Ab initio QM/MM study of class A beta-lactamase acylation: dual participation of Glu166 and Lys73 in a concerted base promotion of Ser70

Beta-lactamase acquisition is the most prevalent basis for Gram-negative bacteria resistance to the beta-lactam antibiotics. The mechanism used by the most common class A Gram-negative beta-lactamases is serine acylation followed by hydrolytic deacylation, destroying the beta-lactam. The ab initio quantum mechanical/molecular mechanical (QM/MM) calculations, augmented by extensive molecular dynamics simulations reported herein, describe the serine acylation mechanism for the class A TEM-1 beta-lactamase with penicillanic acid as substrate. Potential energy surfaces (based on approximately 350 MP2/6-31+G calculations) reveal the proton movements that govern Ser70 tetrahedral formation and then collapse to the acyl-enzyme. A remarkable duality of mechanism for tetrahedral formation is implicated. Following substrate binding, the pathway initiates by a low energy barrier (5 kcal mol(-1)) and an energetically favorable transfer of a proton from Lys73 to Glu166, through the catalytic water molecule and Ser70. This gives unprotonated Lys73 and protonated Glu166. Tetrahedral formation ensues in a concerted general base process, with Lys73 promoting Ser70 addition to the beta-lactam carbonyl. Moreover, the three-dimensional potential energy surface also shows that the previously proposed pathway, involving Glu166 as the general base promoting Ser70 through a conserved water molecule, exists in competition with the Lys73 process. The existence of two routes to the tetrahedral species is fully consistent with experimental data for mutant variants of the TEM beta-lactamase.     

Mechanisms of antibiotic resistance: QM/MM modeling of the acylation reaction of a class A beta-lactamase with benzylpenicillin

Understanding the mechanisms by which beta-lactamases destroy beta-lactam antibiotics is potentially vital in developing effective therapies to overcome bacterial antibiotic resistance. Class A beta-lactamases are the most important and common type of these enzymes. A key process in the reaction mechanism of class A beta-lactamases is the acylation of the active site serine by the antibiotic. We have modeled the complete mechanism of acylation with benzylpenicillin, using a combined quantum mechanical and molecular mechanical (QM/MM) method (B3LYP/6-31G+(d)//AM1-CHARMM22). All active site residues directly involved in the reaction, and the substrate, were treated at the QM level, with reaction energies calculated at the hybrid density functional (B3LYP/6-31+Gd) level. Structures and interactions with the protein were modeled by the AM1-CHARMM22 QM/MM approach. Alternative reaction coordinates and mechanisms have been tested by calculating a number of potential energy surfaces for each step of the acylation mechanism. The results support a mechanism in which Glu166 acts as the general base. Glu166 deprotonates an intervening conserved water molecule, which in turn activates Ser70 for nucleophilic attack on the antibiotic. This formation of the tetrahedral intermediate is calculated to have the highest barrier of the chemical steps in acylation. Subsequently, the acylenzyme is formed with Ser130 as the proton donor to the antibiotic thiazolidine ring, and Lys73 as a proton shuttle residue. The presented mechanism is both structurally and energetically consistent with experimental data. The QM/MM energy barrier (B3LYP/ 6-31G+(d)//AM1-CHARMM22) for the enzymatic reaction of 9 kcal mol(-1) is consistent with the experimental activation energy of about 12 kcal mol(-1). The effects of essential catalytic residues have been investigated by decomposition analysis. The results demonstrate the importance of the "oxyanion hole" in stabilizing the transition state and the tetrahedral intermediate. In addition, Asn132 and a number of charged residues in the active site have been identified as being central to the stabilizing effect of the enzyme. These results will be potentially useful in the development of stable beta-lactam antibiotics and for the design of new inhibitors.     

