxylate group of Asp103; therefore, the water molecule may be less nucleophilic than a water molecule which is coplanar with the carboxylate group.     

Probing the Ser-Ser-Lys catalytic triad mechanism of peptide amidase: computational studies of the ground state, transition state, and intermediate

Peptide amidase (Pam), a hydrolytic enzyme that belongs to the amidase signature (AS) family, selectively catalyzes the hydrolysis of the C-terminal amide bond (CO-NH(2)) of peptides. The recent availability of the X-ray structures of Pam, fatty acid amide hydrolase, and malonamidase E2 has led to the proposal of a novel Ser-Ser-Lys catalytic triad mechanism for the amide hydrolysis by the AS enzymes. The molecular dynamics (MD) simulations using the CHARMM force field were performed to explore the catalytic mechanism of Pam. The 1.8 A X-ray crystal structure of Pam in complex with the amide analogue of chymostatin was chosen for the initial coordinates for the MD simulations. The five systems that were investigated are as follows: (i) enzyme.substrate with Lys123-NH(2), (ii) enzyme.substrate with Lys123-NH(3)(+), (iii) enzyme.substrate with Lys123-NH(3)(+) and Ser226-O(-), (iv) enzyme.transition state, and (v) enzyme.tetrahedral intermediate. Our data support the presence of the hydrogen bonding network among the catalytic triad residues, Ser226, Ser202, and Lys123, where Ser226 acts as the nucleophile and Ser202 bridges Ser226 and Lys123. The MD simulation supports the catalytic role of the crystallographic waters, Wat1 and Wat2. In all the systems that have been studied, the backbone amide nitrogens of Asp224 and Thr223 create an oxyanion hole by hydrogen bonding to the terminal amide oxygen of the substrate, and stabilize the oxyanion tetrahedral intermediate. The results from both our computational investigation and previously published experimental pH profile support two mechanisms. In a mechanism that is relevant at lower pH, the Lys123-NH(3)(+)-Ser202 dyad provides structural support to the catalytic residue Ser226, which in turn carries out a nucleophilic attack at the substrate amide carbonyl in concert with Wat1-mediated deprotonation and stabilization of the tetrahedral transition state by the oxyanion hole. In the mechanism operating at higher pH, the Lys123-NH(2)-Ser202 catalytic dyad acts as a general base to assist addition of Ser226 to the substrate amide carbonyl. The results from the MD simulation of the tetrahedral intermediate state show that both Ser202 and Lys123 are possible candidates for protonation of the leaving group, NH(2), to form the acyl-enzyme intermediate.     

Catalytic role of proton transfers in the formation of a tetrahedral adduct in a serine carboxyl peptidase

Quantum mechanical/molecular mechanical molecular dynamics and 2D free energy simulations are performed to study the formation of a tetrahedral adduct by an inhibitor N-acetyl-isoleucyl-prolyl-phenylalaninal (AcIPF) in a serine-carboxyl peptidase (kumamolisin-As) and elucidate the role of proton transfers during the nucleophilic attack by the Ser278 catalytic residue. It is shown that although the serine-carboxyl peptidases have a fold resembling that of subtilisin, the proton transfer processes during the nucleophilic attack by the Ser residue are likely to be more complex for these enzymes compared to the case in classical serine proteases. The computer simulations demonstrate that both general base and acid catalysts are required for the formation and stabilization of the tetrahedral adduct. The 2D free energy maps further demonstrate that the proton transfer from Ser278 to Glu78 (the general base catalyst) is synchronous with the nucleophilic attack, whereas the proton transfer from Asp164 (the general acid catalyst) to the inhibitor is not. The dynamics of the protons at the active site in different stages of the nucleophilic attack as well as the motions of the corresponding functional groups are also studied. It is found that the side chain of Glu78 is generally rather flexible, consistent with its possible multifunctional role during catalysis. The effects of proton shuffling from Asp82 to Glu78 and from Glu32 to Asp82 are examined, and the results indicate that such proton shuffling may not play an important role in the stabilization of the tetrahedral intermediate analogue.     

