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.     

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 6-phosphogluconate dehydrogenase: a theoretical investigation

Density functional calculations are employed to theoretically explore the mechanism of all elementary reaction steps involved in the catalytic reaction of 6-phosphogluconate dehydrogenase (6PGDH). The model systems we choose for the enzyme contain the essential parts of the cofactor (NADP+), the substrate 6-phosphogluconate (6PG), and some key residues (Lys183 and Glu190) in the active site of sheep liver 6PGDH. The effect of the apoenzyme electrostatic environment on the studied reaction is treated by the self-consistent reaction-field method. Our calculations demonstrate that the first step of the catalytic reaction is the formation of a 3-keto 6PG intermediate, which proceeds through a concerted transition state involving a hydride transfer from 6PG to NADP+, and a proton transfer from 6PG to Lys183. The second step is the elimination of a CO2 molecule from 6-PG, concomitant with a proton transfer from Lys183 to 6-PG. In the final step, a concerted double proton transfer (one from Glu190 to the substrate, another from the substrate to Lys183) results in the final product, the keto form of ribulose 5-phosphate (Ru5P). The rate-limiting step is the formation of a 3-keto 6PG intermediate, with a free energy barrier of 22.7 kcal/mol at room temperature in the protein environment, and all three steps are calculated to be thermodynamically favorable. These results are in good agreement with the general acid/general base mechanism suggested from previous experiments for the 6PGDH reaction.     

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.     

The α-amino group of the threonine substrate as the general base during tRNA aminoacylation: a new version of substrate-assisted catalysis predicted by hybrid DFT

Density functional theory-based methods in combination with large chemical models have been used to investigate the mechanism of the second half-reaction catalyzed by Thr-tRNA synthetase: aminoacyl transfer from Thr-AMP onto the (A76)3'OH of the cognate tRNA. In particular, we have examined pathways in which an active site His309 residue is either protonated or neutral (i.e., potentially able to act as a base). In the protonated His309-assisted mechanism, the rate-limiting step is formation of the tetrahedral intermediate. The barrier for this step is 155.0 kJ mol(-1), and thus, such a pathway is concluded to not be enzymatically feasible. For the neutral His309-assisted mechanism, two models were used with the difference being whether Lys465 was included. For either model, the barrier of the rate-limiting step is below the upper thermodynamic enzymatic limit of ~125 kJ mol(-1). Specifically, without Lys465, the rate-limiting barrier is 122.1 kJ mol(-1) and corresponds to a rotation about the tetrahedral intermediate C(carb)-OH bond. For the model with Lys465, the rate-limiting barrier is slightly lower and corresponds to the formation of the tetrahedral intermediate. Importantly, for both "neutral His309" models, the neutral amino group of the threonyl substrate directly acts as the proton acceptor; in the formation of the tetrahedral intermediate, the (A76)3'OH proton is directly transferred onto the Thr-NH(2). Therefore, the overall mechanism follows a general substrate-assisted catalytic mechanism.     

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     

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.     

Catalytic mechanism of guanine deaminase: an ONIOM and molecular dynamics study

The catalytic mechanism of Bacillus subtilis guanine deaminase (bGD), a Zn metalloenzyme, has been investigated by a combination of quantum mechanical calculations using the multilayered ONIOM method and molecular dynamics simulations. In contrast to a previously proposed catalytic mechanism, which requires the bound guanine to assume a rare tautomeric state, the ONIOM calculations showed that the active-site residues of the enzyme do not affect the tautomeric state of guanine, and consequently the bound guanine is a tautomer that is the most abundant in aqueous solution. Two residues, Glutamate 55 and Aspartate 114, were found to play important roles in proton shuttling in the reaction. The proposed reaction path is initiated by proton transfer from a Zn-bound water to protonate Asp114. This process may be quite complex and rather dynamic in nature, as revealed by the molecular dynamics (MD) simulations, whereby another water may bridge the Zn-bound water and Asp114, which then is eliminated by positioning of guanine in the active site. The binding of guanine stabilizes protonated Asp114 by hydrogen bond formation. Asp114 can then transfer its proton to the N3 of the bound guanine, facilitating the nucleophilic attack on C2 of the guanine by the Zn-bound hydroxide to form a tetrahedral intermediate. This occurs with a rather low barrier. Glu55 then transfers a proton from the Zn-hydroxide to the amino group of the reaction intermediate and, at this point, the C2-N2 bond has lengthened by 0.2 A compared to guanine, making C2-N2 bond cleavage more facile. The C2-N2 bond breaks forming ammonia, with an energy barrier of approximately 8.8 kcal/mol. Ammonia leaves the active site, and xanthine is freed by the cleavage of the Zn-O2 bond, with a barrier approximately 8.4 kcal/mol. Along this reaction path, the highest barrier comes from C2-N2 bond cleavage, while the barrier from the cleavage of the Zn-O2 bond is slightly smaller. The Zn-O2 bond can be broken without the assistance of water during the release of xanthine.     

