Theoretical studies on the deacylation step of serine protease catalysis in the gas phase, in solution, and in elastase

The deacylation step of serine protease catalysis is studied using DFT and ab initio QM/MM calculations combined with MD/umbrella sampling calculations. Free energies of the entire reaction are calculated in the gas phase, in a continuum solvent, and in the enzyme elastase. The calculations show that a concerted mechanism in the gas phase is replaced by a stepwise mechanism when solvent effects or an acetate ion are added to the reference system, with the tetrahedral intermediate being a shallow minimum on the free energy surface. In the enzyme, the tetrahedral intermediate is a relatively stable species ( approximately 7 kcal/mol lower in energy than the transition state), mainly due to the electrostatic effects of the oxyanion hole and Asp102. It is formed in the first step of the reaction, as a result of a proton transfer from the nucleophilic water to His57 and of an attack of the remaining hydroxyl on the ester carbonyl. This is the rate-determining step of the reaction, which requires approximately 22 kcal/mol for activation, approximately 5 kcal/mol less than the reference reaction in water. In the second stage of the reaction, only small energy barriers are detected to facilitate the proton transfer from His57 to Ser195 and the breakdown of the tetrahedral intermediate. Those are attributed mainly to a movement of Ser195 and to a rotation of the His57 side chain. During the rotation, the imidazolium ion is stabilized by a strong H-bond with Asp102, and the C(epsilon)(1)-H...O H-bond with Ser214 is replaced by one with Thr213, suggesting that a "ring-flip mechanism" is not necessary as a driving force for the reaction. The movements of His57 and Ser195 are highly correlated with rearrangements of the binding site, suggesting that product release may be implicated in the deacylation process.     

Theoretical perspectives on the reaction mechanism of serine proteases: the reaction free energy profiles of the acylation process

The reaction mechanism of serine proteases (trypsin), which catalyze peptide hydrolysis, is studied theoretically by ab initio QM/MM electronic structure calculations combined with Molecular Dynamics-Free Energy Perturbation calculations. We have calculated the entire reaction free energy profiles of the first reaction step of this enzyme (acylation process). The present calculations show that the rate-determining step of the acylation is the formation of the tetrahedral intermediate, and the breakdown of this intermediate has a small energy barrier. The calculated activation free energy for the acylation is approximately 17.8 kcal/mol at QM/MM MP2/(aug)-cc-pVDZ//HF/6-31(+)G/AMBER level, and this reaction is an exothermic process. MD simulations of the enzyme-substrate (ES) complex and the free enzyme in aqueous phase show that the substrate binding induces slight conformational changes around the active site, which favor the alignment of the reactive fragments (His57, Asp102, and Ser195) together in a reactive orientation. It is also shown that the proton transfer from Ser195 to His57 and the nucleophilic attack of Ser195 to the carbonyl carbon of the scissile bond of the substrate occur in a concerted manner. In this reaction, protein environment plays a crucial role to lowering the activation free energy by stabilizing the tetrahedral intermediate compared to the ES complex. The polarization energy calculations show that the enzyme active site is in a very polar environment because of the polar main chain contributions of protein. Also, the ground-state destabilization effect (steric strain) is not a major catalytic factor. The most important catalytic factor of stabilizing the tetrahedral intermediate is the electrostatic interaction between the active site and particular regions of protein: the main chain NH groups in Gly193 and Ser195 (so-called oxyanion hole region) stabilize negative charge generated on the carbonyl oxygen of the scissile bond, and the main chain carbonyl groups in Ile212 approximately Ser214 stabilize a positive charge generated on the imidazole ring of His57.     