Mechanism of cyanamide hydration catalyzed by carbonic anhydrase II suggested by cryogenic X-ray diffraction

The three-dimensional structure of a possible intermediate in the hydration reaction of cyanamide to urea catalyzed by human carbonic anhydrase II (hCAII) has been determined by cryocrystallographic techniques. The crystal structure shows that two different adducts are formed under the experimental conditions and that they have different occupancy in the crystal. The high occupancy form consists of a binary hCAII-cyanamide complex where the substrate has replaced the zinc-bound hydroxide anion present in the native enzyme, maintaining the tetrahedral geometry around the metal ion. The second, low-occupancy form consists of a hCAII-cyanamide-water ternary complex where the catalytic zinc ion, still being bound to cyanamide, is approached by a water molecule in a five-coordinate adduct. While the first form can be considered a nonproductive complex, the second form may represent an intermediate state of the catalyzed reaction where the water molecule is about to perform a nucleophilic attack on the zinc-activated cyanamide substrate. The structural evidence is consistent with the kinetic data previously reported about this recently described hydrolytic reaction catalyzed by hCAII, and indicates that a different mechanism with respect to that generally accepted for the physiologic carbon dioxide hydration reaction may be adopted by the enzyme, depending on the substrate chemical properties.     

Enolpyruvyl activation by enolpyruvylshikimate-3-phosphate synthase

Enolpyruvylshikimate-3-phosphate synthase (AroA, also called EPSP synthase) is a carboxyvinyl transferase involved in aromatic amino acid biosynthesis, forming EPSP from shikimate 3-phosphate and phosphoenolpyruvate. Upon extended incubation, EPSP ketal, a side product, forms by intramolecular nucleophilic addition of O4 to C2' of the enolpyruvyl group. The catalytic significance of this reaction was unclear, as it was initially proposed to arise from nonenzymatic breakdown of tetrahedral intermediate that had dissociated from AroA. This study shows that EPSP ketal formed in AroA's active site, not nonenzymatically, by demonstrating its formation in the presence of excess AroA. It formed both in the normal reaction and during AroA-catalyzed EPSP hydrolysis. In addition, nonenzymatic EPSP hydrolysis was studied to elucidate the catalytic imperative for enolpyruvyl reactions. Hydrolysis was acid-catalyzed, with a rate enhancement of >5 x 10(8)-fold. There was no detectable EPSP breakdown after 16 days at 90 degrees C in 1 M KOH, a solution that is 1000-fold more nucleophilic than neutral aqueous solutions. Thus, an unactivated enolpyruvyl group is not susceptible to nucleophilic attack. Enzymatic EPSP ketal formation therefore requires enolpyruvyl activation through protonation of C3' to form either a cationic intermediate or a highly cation-like transition state. Forming an EPSP cation requires the investment of considerable catalytic power by AroA. Such an intermediate is a potential target motif for inhibitor design.     

High-resolution X-ray structure of isoaspartyl dipeptidase from Escherichia coli

Isoaspartyl dipeptidase from Escherichia coli functions in protein degradation by catalyzing the hydrolysis of beta-L-isoaspartyl linkages in dipeptides. The best substrate for the enzyme reported thus far is iso-Asp-Leu. Here we report the X-ray analysis of the enzyme in its resting state and complexed with aspartate to 1.65 and 2.1 A resolution, respectively. The quaternary structure of the enzyme is octameric and can be aptly described as a tetramer of dimers. Each subunit folds into two distinct domains: the N-terminal region containing eight strands of mixed beta-sheet and the C-terminal motif that is dominated by a (beta,alpha)(8)-barrel. A binuclear zinc center is located in each subunit at the C-terminal end of the (beta,alpha)(8)-barrel. Ligands to the binuclear metal center include His 68, His 70, His 201, His 230, and Asp 285. The two zincs are bridged by a carboxylated lysine residue (Lys 162) and a solvent molecule, most likely a hydroxide ion. The product of the reaction, aspartate, binds to the enzyme by displacing the bridging solvent with its side chain functional group. From this investigation it is proposed that the reaction mechanism of the enzyme proceeds through a tetrahedral intermediate and that the bridging solvent attacks the re face of the carbonyl carbon of the scissile peptide bond. This structural analysis confirms the placement of isoaspartyl dipeptidase into the urease-related amidohydrolase superfamily.     