Role of protein flexibility in enzymatic catalysis: quantum mechanical-molecular mechanical study of the deacylation reaction in class A beta-lactamases

We present a theoretical study of a mechanism for the hydrolysis of the acyl-enzyme complex formed by a class A beta-lactamase (TEM1) and an antibiotic (penicillanate), as a part of the process of antibiotic's inactivation by this type of enzymes. In the presented mechanism the carboxylate group of a particular residue (Glu166) activates a water molecule, accepting one of its protons, and afterward transfers this proton directly to the acylated serine residue (Ser70). In our study we employed a quantum mechanics (AM1)-molecular mechanics partition scheme (QM/MM) where all the atoms of the system were allowed to relax. For this purpose we used the GRACE procedure in which part of the system is used to define the Hessian matrix while the rest is relaxed at each step of the stationary structures search. By use of this computational scheme, the hydrolysis of the acyl-enzyme is described as a three-step process: The first step corresponds to the proton transfer from the hydrolytic water molecule to the carboxylate group of Glu166 and the subsequent formation of a tetrahedral adduct as a consequence of the attack of this activated water molecule to the carbonyl carbon atom of the beta-lactam. In the second step, the acyl-enzyme bond is broken, obtaining a negatively charged Ser70. In the last step this residue is protonated by means of a direct proton transfer from Glu166. The large mobility of Glu166, a residue that is placed in a Ohms-loop, is essential to facilitate this mechanism. The geometry of the acyl-enzyme complex shows a large distance between Glu166 and Ser70 and thus, if protein coordinates were kept frozen during the reaction path, it would be difficult to get a direct proton transfer between these two residues. This computational study shows how a flexible treatment suggests the feasibility of a mechanism that could have been discounted on the basis of crystallographic positions.     

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).     

Structure, function, and inhibition along the reaction coordinate of CTX-M beta-lactamases

CTX-M enzymes are an emerging group of extended spectrum beta-lactamases (ESBLs) that hydrolyze not only the penicillins but also the first-, second-, and third-generation cephalosporins. Although they have become the most frequently observed ESBLs in certain areas, there are few effective inhibitors and relatively little is known about their detailed mechanism. Here we describe the X-ray crystal structures of CTX-M enzymes in complex with different transition-state analogues and beta-lactam inhibitors, representing the enzyme as it progresses from its acylation transition state to its acyl enzyme complex to the deacylation transition state. As the enzyme moves along this reaction coordinate, two key catalytic residues, Lys73 and Glu166, change conformations, tracking the state of the reaction. Unexpectedly, the acyl enzyme complex with the beta-lactam inhibitor cefoxitin still has the catalytic water bound; this water had been predicted to be displaced by the unusual 7alpha-methoxy of the inhibitor. Instead, the 7alpha-group appears to inhibit by preventing the formation of the deacylation transition state through steric hindrance. From an inhibitor design standpoint, we note that the best of the reversible inhibitors, a ceftazidime-like boronic acid compound, binds to CTX-M-16 with a K(i) value of 4 nM. When used together in cell culture, this inhibitor reversed cefotaxime resistance in CTX-M-producing bacteria. The structure of its complex with CTX-M enzyme and the structural view of the reaction coordinate described here provide templates for inhibitor design and intervention to combat this family of antibiotic resistance enzymes.     

An ultrahigh resolution structure of TEM-1 beta-lactamase suggests a role for Glu166 as the general base in acylation

Although TEM-1 beta-lactamase is among the best studied enzymes, its acylation mechanism remains controversial. To investigate this problem, the structure of TEM-1 in complex with an acylation transition-state analogue was determined at ultrahigh resolution (0.85 A) by X-ray crystallography. The quality of the data was such as to allow for refinement to an R-factor of 9.1% and an R(free) of 11.2%. In the resulting structure, the electron density features were clear enough to differentiate between single and double bonds in carboxylate groups, to identify multiple conformations that are occupied by residues and loops, and to assign 70% of the protons in the protein. Unexpectedly, even at pH 8.0 where the protein was crystallized, the active site residue Glu166 is clearly protonated. This supports the hypothesis that Glu166 is the general base in the acylation half of the reaction cycle. This structure suggests that Glu166 acts through the catalytic water to activate Ser70 for nucleophilic attack on the beta-lactam ring of the substrate. The hydrolytic mechanism of class A beta-lactamases, such as TEM-1, appears to be symmetrical, as are the serine proteases. Apart from its mechanistic implications, this atomic resolution structure affords an unusually detailed view of the structure, dynamics, and hydrogen-bonding networks of TEM-1, which may be useful for the design of inhibitors against this key antibiotic resistance target.     