Why substituting the asparagine at position 35 in Bacillus circulans xylanase with an aspartic acid remarkably improves the enzymatic catalytic activity? A quantum chemistry-based calculation study

Xylanases from Bacillus circulans (BCX) are known as configuration-retaining glycoside hydrolases, which hydrolyze xylans with two glutamic acid residues (Glu78 and Glu172) serving as catalytic active residues according to a double displacement mechanism. Existing experimental researches show that mutating the asparagines (Asn) to aspartic acid (Asp) at position 35 next to Glu172 can obviously improve the catalytic activity of BCX. To better understand the inherent mechanism for the experimental finding, we performed quantum chemistry calculations on two model systems to mimic the catalyses of wild-type and mutant BCXs. Geometrical structures and relative energies of intermediates and transition states involved in the hydrolysis reactions are given in detail. It is found that in the wild-type model system Asn35 interacts with Glu172 via a loose hydrogen bond, while in the mutant model system Asp35 forms a very tight hydrogen bond with Glu172. The glycosidic bond cleavage is proposed to be the rate-determining step for the hydrolysis reaction, whose barrier varies from 98 to 65 kJ mol⁻¹ when Asn35 is replaced by Asp35, showing the presence of Asp35 remarkably reduces the energy demand for the hydrolysis reaction. The present result provides a theoretical elucidation for why a single amino acid substitution can importantly influences catalytic activity of BCX.     

Quantum and molecular mechanical study of the first proton transfer in the catalytic cycle of cytochrome P450cam and its mutant D251N

In the catalytic cycle of cytochrome P450cam, the hydroperoxo intermediate (Cpd 0) is formed by proton transfer from a reduced oxyheme complex (S5). This process is drastically slowed down when Asp251 is mutated to Asn (D251N). We report quantum mechanical/molecular mechanical (QM/MM) calculations that address this proton delivery in the doublet state through a hydrogen-bond network in the Asp251 channel, both for the wild-type enzyme and the D251N mutant, using four different active-site models. For the wild-type, we find a facile concerted mechanism for proton transfer from protonated Asp251 via Wat901 and Thr252 to the FeOO moiety, with a barrier of about 1 kcal/mol and a high exothermicity of more than 20 kcal/mol. In the D251N mutant with a neutral Asn251 residue, the proton transfer is almost thermoneutral or slightly exothermic in the three models considered. It is still very facile when the Asn251 residue adopts a conformation analogous to Asp251 in the wild-type enzyme, but the barrier increases significantly when the Asn251 side chain flips (as indicated by classical molecular dynamics simulations). This flip disrupts the hydrogen-bond network and hence the proton-transfer pathway, which causes a longer lifetime of S5 in the D251N mutant (consistent with experimental observations). The entry of an additional water molecule into the active site of D251N with flipped Asn251 regenerates the hydrogen-bond network and provides a viable mechanism for proton delivery in the mutant, with a moderate barrier of about 7 kcal/mol.     

Quantum chemical calculations on structural models of the catalytic site of chymotrypsin: comparison of calculated results with experimental data from NMR spectroscopy

Hybrid density functional quantum mechanical calculations were used to study the strength of the hydrogen bond between His(57) N(delta)(1) and Asp(102) O(delta)(1) in chymotrypsin and how it changes along the reaction coordinate. Comparison of experimental shifts with the results of chemical shift calculations on a variety of small molecules, including species containing very strong hydrogen bonds, has validated the overall approach and provided the means for calibrating and correcting the calculated values. Models of the active site of chymotrypsin in its resting state and tetrahedral intermediate state were derived from high-resolution X-ray structures. The distance between His(57) N(delta)(1) and Asp(102) O(delta)(1) in each model was varied between 2.77 A (weak hydrogen bond) and 2.50 A (extremely strong hydrogen bond), and the one-dimensional potential energy surface of the hydrogen-bonded proton (or deuteron/triton) was determined. The zero-point energy, probability distribution, and chemical shift were determined for each distance. Calculated values for NMR chemical shifts, NMR chemical shift differences between (1)H and (3)H, and (2)H/(1)H fractionation factors were compared with published experimental values. Energies provided by the calculations indicated that the hydrogen bond between His(57) N(delta)(1) and Asp(102) O(delta)(1) in the chymotrypsin active site increases in strength by 11 kcal mol(-)(1) in going from the resting state of the enzyme to the tetrahedral intermediate state. This result confirms the hypothesis that the strengthened hydrogen bond plays an important role in lowering the energy of the transition state and, hence, in the catalytic efficiency of the enzyme. Models of the transition state that best fit the experimental data are consistent with a "strong" hydrogen bond between His(57) N(delta)(1) and Asp(102) O(delta)(1) but apparently not a "low-barrier" or "very strong" hydrogen bond.     