5-Enolpyruvylshikimate-3-phosphate synthase: determination of the protonation state of active site residues by the semiempirical method

EPSP synthase (EPSPS) catalyzes the addition of shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form a tetrahedral intermediate (TI) that is converted to 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. A semiempirical molecular modeling study of the EPSPS active site containing the TI was implemented for the assignment of the protonation states of four basic residues, Lys22, Lys340, His385, and Lys411, based on the evaluation of 16 different protonation states and comparison of the resulting energy minimized heavy atoms coordinates with available X-ray crystallographic data of the D313A mutant of EPSPS. The results, employing both gas phase and continuum solvent models, are indicative that after the TI formation the histidine residue is most probably in neutral form (N^ε-protonated) and the lysine residues are in protonated form, which suggests that none of the presently proposed assignments of aminoacid residues involved in the reaction mechanism could be completely correct. The protonated state of Lys22 in the presence of the TI supports the proposal that this residue is a general acid catalyst for TI breakdown. Modeling of the native enzyme active site suggests that Asp313 residue has only minor effects on the definition of the TI position inside the active site. Hydrogen-bonds distances suggest that, in order to act as a base, Asp313 needs the intermediacy of a hydroxyl group of the TI for effecting the attack on the TI methyl group in the elimination step leading to EPSP, as suggested previously in the literature.     

Mechanistic characterization of N-formimino-L-glutamate iminohydrolase from Pseudomonas aeruginosa

N-Formimino-l-glutamate iminohydrolase (HutF) from Pseudomonas aeruginosa catalyzes the deimination of N-formimino-l-glutamate in the histidine degradation pathway. An amino acid sequence alignment between HutF and members of the amidohydrolase superfamily containing mononuclear metal centers indicated that residues Glu-235, His-269, and Asp-320 are involved in substrate binding and activation of the nucleophilic water molecule. The purified enzyme contained up to one equivalent of zinc. The metal was removed by dialysis against the metal chelator dipicolinate with the complete loss of catalytic activity. Enzymatic activity was restored by incubation of the apoprotein with Zn2+, Cd2+, Ni2+, or Cu2+. The mutation of Glu-235, His-269, or Asp-320 resulted in the diminution of catalytic activity by two to six orders of magnitude. Bell-shaped profiles were observed for kcat and kcat/Km as a function of pH. The pKa of the group that must be unprotonated for catalytic activity was consistent with the ionization of His-269. This residue is proposed to function as a general base in the abstraction of a proton from the metal-bound water molecule. In the proposed catalytic mechanism, the reaction is initiated by the abstraction of a proton from the metal-bound water molecule by the side chain imidazole of His-269 to generate a tetrahedral intermediate of the substrate. The collapse of the tetrahedral intermediate commences with the abstraction of a second proton via the side chain carboxylate of Asp-320. The C-N bond of the substrate is subsequently cleaved with proton transfer from His-269 to form ammonia and the N-formyl product. The postulated role of the invariant Glu-235 is to ion pair with the positively charged formimino group of the substrate.     

Structural analyses on intermediates in serine protease catalysis

Although the subject of many studies, detailed structural information on aspects of the catalytic cycle of serine proteases is lacking. Crystallographic analyses were performed in which an acyl-enzyme complex, formed from elastase and a peptide, was reacted with a series of nucleophilic dipeptides. Multiple analyses led to electron density maps consistent with the formation of a tetrahedral species. In certain cases, apparent peptide bond formation at the active site was observed, and the electron density maps suggested production of a cis-amide rather than a trans-amide. Evidence for a cis-amide configuration was also observed in the noncovalent complex between elastase and an alpha1-antitrypsin-derived tetrapeptide. Although there are caveats on the relevance of the crystallographic data to solution catalysis, the results enable detailed proposals for the pathway of the acylation step to be made. At least in some cases, it is proposed that the alcohol of Ser-195 may preferentially attack the carbonyl of the cis-amide form of the substrate, in a stereoelectronically favored manner, to give a tetrahedral oxyanion intermediate, which undergoes N-inversion and/or C-N bond rotation to enable protonation of the leaving group nitrogen. The mechanistic proposals may have consequences for protease inhibition, in particular for the design of high energy intermediate analogues.     

Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.     

Quantum chemistry analysis of the mechanism of action of proteolytic enzymes. III. Proton transport in serine proteases

The model system for the proton transfer on the amide atom of the substrate leaving group based on the existence of "charge relay system" in the serine type proteases was analysed by the CNDO/2 method. The unfitness of this model to explain the action mechanism of serine proteases was shown. The model system for proton transfer with the water molecule as the intermediate acceptor of the Ser-195 proton was suggested and analysed by the same method. The acylation activation barrier of this system was shown to localize on the stage of synchronous transfer of the Ser-195 alcoholic proton and the water molecule proton hydrogen bound to the His-57 N epsilon 2-atom on the water molecule oxygen atom and the N epsilon 2-atom, respectively. The protonation of substrate in the case of the model system with the water molecule as the intermediate acceptor of proton was demonstrated to begin before the completion of the tetrahedral intermediate substance and the protonated from of the tetrahedral intermediate was shown to form only. A hypothesis considering the role of this water molecule as the nucleophilic reagent on the deacylation stage is presented.     