Investigation of the mechanism of the cell wall DD-carboxypeptidase reaction of penicillin-binding protein 5 of Escherichia coli by quantum mechanics/molecular mechanics calculations

Penicillin-binding protein 5 (PBP 5) of Escherichia coli hydrolyzes the terminal D-Ala-D-Ala peptide bond of the stem peptides of the cell wall peptidoglycan. The mechanism of PBP 5 catalysis of amide bond hydrolysis is initial acylation of an active site serine by the peptide substrate, followed by hydrolytic deacylation of this acyl-enzyme intermediate to complete the turnover. The microscopic events of both the acylation and deacylation half-reactions have not been studied. This absence is addressed here by the use of explicit-solvent molecular dynamics simulations and ONIOM quantum mechanics/molecular mechanics (QM/MM) calculations. The potential-energy surface for the acylation reaction, based on MP2/6-31+G(d) calculations, reveals that Lys47 acts as the general base for proton abstraction from Ser44 in the serine acylation step. A discrete potential-energy minimum for the tetrahedral species is not found. The absence of such a minimum implies a conformational change in the transition state, concomitant with serine addition to the amide carbonyl, so as to enable the nitrogen atom of the scissile bond to accept the proton that is necessary for progression to the acyl-enzyme intermediate. Molecular dynamics simulations indicate that transiently protonated Lys47 is the proton donor in tetrahedral intermediate collapse to the acyl-enzyme species. Two pathways for this proton transfer are observed. One is the direct migration of a proton from Lys47. The second pathway is proton transfer via an intermediary water molecule. Although the energy barriers for the two pathways are similar, more conformers sample the latter pathway. The same water molecule that mediates the Lys47 proton transfer to the nitrogen of the departing D-Ala is well positioned, with respect to the Lys47 amine, to act as the hydrolytic water in the deacylation step. Deacylation occurs with the formation of a tetrahedral intermediate over a 24 kcal x mol(-1) barrier. This barrier is approximately 2 kcal x mol(-1) greater than the barrier (22 kcal x mol(-1)) for the formation of the tetrahedral species in acylation. The potential-energy surface for the collapse of the deacylation tetrahedral species gives a 24 kcal x mol(-1) higher energy species for the product, signifying that the complex would readily reorganize and pave the way for the expulsion of the product of the reaction from the active site and the regeneration of the catalyst. These computational data dovetail with the knowledge on the reaction from experimental approaches.     

Catalytic mechanism of class A beta-lactamase. I. The role of Glu166 and Serl30 in the deacylation reaction

The tetrahedral intermediate formation process, which is the first step in the deacylation reaction by class A beta-lactamase, was investigated by the ab initio molecular orbital method. In this study, benzyl penicillin was used as the substrate. From the results of our molecular dynamics study of the structure of beta-lactam antibiotics-beta-lactamase complex, the substrate, Ser70, Lys73, Ser130, Glu166 and a water molecule for the deacylation reaction were considered for construction of a model for calculation. The calculation results indicated that Glu166 plays a role in holding a water molecule, which is necessary for the deacylation reaction, and that the hydrogen bond network among Lys73Nzeta, Ser130Ogamma, and the carboxyl group of the beta-lactam antibiotics was formed by the uptake of beta-lactam antibiotics by beta-lactamase. The activation energy for this reaction was 33.3 kcal/mol, and it is very likely that the reaction occurred at body temperature. Subsequent calculation results obtained by using the model excluding Ser130 and the carboxyl group of the substrate indicated that the activation energy for this reaction was 40.8 kcal/mol, which is 7.5 kcal/mol higher than that of the previous reaction. It was found that the hydrogen bond network plays an important role in decreasing the activation energy for the tetrahedral intermediate formation reaction. Lys73Nzeta, which is located at the edge of the hydrogen bond network, played a role in forming a hydrogen bond with Glu166Oepsilon in order to help the deacylation reaction. The role of amino acid residues around the active site of class A beta-lactamase was also discussed.     

High-resolution X-ray structure of an acyl-enzyme species for the class D OXA-10 beta-lactamase

Beta-lactamases are resistance enzymes for beta-lactam antibiotics. These enzymes hydrolyze the beta-lactam moieties of these antibiotics, rendering them inactive. Of the four classes of known beta-lactamases, the enzymes of class D are the least understood. We report herein the high-resolution (1.9 A) crystal structure of the class D OXA-10 beta-lactamase inhibited by a penicillanate derivative. The structure provides evidence that the carboxylated Lys-70 (a carbamate) is intimately involved in the mechanism of the enzyme.     

