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.     

Investigation of the catalytic mechanism of farnesyl pyrophosphate synthase by computer simulation

Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways, farnesyl pyrophosphate, by the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP). Recently, FPPS has been shown to represent an important target for the treatment of parasitic diseases such as Chagas disease and African trypanosomiasis. Bisphosphonates, pyrophosphate analogues in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with different side chains, are able to inhibit the FPPS enzyme. Moreover, nitrogen-containing bisphosphonates have been proposed as carbocation transition state analogues of FPPS. On the basis of structural and kinetic data, different catalytic mechanisms have been proposed for FPPS. By analyzing different reaction coordinates we propose that the reaction occurs in one step through a carbocationic transition state and the subsequent transfer of a hydrogen atom from IPP to the pyrophosphate moiety of DMAPP. Moreover, we have analyzed the role of the active site amino acids on the activation barrier and the reaction mechanism. The structure of the active site is well conserved in the isoprenyl diphosphate synthase family; thus, our results are relevant for the understanding of this important class of enzymes and for the design of more potent and specific inhibitors for the treatment of parasitic diseases.     

3-芳胺甲烯基-5,6-二氢-二氢吡喃-2,4-二酮类化合物催化氢化反应选择性的理论研究

用分子力学方法、AM1半经验方法以及从头算密度泛函B3LYP/6-311G^**方法研究了3-芳胺甲烯基-5,6-二氢-二氢吡喃-2,4-二酮类化合物在催化氢化反应中影响反应选择性的因素,结果表明,R基团对自由基中间物稳定性的影响决定了反应选择性.     

Application of computational chemistry to understanding the structure and mechanism of the Mn catalytic site in photosystem II--a review

Applications of Density Functional Theory (DFT) computational techniques to studies of the molecular structure and mechanism of the oxygen evolving, water oxidising Mn(4)/Ca catalytic site in Photosystem II are reviewed. We summarise results from the earlier studies (pre 2000) but concentrate mainly on those developments which have occurred since publication of several PS II crystal structures of progressively increasing resolution, starting in 2003. The work of all computational groups actively involved in PS II studies is examined, in the light of direct PS II structural information from X-ray diffraction crystallography and EXAFS on the metals in the catalytic site. We further address the consistency of the various computational models with results from a range of spectroscopic studies on the PS II site, in all of those functionally intermediate states (S-states) amenable to study. Experimental data considered include Mn K-edge XANES studies, hyperfine coupling of Mn nuclei and various ligand nuclei (including those from substrate water) seen by several EPR techniques applied to the net spin half intermediates, S(0) and S(2), at low temperatures. Finally we consider proposed catalytic mechanisms for the O-O bond formation step, from two groups, in the light of the available experimental evidence bearing on this process, which we also summarise.     

The 4-oxalocrotonate tautomerase- and YwhB-catalyzed hydration of 3E-haloacrylates: implications for the evolution of new enzymatic activities

4-Oxalocrotonate tautomerase (4-OT) catalyzes the conversion of 2-oxo-4E-hexenedioate to 2-oxo-3E-hexenedioate through the intermediate, 2-hydroxy-2,4E-hexadienedioate. 4-OT and a homologue found in Bacillus subtilis (designated YwhB) share sequence identity and two key catalytic groups, Pro-1 and Arg-11, with the two subunits comprising trans-3-chloroacrylic acid dehalogenase (CaaD). 4-OT and YwhB have now been found to display a low-level hydratase activity, resulting in the dehalogenation of 3E-haloacrylates. The enzymes are highly selective for the (E)-isomer, and Pro-1 is critical for the activity while an arginine is likely required. Two mechanisms are proposed in which Pro-1 functions as a general base or a general acid catalyst and, along with the arginine, facilitates the Michael addition of water. Both mechanisms suggest an intriguing route for the evolution of the CaaD activity. One or more mutations could decrease the hydrophobic environment of the active site, which would make it more favorable for a hydrolytic reaction, thereby raising the pKa of Pro-1 and increasing the concentration of enzyme in the reactive form.     