Computational evidence for the catalytic mechanism of glutaminyl cyclase. A DFT investigation

The results of a DFT theoretical investigation on the catalytic mechanism of the QC enzyme are presented. A rather large model-system is used. It includes the most important residues that are believed to play a key-role in the catalysis. The computational results show that the rate-determining step of the catalytic process is not the nucleophilic attack leading to the cycle formation (a very easy and fast process with a negligible barrier of 0.8 kcal mol(-1)), but a proton transfer, which is assisted by the Glu201 residue acting as a proton shuttle (general base and general acid). A complex network of hydrogen bonds (involving Asp248 and other residues) contribute to lower the activation barrier for the proton shift which affords the formation of an ammonia molecule bonded to the substrate. The ammonia molecule is a good leaving group which is easily expelled from the substrate in the last step of the catalytic cycle, but remains anchored to the enzyme as a ligand of the zinc cation. The metal plays a key-role in assisting the nucleophilic attack (electrostatic catalysis) since it polarizes the substrate gamma-amide carbonyl group (its electrophilic character increases). Also, the strength of the nucleophilic nitrogen (substrate alpha-amino group) is enhanced by hydrogen bonds involving the Glu201 residue. The computations outline the important role of Trp329 in helping the substrate binding process and stabilizing the cyclization transition state.     

Mechanism of hydrolyses of phenyl alpha-maltosides catalyzed by taka-amylase A.

Michaelis constants (Kms) and molecular activities (kos) of phenyl, p-nitrophenyl and p-methylphenyl alpha-maltoside for taka-amylase A catalyzed hydrolyses were determined in H2O and in D2O at pH or pD 5.3 and at 25 degrees C. Production of alpha-maltose in the hydrolysis was confirmed by 1H NMR. Neither substituent nor solvent deuterium isotope effects on Kms for phenyl, p-nitrophenyl and p-methylphenyl alpha-maltosides were detected. On the other hand, substituent effects on kos of these compounds were evident, but the isotope effects on kos were not marked, so that protonation of the substrate in the catalytic reaction might not be rate-limiting. The result indicates that nucleophilic attack of a carboxylate anion of the enzyme upon the protonated substrate is the rate-limiting step in the hydrolysis proceeding through the nucleophilic double displacement mechanism, which involves a covalently bonded glycosyl intermediate. The molecular orbitals of phenyl alpha-D-glucosides as model compounds of phenyl alpha-maltosides were calculated by the AM1 method. From the results, it was concluded that the lowering of the lowest unoccupied molecular orbital (LUMO) energy levels and the increase of distribution of LUMO on the anomeric carbon, C-1, of the compounds are caused by protonation at the glycosidic oxygen from the protonated carboxyl group of the enzyme. This causes acceleration of the hydrolysis of a substrate by the enzyme.     

Snapshots of enzymatic Baeyer-Villiger catalysis: oxygen activation and intermediate stabilization

Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP(+) ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and "Criegee-stabilizing" catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.     

Mechanism of substrate dephosphorylation in low Mr protein tyrosine phosphatase.

Substrate dephosphorylation by the low molecular weight protein tyrosine phosphatases proceeds via nucleophilic substitution at the phosphorous atom yielding a cysteinyl phosphate intermediate. However, several questions regarding the exact reaction mechanism remain unanswered. Starting from the crystal structure of the enzyme we study the energetics of this reaction, using the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The free energy profiles of two mechanisms corresponding to different protonation states of the reacting groups are examined along stepwise and concerted pathways. The activation barriers calculated relative to the enzyme-substrate complex are very similar for both monoanionic and dianionic substrates, but taking the substrate binding step into account shows that hydrolysis of monoanionic substrates is strongly favored by the enzyme, because a dianionic substrate will not bind when the reacting cysteine is ionized. The calculated activation barrier for dephosphorylation of monoanionic phenyl phosphate according to this novel mechanism is 14 kcal mol(-1), which is in good agreement with experimental data. Proteins 1999;36:370-379.     

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.     

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J.Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to (18)O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from (31)P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.