Water-assisted reaction mechanism of monozinc beta-lactamases

Hybrid Car-Parrinello QM/MM calculations are used to investigate the reaction mechanism of hydrolysis of a common beta-lactam substrate (cefotaxime) by the monozinc beta-lactamase from Bacillus cereus (BcII). The calculations suggest a fundamental role for an active site water in the catalytic mechanism. This water molecule binds the zinc ion in the first step of the reaction, expanding the zinc coordination number and providing a proton donor adequately oriented for the second step. The free energy barriers of the two reaction steps are similar and consistent with the available experimental data. The conserved hydrogen bond network in the active site, defined by Asp120, Cys221, and His263, not only contributes to orient the nucleophile (as already proposed), but it also guides the second catalytic water molecule to the zinc ion after the substrate is bound. The hydrolysis reaction in water has a relatively high free energy barrier, which is consistent with the stability of cefotaxime in water solution. The modeled Michaelis complexes for other substrates are also characterized by the presence of an ordered water molecule in the same position, suggesting that this mechanism might be general for the hydrolysis of different beta-lactam substrates.     

Reactions of pyrazolylborate-zinc-hydroxide complexes related to beta-lactamase activity

Simple beta-lactams and their hydrolysis products, the beta-amino acids, react with TpZn-OH under deprotonation. The latter become semibidentate carboxylate ligands with a NH...O hydrogen bond, and the former become N-bound beta-lactamide ligands. Likewise the antibiotic derivatives 6-aminopenicillanic acid and 7-aminocephalosporanic acid are incorporated as carboxylate ligands. beta-Lactams bearing nitrophenyl or acyl substituents at the nitrogen atoms are opened hydrolytically by TpZn-OH, and the resulting N-substituted beta-amino acids are attached to zinc by their carboxylate functions. Only with trifluoroacetyl as the N-substituent does the hydrolytic cleavage occur at the external amide bond, yielding the free beta-lactam and TpZn-trifluoroacetate. The kinetic investigation of the opening reactions has shown them to be of second order like all other TpZn-OH-induced hydrolytic cleavages, thereby supporting the four-center mechanism for the monozinc beta-lactamases.     

Structural aspects for evolution of beta-lactamases from penicillin-binding proteins

Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and beta-lactamases, resistance enzymes to beta-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for beta-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7beta-[N-Acetyl-L-alanyl-gamma-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporin bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC beta-lactamase from Escherichia coli was solved at 1.71 A resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the -lactamase active site. Furthermore, insertion of a peptide in the beta-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the beta-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.     

Acylation and deacylation mechanism and kinetics of penicillin G reaction with Streptomyces R61 DD-peptidase

Two quantum mechanical (QM)-cluster models are built for studying the acylation and deacylation mechanism and kinetics of Streptomyces R61 DD-peptidase with the penicillin G at atomic level detail. DD-peptidases are bacterial enzymes involved in the cross-linking of peptidoglycan to form the cell wall, necessary for bacterial survival. The cross-linking can be inhibited by antibiotic beta-lactam derivatives through acylation, preventing the acyl-enzyme complex from undergoing further deacylation. The deacylation step was predicted to be rate-limiting. Transition state and intermediate structures are found using density functional theory in this study, and thermodynamic and kinetic properties of the proposed mechanism are evaluated. The acyl-enzyme complex is found lying in a deep thermodynamic sink, and deacylation is indeed the severely rate-limiting step, leading to suicide inhibition of the peptidoglycan cross-linking. The usage of QM-cluster models is a promising technique to understand, improve, and design antibiotics to disrupt function of the Streptomyces R61 DD-peptidase.     

Insights into the acylation mechanism of class A beta-lactamases from molecular dynamics simulations of the TEM-1 enzyme complexed with benzylpenicillin

Herein, we present results from molecular dynamics MD simulations ( approximately 1 ns) of the TEM-1 beta-lactamase in aqueous solution. Both the free form of the enzyme and its complex with benzylpenicillin were studied. During the simulation of the free enzyme, the conformation of the Omega loop and the interresidue contacts defining the complex H-bond network in the active site were quite stable. Most interestingly, the water molecule connecting Glu166 and Ser70 does not exchange with bulk solvent, emphasizing its structural and catalytic relevance. In the presence of the substrate, Ser130, Ser235, and Arg244 directly interact with the beta-lactam carboxylate via H-bonds, whereas the Lys234 ammonium group has only an electrostatic influence. These interactions together with other specific contacts result in a very short distance ( approximately 3 A) between the attacking hydroxyl group of Ser70 and the beta-lactam ring carbonyl group, which is a favorable orientation for nucleophilic attack. Our simulations also gave insight into the possible pathways for proton abstraction from the Ser70 hydroxyl group. We propose that either the Glu166 carboxylate-Wat1 or the substrate carboxylate-Ser130 moieties could abstract a proton from the nucleophilic Ser70.     