A theoretical study on the mechanism of camphor hydroxylation by compound I of cytochrome p450

Mechanistic and energetic aspects for the conversion of camphor to 5-exo-hydroxycamphor by the compound I iron-oxo species of cytochrome P450 are discussed from B3LYP DFT calculations. This reaction occurs in a two-step manner along the lines that the oxygen rebound mechanism suggests. The activation energy for the first transition state of the H atom abstraction at the C5 atom of camphor is computed to be more than 20 kcal/mol. This H atom abstraction is the rate-determining step in this hydroxylation reaction, leading to a reaction intermediate that involves a carbon radical species and the iron-hydroxo species. The second transition state of the rebound step that connects the reaction intermediate and the product alcohol complex lies a few kcal/mol below that for the H atom abstraction on the doublet and quartet potential energy surfaces. This energetic feature allows the virtually barrierless recombination in both spin states, being consistent with experimentally observed high stereoselectivity and brief lifetimes of the reaction intermediate. The overall energetic profile of the catalytic mechanism of camphor hydroxylation particularly with respect to why the high activation energy for the H atom abstraction is accessible under physiological conditions is also considered and calculated. According to a proton source model involving Thr252, Asp251, and two solvent water molecules (Biochemistry 1998, 37, 9211), the energetics for the conversion of the iron-peroxo species to compound I is studied. A significant energy over 50 kcal/mol is released in the course of this dioxygen activation process. The energy released in this chemical process is an important driving force in alkane hydroxylation by cytochrome P450. This energy is used for the access to the high activation energy for the H atom abstraction.     

New insights into the mechanism of the Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase from S. enterica: a theoretical study

The reaction pathway of Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase (DHQD) from S. enterica has been studied by performing molecular dynamics (MD) simulations and density functional theory (DFT) calculations and the corresponding potential energy profile has also been identified. On the basis of the results, the catalytic hydrolysis process for the wild-type enzyme consists of three major reaction steps, including nucleophilic attack on the carbon atom involved in the carbon-nitrogen double bond of the Schiff base intermediate by a water molecule, deprotonation of the His143 residue, and dissociation between the product and the Lys170 residue of the enzyme. The remarkable difference between this and the previously proposed reaction mechanism is that the second step here, absent in the previously proposed reaction mechanism, plays an important role in facilitating the reaction through a key proton transfer by the His143 residue, resulting in a lower energy barrier. Comparison with our recently reported results on the Schiff base formation and dehydration processes clearly shows that the Schiff base hydrolysis is rate-determining in the overall reaction catalyzed by type I DHQD, consistent with the experimental prediction, and the calculated energy barrier of ～16.0 kcal mol(-1) is in good agreement with the experimentally derived activation free energy of ～14.3 kcal mol(-1). When the imidazole group of His143 residue is missing, the Schiff base hydrolysis is initiated by a hydroxide ion in the solution, rather than a water molecule, and both the reaction mechanism and the kinetics of Schiff base hydrolysis have been remarkably changed, clearly elucidating the catalytic role of the His143 residue in the reaction. The new mechanistic insights obtained here will be valuable for the rational design of high-activity inhibitors of type I DHQD as non-toxic antimicrobials, anti-fungals, and herbicides.     

Theoretical modeling study for the phosphonylation mechanisms of the catalytic triad of acetylcholinesterase by sarin

Potential energy surfaces for the process of phosphonylation of the catalytic triad of acetylcholinesterase by sarin have been explored at the B3LYP/6-311G(d,p) level of theory through a computational study. It is concluded that the phosphonylation process involves a critical addition-elimination mechanism. The first nucleophilic addition process is the rate-determining step. The following elimination process of the fluoride ion comprises a composite reaction that includes several steps, and it occurs rapidly by comparison with the rate-determining step. The mobility characteristics of histidine play an important role in the reaction. A double proton-transfer mechanism is proposed for the catalytic triad during the phosphonylation process of sarin on AChE. The effect of aqueous solvation has been considered via the polarizable continuum model (PCM). One concludes that the energy barriers are generally lowered in solvent, compared to the gas-phase reactions.     