Ab initio model study on acetylcholinesterase catalysis: potential energy surfaces of the proton transfer reactions

Ab initio molecular orbital (MO) and hybrid density functional theory (DFT) calculations have been applied to the initial step of the acylation reaction catalyzed by acetylcholinesterase (AChE), which is the nucleophiric addition of Ser200 in catalytic triads to a neurotransmitter acetylcholine (ACh). We focus our attention mainly on the effects of oxyanion hole and Glu327 on the potential energy surfaces (PESs) for the proton transfer reactions in the catalytic triad Ser200-His440-Glu327. The activation barrier for the addition reaction of Ser200 to ACh was calculated to be 23.4 kcal/mol at the B3LYP/6-31G(d)//HF/3-21G(d) level of theory. The barrier height under the existence of oxyanion hole, namely, Ser200-His440-Glu327-ACh-(oxyanion hole) system, decreased significantly to 14.2 kcal/mol, which is in reasonable agreement with recent experimental value (12.0 kcal/mol). Removal of Glu327 from the catalytic triad caused destabilization of both energy of transition state for the reaction and tetrahedral intermediate (product). PESs calculated for the proton transfer reactions showed that the first proton transfer process is the most important in the stabilization of tetrahedral intermediate complex. The mechanism of addition reaction of ACh was discussed on the basis of theoretical results.     

Class I ribonucleotide reductase revisited: the effect of removing a proton on Glu441

The substrate mechanism of class I ribonucleotide reductase has been revisited using the hybrid density functional B3LYP method. The molecular model used is based on the X-ray structure and includes all the residues of the R1 subunit commonly considered in the RNR substrate conversion scheme: Cys439 initiating the reaction as a thiyl radical, the redox-active cysteines Cys225 and Cys462, and the catalytically important Glu441 and Asn437. In contrast to previous theoretical studies of the overall mechanism, Glu441 is added as an anion. All relevant transition states have been optimized, including one where an electron is transferred 8 A from the disulfide to the substrate simultaneously with a proton transfer from Glu441. The calculated barrier for this step is 19.1 kcal/mol, which can be compared to the rate-limiting barrier indicated by experiments of about 17 kcal/mol. Even though the calculated barrier is somewhat higher than the experimental limit, the discrepancy is within the normal error bounds of B3LYP. The suggestion from the present modeling study is thus that a protonated Glu441 does not need to be present at the active site from the beginning of the catalytic cycle. However, the previously suggested mechanism with an initial protonation of Glu441 cannot be ruled out, because even with the cost added for protonation of Glu441 with a typical pK(a) of 4, the barrier for that mechanism is lower than the one obtained for the present mechanism. The results are compared to experimental results and suggestions.     