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.     

Role of the catalytic triad and oxyanion hole in acetylcholinesterase catalysis: an ab initio QM/MM study

The initial step of the acylation reaction catalyzed by acetylcholinesterase (AChE) has been studied by a combined ab initio quantum mechanical/molecular mechanical (QM/MM) approach. The reaction proceeds through the nucleophilic addition of the Ser203 O to the carbonyl C of acetylcholine, and the reaction is facilitated by simultaneous proton transfer from Ser203 to His447. The calculated potential energy barrier at the MP2(6-31+G) QM/MM level is 10.5 kcal/mol, consistent with the experimental reaction rate. The third residue of the catalytic triad, Glu334, is found to be essential in stabilizing the transition state through electrostatic interactions. The oxyanion hole, formed by peptidic NH groups from Gly121, Gly122, and Ala204, is also found to play an important role in catalysis. Our calculations indicate that, in the AChE-ACh Michaelis complex, only two hydrogen bonds are formed between the carbonyl oxygen of ACh and the peptidic NH groups of Gly121 and Gly122. As the reaction proceeds, the distance between the carbonyl oxygen of ACh and NH group of Ala204 becomes smaller, and the third hydrogen bond is formed both in the transition state and in the tetrahedral intermediate.     

Origin of tight binding of a near-perfect transition-state analogue by cytidine deaminase: implications for enzyme catalysis

Cytidine deaminase (CDA) is a zinc metalloenzyme that catalyzes the hydrolytic deamination of cytidine to uridine. Zebularine (ZEB) binds to CDA, and the binding process leads to a near-perfect transition-state analogue (TSA) inhibitor at the active site with an estimated K(i) value of 1.2 x 10(-)(12) M. The interaction of CDA with the TSA inhibitor has become a paradigm for studying the tight TSA binding by enzymes. The formation of the TSA is catalyzed by CDA by a mechanism that is similar to the formation of the tetrahedral intermediate during the CDA-catalyzed reaction (i.e., through the nucleophilic attack of a Zn-hydroxide group on C(4)). It is believed that the TSA formed at the active site is zebularine 3,4-hydrate. In this paper, it is shown from QM/MM molecular dynamics and free energy simulations that zebularine 3,4-hydrate may in fact be unstable in the enzyme and that a proton transfer from the Zn-hydroxide group to Glu-104 during the nucleophilic attack could be responsible for the very high affinity. The nucleophilic attack by the Zn-hydroxide on C(4) is found to be concerted with two proton transfers. Such concerted process allows the TSA, an alkoxide-like inhibitor, to be stabilized through a mechanism that is similar to the transition-state stabilization in the general acid-base catalysis. It is suggested that the proton transfer from the Zn-hydroxide to Glu-104, which is required to generate the general acid for protonating the leaving ammonia, may play an important role in lowering the activation barrier during the catalysis.     

Combined QM(DFT)/MM molecular dynamics simulations of the deamination of cytosine by yeast cytosine deaminase (yCD)

Extensive combined quantum mechanical (B3LYP/6‐31G*) and molecular mechanical (QM/MM) molecular dynamics simulations have been performed to elucidate the hydrolytic deamination mechanism of cytosine to uracil catalyzed by the yeast cytosine deaminase (yCD). Though cytosine has no direct binding to the zinc center, it reacts with the water molecule coordinated to zinc, and the adjacent conserved Glu64 serves as a general acid/base to shuttle protons from water to cytosine. The overall reaction consists of several proton‐transfer processes and nucleophilic attacks. A tetrahedral intermediate adduct of cytosine and water binding to zinc is identified and similar to the crystal structure of yCD with the inhibitor 2‐pyrimidinone. The rate‐determining step with the barrier of 18.0 kcal/mol in the whole catalytic cycle occurs in the process of uracil departure where the proton transfer from water to Glu64 and nucleophilic attack of the resulting hydroxide anion to C2 of the uracil ring occurs synchronously. © 2016 Wiley Periodicals, Inc.     

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.