Ab initio QM/MM dynamics simulation of the tetrahedral intermediate of serine proteases: insights into the active site hydrogen-bonding network

Ab initio QM/MM dynamics simulation is employed to examine the stability of the tetrahedral intermediate during the deacylation step in elastase-catalyzed hydrolysis of a simple peptide. An extended quantum region includes the catalytic triad, the tetrahedral structure, and the oxyanion hole. The calculations indicate that the tetrahedral intermediate of serine proteases is a stable species on the picosecond time scale. On the basis of geometrical and dynamical properties, and in agreement with many experimental and theoretical studies, it is suggested that the crucial hydrogen bonds involved in stabilizing this intermediate are between Asp-102 and His-57 and between the charged oxygen of the intermediate and the backbone N-H group of Gly-193 in the oxyanion hole. The mobility of the imidazolium ring between O(w) and O(gamma), two of the oxygens of the tetrahedral structure, shows how the intermediate could proceed toward the product state without a "ring-flip mechanism", proposed earlier on the basis of NMR data. In addition to the proposed C(epsilon)(1)-H.O hydrogen bond between the imidazolium ring and the backbone carbonyl of Ser-214, we observe an alternative C(epsilon)(1)-H.O hydrogen bond with the backbone carbonyl of Thr-213, that can stabilize the intermediate during the imidazolium movement. Proton hopping occurs between Asp-102 and His-57 during the simulation. The proton is, however, largely localized on the nitrogen, and hence it does not participate in a low-barrier hydrogen bond. The study also suggests factors that may be implicated in product release: breaking the hydrogen bond of the charged oxygen with the backbone of Ser-195 in the oxyanion hole and a loop opening between residues 216-225 that enables the breaking of a hydrogen bond in subsite S(3).     

Hybrid QM/MM and DFT investigations of the catalytic mechanism and inhibition of the dinuclear zinc metallo-beta-lactamase CcrA from Bacteroides fragilis

Based on hybrid QM/MM molecular dynamics simulation and density functional theoretical (DFT) calculations, we investigate the mechanistic and energetic features of the catalytic action of dizinc metallo-beta-lactamase CcrA from Bacteroides fragilis. The 200 ps QM/MM simulation of the CcrA enzyme in complex with nitrocefin shows that the substrate beta-lactam moiety is directed toward the active site dizinc center through the interactions of aminocarbonyl and carboxylate groups with the two active site zinc ions and the two conserved residues, Lys167 and Asn176. From the determination of the potential energy profile of a relevant enzymatic reaction model, it is found that the nucleophilic displacement reaction step proceeds with a low-barrier height, leading to the formation of an energetically favored reaction intermediate. The results also show that the high catalytic activity of the CcrA enzyme stems from a simultaneous operation of three catalytic components: activation of the bridging hydroxide nucleophile by zinc-coordinated Asp86; polarization of the substrate aminocarbonyl group by the first zinc ion; stabilization of the negative charge developed on the departing amide nitrogen by the second zinc ion. Consistent with the previous experimental finding that the proton-transfer reaction step is rate-limiting, the activation energy of the second step is found to be 1.6 kcal/mol higher than that of the first step. Finally, through an examination of the structural and energetic features of binding of a thiazolidinecarboxylic acid inhibitor to the active site dizinc center, a two-step inhibition mechanism involving a protonation-induced ligand exchange reaction is proposed for the inhibitory action of a tight-binding inhibitor possessing a thiol group.     