Theoretical study of oxidation of cyclohexane diol to adipic anhydride by [Ru(IV)(O)(tpa)(H2O)]2+ complex (tpa ═ tris(2-pyridylmethyl)amine)

The catalytic conversion of 1,2-cyclohexanediol to adipic anhydride by Ru(IV)O(tpa) (tpa ═ tris(2-pyridylmethyl)amine) is discussed using density functional theory calculations. The whole reaction is divided into three steps: (1) formation of α-hydroxy cyclohexanone by dehydrogenation of cyclohexanediol, (2) formation of 1,2-cyclohexanedione by dehydrogenation of α-hydroxy cyclohexanone, and (3) formation of adipic anhydride by oxygenation of cyclohexanedione. In each step the two-electron oxidation is performed by Ru(IV)O(tpa) active species, which is reduced to bis-aqua Ru(II)(tpa) complex. The Ru(II) complex is reactivated using Ce(IV) and water as an oxygen source. There are two different pathways of the first two steps of the conversion depending on whether the direct H-atom abstraction occurs on a C-H bond or on its adjacent oxygen O-H. In the first step, the C-H (O-H) bond dissociation occurs in TS1 (TS2-1) with an activation barrier of 21.4 (21.6) kcal/mol, which is followed by abstraction of another hydrogen with the spin transition in both pathways. The second process also bifurcates into two reaction pathways. TS3 (TS4-1) is leading to dissociation of the C-H (O-H) bond, and the activation barrier of TS3 (TS4-1) is 20.2 (20.7) kcal/mol. In the third step, oxo ligand attack on the carbonyl carbon and hydrogen migration from the water ligand occur via TS5 with an activation barrier of 17.4 kcal/mol leading to a stable tetrahedral intermediate in a triplet state. However, the slightly higher energy singlet state of this tetrahedral intermediate is unstable; therefore, a spin crossover spontaneously transforms the tetrahedral intermediate into a dione complex by a hydrogen rebound and a C-C bond cleavage. Kinetic isotope effects (k(H)/k(D)) for the electronic processes of the C-H bond dissociations calculated to be 4.9-7.4 at 300 K are in good agreement with experiment values of 2.8-9.0.     

The catalytic mechanism of mouse renin studied with QM/MM calculations

Hypertension is a chronic condition that affects nearly 25% of adults worldwide. As the Renin-Angiotensin-Aldosterone System is implicated in the control of blood pressure and body fluid homeostasis, its combined blockage is an attractive therapeutic strategy currently in use for the treatment of several cardiovascular conditions. We have performed QM/MM calculations to study the mouse renin catalytic mechanism in atomistic detail, using the N-terminal His6-Asn14 segment of angiotensinogen as substrate. The enzymatic reaction (hydrolysis of the peptidic bond between residues in the 10th and 11th positions) occurs through a general acid/base mechanism and, surprisingly, it is characterized by three mechanistic steps: it begins with the creation of a first very stable tetrahedral gem-diol intermediate, followed by protonation of the peptidic bond nitrogen, giving rise to a second intermediate. In a final step the peptidic bond is completely cleaved and both gem-diol hydroxyl protons are transferred to the catalytic dyad (Asp32 and Asp215). The final reaction products are two separate peptides with carboxylic acid and amine extremities. The activation energy for the formation of the gem-diol intermediate was calculated as 23.68 kcal mol(-1), whereas for the other steps the values were 15.51 kcal mol(-1) and 14.40 kcal mol(-1), respectively. The rate limiting states were the reactants and the first transition state. The associated barrier (23.68 kcal mol(-1)) is close to the experimental values for the angiotensinogen substrate (19.6 kcal mol(-1)). We have also tested the influence of the density functional on the activation and reaction energies. All eight density functionals tested (B3LYP, B3LYP-D3, X3LYP, M06, B1B95, BMK, mPWB1K and B2PLYP) gave very similar results.     

A theoretical study on the substrate deacylation mechanism of class C beta-lactamase