Ab initio and density functional theory studies of the catalytic mechanism for ester hydrolysis in serine hydrolases

We present results from ab initio and density functional theory studies of the mechanism for serine hydrolase catalyzed ester hydrolysis. A model system containing both the catalytic triad and the oxyanion hole was studied. The catalytic triad was represented by formate anion, imidazole, and methanol. The oxyanion hole was represented by two water molecules. Methyl formate was used as the substrate. In the acylation step, our computations show that the cooperation of the Asp group and oxyanion hydrogen bonds is capable of lowering the activation barrier by about 15 kcal/mol. The transition state leading to the first tetrahedral intermediate in the acylation step is rate limiting with an activation barrier (ΔE₀) of 13.4 kcal/mol. The activation barrier in the deacylation step is smaller. The double-proton-transfer mechanism is energetically unfavorable by about 2 kcal/mol. The bonds between the Asp group and the His group, and the hydrogen bonds in the oxyanion hole, increase in strength going from the Michaelis complex toward the transition state and the tetrahedral intermediate. In the acylation step, the tetrahedral intermediate is a very shallow minimum on the energy surface and is not viable when molecular vibrations are included. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 69: 89–103, 1998     

A conserved asparagine in a ubiquitin-conjugating enzyme positions the substrate for nucleophilic attack

The mechanism used by the ubiquitin-conjugating enzyme, Ubc13, to catalyze ubiquitination is probed with three computational techniques: Born–Oppenheimer molecular dynamics, single point quantum mechanics/molecular mechanics energies, and classical molecular dynamics. These simulations support a long-held hypothesis and show that Ubc13-catalyzed ubiquitination uses a stepwise, nucleophilic attack mechanism. Furthermore, they show that the first step—the formation of a tetrahedral, zwitterionic intermediate—is rate limiting. However, these simulations contradict another popular hypothesis that supposes that the negative charge on the intermediate is stabilized by a highly conserved asparagine (Asn79 in Ubc13). Instead, calculated reaction profiles of the N79A mutant illustrate how charge stabilization actually increases the barrier to product formation. Finally, an alternate role for Asn79 is suggested by simulations of wild-type, N79A, N79D, and H77A Ubc13: it stabilizes the motion of the electrophile prior to the reaction, positioning it for nucleophilic attack. © 2019 Wiley Periodicals, Inc.     

Origins of enhanced proton transport in the Y7F mutant of human carbonic anhydrase II

Human carbonic anhydrase II (HCA II), among the fastest enzymes known, catalyzes the reversible hydration of CO 2 to HCO 3 (-). The rate-limiting step of this reaction is believed to be the formation of an intramolecular water wire and transfer of a proton across the active site cavity from a zinc-bound solvent to a proton shuttling residue (His64). X-ray crystallographic studies have shown this intramolecular water wire to be directly stabilized through hydrogen bonds via a small well-defined set of amino acids, namely, Tyr7, Asn62, Asn67, Thr199, and Thr200. Furthermore, X-ray crystallographic and kinetic studies have shown that the mutation of tyrosine 7 to phenylalanine, Y7F HCA II, has the effect of increasing the proton transfer rate by 7-fold in the dehydration direction of the enzyme reaction compared to wild-type (WT). This increase in the proton transfer rate is postulated to be linked to the formation of a more directional, less branched, water wire. To evaluate this proposal, molecular dynamics simulations have been employed to study water wire formation in both the WT and Y7F HCA II mutant. These studies reveal that the Y7F mutant enhances the probability of forming small water wires and significantly extends the water wire lifetime, which may account for the elevated proton transfer seen in the Y7F mutant. Correlation analysis of the enzyme and intramolecular water wire indicates that the Y7F mutant significantly alters the interaction of the active site waters with the enzyme while occupancy data of the water oxygens reveals that the Y7F mutant stabilizes the intramolecular water wire in a manner that maximizes smaller water wire formation. This increase in the number of smaller water wires is likely to elevate the catalytic turnover of an already very efficient enzyme.     