The whole reaction of the deacylation of class C beta-lactamase was investigated by performing quantum chemical calculations under physiological conditions. In this study, the X-ray crystallographic structure of the inhibitor moxalactam-bound class C beta-lactamase (Patera et al. J. Am. Chem. Soc. 2000, 122, 10504-10512.) was utilized and moxalactam was changed into the substrate cefaclor. A model for quantum chemical calculations was constructed using an energy-minimized structure of the substrate-bound enzyme obtained by molecular mechanics calculation, in which the enzyme was soaked in thousands of TIP3P water molecules. It was found that the deacylation reaction consisted of two elementary processes. The first process was formation of a tetrahedral intermediate, which was initiated by the activation of catalytic water by Tyr150, and the second process was detachment of the hydroxylated substrate from the enzyme, which associated with proton transfer from the side chain of Lys67 to Ser64O(gamma). The first process is a rate-determining process, and the activation energy was estimated to be 30.47 kcal/mol from density functional theory calculations considering electron correlation (B3LYP/6-31G**). The side chain of Tyr150 was initially in a deprotonated state and was stably present in the active site of the acyl-enzyme complex, being held by Lys67 and Lys315 cooperatively.     

Hydrolysis of chemically distinct sites of human serum albumin by polyoxometalate: A hybrid QM/MM (ONIOM) study

In this study, mechanisms of hydrolysis of all four chemically diverse cleavage sites of human serum albumin (HSA) by [Zr(OH)(PW₁₁O₃₉)]⁴⁻ (ZrK) have been investigated using the hybrid two-layer QM/MM (ONIOM) method. These reactions have been proposed to occur through the following two mechanisms: internal attack (IA) and water assisted (WA). In both mechanisms, the cleavage of the peptide bond in the Cys392-Glu393 site of HSA is predicted to occur in the rate-limiting step of the mechanism. With the barrier of 27.5 kcal/mol for the hydrolysis of this site, the IA mechanism is found to be energetically more favorable than the WA mechanism (barrier = 31.6 kcal/mol). The energetics for the IA mechanism are in line with the experimentally measured values for the cleavage of a wide range of dipeptides. These calculations also suggest an energetic preference (Cys392-Glu393, Ala257-Asp258, Lys313-Asp314, and Arg114-Leu115) for the hydrolysis of all four sites of HSA. © 2018 Wiley Periodicals, Inc.     

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.     

Catalytic mechanism of dopamine beta-monooxygenase mediated by Cu(III)-oxo

Mechanisms of dopamine hydroxylation by the Cu(II)-superoxo species and the Cu(III)-oxo species of dopamine beta-monooxygenase (DBM) are discussed using QM/MM calculations for a whole-enzyme model of 4700 atoms. A calculated activation barrier for the hydrogen-atom abstraction by the Cu(II)-superoxo species is 23.1 kcal/mol, while that of the Cu(III)-oxo, which can be viewed as Cu(II)-O*, is 5.4 kcal/mol. Energies of the optimized radical intermediate in the superoxo- and oxo-mediated pathways are 18.4 and -14.2 kcal/mol, relative to the corresponding reactant complexes, respectively. These results demonstrate that the Cu(III)-oxo species can better mediate dopamine hydroxylation in the protein environment of DBM. The side chains of three amino acid residues (His415, His417, and Met490) coordinate to the Cu(B) atom, one of the copper sites in the catalytic core that plays a role for the catalytic function. The hydrogen-bonding network between dopamine and the three amino acid residues (Glu268, Glu369, and Tyr494) plays an essential role in substrate binding and the stereospecific hydroxylation of dopamine to norepinephrine. The dopamine hydroxylation by the Cu(III)-oxo species is a downhill and lower-barrier process toward the product direction with the aid of the protein environment of DBM. This enzyme is likely to use the high reactivity of the Cu(III)-oxo species to activate the benzylic C-H bond of dopamine; the enzymatic reaction can be explained by the so-called oxygen rebound mechanism.     

A DFT study of the mechanism of Ni superoxide dismutase (NiSOD): role of the active site cysteine-6 residue in the oxidative half-reaction

In the present DFT study, the catalytic mechanism of H2O2 formation in the oxidative half-reaction of NiSOD, E-Ni(II) + O2- + 2H+ --> E-Ni(III) + H2O2, has been investigated. The main objective of this study is to investigate the source of two protons required in this half-reaction. The proposed mechanism consists of two steps: superoxide coordination and H2O2 formation. The effect of protonation of Cys6 and the proton donating roles of side chains (S) and backbones (B) of His1, Asp3, Cys6, and Tyr9 residues in these two steps have been studied in detail. For protonated Cys6, superoxide binding generates a Ni(III)-O2H species in a process that is exothermic by 17.4 kcal/mol (in protein environment using the continuum model). From the Ni(III)-O2H species, H2O2 formation occurs through a proton donation by His1 via Tyr9, which relative to the resting position of the enzyme is exothermic by 4.9 kcal/mol. In this pathway, a proton donating role of His1 residue is proposed. However, for unprotonated Cys6, a Ni(II)-O2- species is generated in a process that is exothermic by 11.3 kcal/mol. From the Ni(II)-O2- species, the only feasible pathway for H2O2 formation is through donation of protons by the Tyr9(S)-Asp3(S) pair. The results discussed in this study elucidate the role of the active site residues in the catalytic cycle and provide intricate details of the complex functioning of this enzyme.     

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.     

A QM/MM simulation study of transamination reaction at the active site of aspartate aminotransferase: Free energy landscape and proton transfer pathways

Transaminase is a key enzyme for amino acid metabolism, which reversibly catalyzes the transamination reaction with the help of PLP (pyridoxal 5^' -phosphate) as its cofactor. Here we have investigated the mechanism and free energy landscape of the transamination reaction involving the aspartate transaminase (AspTase) enzyme and aspartate-PLP (Asp-PLP) complex using QM/MM simulation and metadynamics methods. The reaction is found to follow a stepwise mechanism where the active site residue Lys258 acts as a base to shuttle a proton from α -carbon (CA) to imine carbon (C4A) of the PLP-Asp Schiff base. In the first step, the Lys258 abstracts the CA proton of the substrate leading to the formation of a carbanionic intermediate which is followed by the reprotonation of the Asp-PLP Schiff base at C4A atom by Lys258. It is found that the free energy barrier for the proton abstraction by Lys258 and that for the reprotonation are 17.85 and 3.57 kcal/mol, respectively. The carbanionic intermediate is 7.14 kcal/mol higher in energy than the reactant. Hence, the first step acts as the rate limiting step. The present calculations also show that the Lys258 residue undergoes a conformational change after the first step of transamination reaction and becomes proximal to C4A atom of the Asp-PLP Schiff base to favor the second step. The active site residues Tyr70* and Gly38 anchor the Lys258 in proper position and orientation during the first step of the reaction and stabilize the positive charge over Lys258 generated at the intermediate step.     

Peptide hydrolysis catalyzed by matrix metalloproteinase 2: a computational study

The MMP-2 reaction mechanism is investigated by using different computational methodologies. First, quantum mechanical (QM) calculations are carried out on a cluster model of the active site bound to an Ace-Gly approximately Ile-Nme peptide. Along the QM reaction path, a Zn-bound water molecule attacks the Gly carbonyl group to give a tetrahedral intermediate. The breaking of the C-N bond is completed thanks to the Glu 404 residue that shuttles a proton from the water molecule to Ile-N atom. The gas-phase QM energy barrier is quite low ( approximately 14 kcal/mol), thus suggesting that the essential catalytic machinery is included in the cluster model. A similar reaction path occurs in the MMP-2 catalytic domain bound to an octapeptide substrate according to hybrid QM and molecular mechanical (QM/MM) geometry optimizations. However, the rupture of the Gly( P 1) approximately Ile( P 1') amide bond is destabilized in the static QM/MM calculations, owing to the positioning of the Ile( P 1') side chain inside the MMP-2 S 1' pocket and to the inability of simple energy miminization methodologies to properly relax complex systems. Molecular dynamics simulations show that these steric limitations are overcome easily through structural fluctuations. The energetic effect of structural fluctuations is taken into account by combining QM energies with average MM Poisson-Boltzmann free energies, resulting in a total free energy barrier of 14.8 kcal/mol in good agreement with experimental data. The rate-determining event in the MMP-2 mechanism corresponds to a H-bond rearrangement involving the Glu 404 residue and/or the Glu 404-COOH --> N-Ile( P 1') proton transfer. Overall, the present computational results and previous experimental data complement each other well in order to provide a detailed view of the MMPs catalytic mechanism.