Theoretical study on the hydrolysis mechanism of N,N-dimethyl-N'-(2-oxo-1, 2-dihydro-pyrimidinyl)formamidine: water-assisted mechanism and cluster-continuum model

The hydrolysis reaction of N,N-dimethyl-N'-(2-oxo-1, 2-dihydro-pyrimidinyl)formamidine (DMPFA), a model compound of the antivirus drug amidine-3TC (3TC = 2', 3'-dideoxy-3'-thiacytidine), is investigated by the hybrid density functional theory B3LYP/6-31+G (d,p) method. The hydrolysis reaction of the title compound is predicted to undergo via two pathways, each of which is a stepwise process. Path A is the addition of H2O to the C=N double bond in the amidine group to form a tetrahedral structure in its first step, and then the transfer of the H atom of hydroxyl leads to the corresponding products via four possible channels. Path B simultaneously involves the nucleophilic attack of H2O to the C atom of the C=N bond and the proton transfer to the N atom of amino group leading to the cleavage of the C-N single bond in the amidine group. The results indicate that path A is more favorable than path B in the gas phase. Moreover, to simulate the title reaction in aqueous solution, water-assisted mechanism and the cluster-continuum model, based on the SCRF/CPCM model, are taken into account in our work. The results indicate that it is rational for two water molecules served as a bridge to assist in the first step of path A and that cytosine rather than the cytosine-substituted formamide should be released from the tetrahedral intermediate via s six-membered cycle transition state (channel 2). Our calculations exhibit that the process toward the tetrahedral intermediate is the rate-determining step both in the gas phase and in aqueous solution.     

A theoretical study on the catalytic mechanism of Mus musculus adenosine deaminase

The catalytic mechanism of Mus musculus adenosine deaminase (ADA) has been studied by quantum mechanics and two‐layered ONIOM calculations. Our calculations show that the previously proposed mechanism, involving His238 as the general base to activate the Zn‐bound water, has a high activation barrier of about 28 kcal/mol at the proposed rate‐determining nucleophilic addition step, and the corresponding calculated kinetic isotope effects are significantly different from the recent experimental observations. We propose a revised mechanism based on calculations, in which Glu217 serves as the general base to abstract the proton of the Zn‐bound water, and the protonated Glu217 then activates the substrate for the subsequent nucleophilic addition. The rate‐determining step is the proton transfer from Zn‐OH to 6‐NH₂ of the tetrahedral intermediate, in which His238 serves as a proton shuttle for the proton transfer. The calculated kinetic isotope effects agree well with the experimental data, and calculated activation energy is also consistent with the experimental reaction rate. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Molecular dynamics simulations of the catalytic pathway of a cysteine protease: a combined QM/MM study of human cathepsin K

Molecular dynamics simulations using a combined QM/MM potential have been performed to study the catalytic mechanism of human cathepsin K, a member of the papain family of cysteine proteases. We have determined the two-dimensional free energy surfaces of both acylation and deacylation steps to characterize the reaction mechanism. These free energy profiles show that the acylation step is rate limiting with a barrier height of 19.8 kcal/mol in human cathepsin K and of 29.3 kcal/mol in aqueous solution. The free energy of activation for the deacylation step is 16.7 kcal/mol in cathepsin K and 17.8 kcal/mol in aqueous solution. The reduction of free energy barrier is achieved by stabilization of the oxyanion in the transition state. Interestingly, although the "oxyanion hole" has been formed in the Michaelis complex, the amide units do not donate hydrogen bonds directly to the carbonyl oxygen of the substrate, but they stabilize the thiolate anion nucleophile. Hydrogen-bonding interactions are induced as the substrate amide group approaches the nucleophile, moving more than 2 A and placing the oxyanion in contact with Gln19 and the backbone amide of Cys25. The hydrolysis of peptide substrate shares a common mechanism both for the catalyzed reaction in human cathepsin K and for the uncatalyzed reaction in water. Overall, the nucleophilic attack by Cys25 thiolate and the proton-transfer reaction from His162 to the amide nitrogen are highly coupled, whereas a tetrahedral intermediate is formed along the nucleophilic reaction pathway.     

Molecular dynamics study of the active site of methylamine dehydrogenase

We have obtained AMBER94 force-field parameters for the TTQ cofactor of the enzyme methylamine dehydrogenase (MADH). This enzyme catalyzes the oxidation of methylamine to produce formaldehyde and ammonia. In the rate-determining step of the catalyzed reaction, a proton is transferred from the methyl group of the substrate to residue Asp76. We used the new parameters to perform molecular dynamics simulations of MADH in order to characterize the dynamics of the active site prior to the proton-transfer step. We found that only one of the oxygen atoms of Asp76 can act as an acceptor of the proton. The other oxygen interacts with Thr122 via a strong hydrogen bond. In contrast, because of the rotation the methyl group of the substrate, the three methyl hydrogen atoms are alternately in position to be transferred. The distance that the proton has to travel presents a broad distribution with a peak between 1.0 and 1.1 A and reaches values as short as 0.8 A. The fluctuation of the distance between the donor and the acceptor has the largest frequency component at 50 cm(-1), but the spectrum presents a rich structure between 10 and 400 cm(-1). The more important peaks appear below 250 cm(-1).     

Subangstrom crystallography reveals that short ionic hydrogen bonds, and not a His-Asp low-barrier hydrogen bond, stabilize the transition state in serine protease catalysis

To address questions regarding the mechanism of serine protease catalysis, we have solved two X-ray crystal structures of alpha-lytic protease (alphaLP) that mimic aspects of the transition states: alphaLP at pH 5 (0.82 A resolution) and alphaLP bound to the peptidyl boronic acid inhibitor, MeOSuc-Ala-Ala-Pro-boroVal (0.90 A resolution). Based on these structures, there is no evidence of, or requirement for, histidine-flipping during the acylation step of the reaction. Rather, our data suggests that upon protonation of His57, Ser195 undergoes a conformational change that destabilizes the His57-Ser195 hydrogen bond, preventing the back-reaction. In both structures the His57-Asp102 hydrogen bond in the catalytic triad is a normal ionic hydrogen bond, and not a low-barrier hydrogen bond (LBHB) as previously hypothesized. We propose that the enzyme has evolved a network of relatively short hydrogen bonds that collectively stabilize the transition states. In particular, a short ionic hydrogen bond (SIHB) between His57 Nepsilon2 and the substrate's leaving group may promote forward progression of the TI1-to-acylenzyme reaction. We provide experimental evidence that refutes use of either a short donor-acceptor distance or a downfield 1H chemical shift as sole indicators of a LBHB.     

Theoretical study toward understanding the catalytic mechanism of pyruvate decarboxylase

Density functional calculations are employed to explore the mechanisms of all elementary reaction steps involved in the catalytic cycle of pyruvate decarboxylase (PDC). Different models are constructed for mimicking the involvement of some key residues in a certain step. The effect of the protein framework on the potential energy profiles of active site models is approximately modeled by fixing some freedoms, based on the crystal structure of the PDC enzyme from Saccharomyces cerevisiae (ScPDC). Our calculations confirm that Glu51 is the most important residue in the formation of the ylide and the release of acetaldehyde via the proton relay between Glu51, N1', and the 4'-amino group of thiamine diphosphate. The presence of Glu477 and Asp28 residues makes the decarboxylation of lactylthiamin diphosphate (LThDP) an endothermic process with a significant free energy barrier. The protonation of the alpha-carbanion to form 2-(1-hydroxyethyl)-thiamin diphosphate is found to go through a concerted double proton transfer transition state involving both Asp28 and His115 residues. The final step, acetaldehyde release, is likely to proceed through a concerted transition state involving carbon-carbon bond-breaking and the deprotonation of the alpha-hydroxyl group. The decarboxylation of LThDP and the protonation of the alpha-carbanion are two rate-limiting steps, relative to the facile occurrence of the ylide formation and acetaldehyde release. The catalytic roles of residues Glu51, Glu477, Asp28, and Gly417 in the active site of ScPDC in individual steps elucidated from the present study are in good agreement with those derived from site-directed mutagenesis.