Initial characterization of a type I fatty acid synthase and polyketide synthase multienzyme complex NorS in the biosynthesis of aflatoxin B(1)

The biosynthesis of the potent environmental carcinogen aflatoxin B(1) is initiated by norsolorinic acid synthase (NorS), a complex of an iterative type I polyketide synthase and a specialized yeast-like pair of fatty acid synthases. NorS has been partially purified from Aspergillus parasiticus, has been found to have a mass of approximately 1.4 x 10(6) Da, and carries out the synthesis of norsolorinic acid in the presence of acetylCoA, malonylCoA, and NADPH where hexanoylCoA is not a free intermediate. The N-acetylcysteamine thioester of hexanoic acid can substitute for the catalytic functions of HexA/B to initiate norsolorinic acid synthesis by the complex in the presence of only malonylCoA. An alpha(2)beta(2)gamma(2) stoichiometry is proposed for NorS in keeping with its estimated mass and the observed dimeric or higher-order quarternary structures of PKS and FAS enzymes.     

Mechanism for catechol ring cleavage by non-heme iron intradiol dioxygenases: a hybrid DFT study

The mechanism of the catalytic reaction of protocatechuate 3,4-dioxygenase (3,4-PCD), a representative intradiol dioxygenase, was studied with the hybrid density functional method B3LYP. First, a smaller model involving only the iron first-shell ligands (His460, His462, and Tyr408) and the substrates (catechol and dioxygen) was used to probe various a priori plausible reaction mechanisms. Then, an extended model involving also the most important second-shell groups (Arg457, Gln477, and Tyr479) was used for the refinement of the preselected mechanisms. The computational results suggest that the chemical reactions constituting the catalytic cycle of intradiol dioxygenases involve: (1) binding of the substrate as a dianion, in agreement with experimental suggestions, (2) binding of dioxygen to the metal aided by an electron transfer from the substrate to O(2), (3) formation of a bridging peroxo intermediate and its conformational change, which opens the coordination site trans to His462, (4) binding of a neutral XOH ligand (H(2)O or Tyr447) at the open site, (5) proton transfer from XOH to the neighboring peroxo ligand yielding the hydroperoxo intermediate, (6) a Criegee rearrangement leading to the anhydride intermediate, and (7) hydrolysis of the anhydride to the final acyclic product. One of the most important results obtained is that the Criegee mechanism requires an in-plane orientation of the four atoms (two oxygen and two carbon atoms) mainly involved in the reaction. This orientation yields a good overlap between the two sigma orbitals involved, C-C sigma and O-O sigma, allowing an efficient electron flow between them. Another interesting result is that under some conditions, a homolytic O-O bond cleavage might compete with the Criegee rearrangement. The role of the second-shell residues and the substituent effects are also discussed.     

Theoretical studies on thermochemistry for conversion of 5-chloromethylfurfural into valuable chemicals

Recently, 5-chloromethylfurfural (CMF) was proposed as a central intermediate in the conversion of carbohydrate-based material into useful organic commodities. In the present work, we have calculated the thermochemistry using the highly accurate G4 theory and several state-of-art density functional theory (DFT) methods (e.g., X1, M06-2X, B2PLYP-D, and XYG3) for the conversion from CMF to 5-hydroxymethylfurfural (HMF) and levulinic acid (LA) in water, and that to biofuels 5-ethoxymethylfurfural (EMF) and ethyllevulinate (EL) in alcohol. New reaction mechanisms have been proposed, which complement the well-recognized Horvat mechanisms. The assessment of DFT methods suggested that XYG3 be a viable method for biomass related thermochemistry calculations.     

Enzyme ribonucleotide reductase: unraveling an enigmatic paradigm of enzyme inhibition by furanone derivatives

Several 2'-substituted-2'-deoxyribonucleotides are potent inactivators of the enzyme ribonucleotide reductase (RNR), by destroying the essential tyrosyl radical located in subunit R2 or/and covalently alkylating the subunit R1. In the absence of external reductants, the inactivation is achieved by alkylation of subunit R1 by a methylene-3(2H)-furanone. The furanone is generated in solution through degradation of a keto-deoxyribonucleotide intermediate, produced during the inhibitory mechanism of a wide group of 2'-substituted inhibitors, and is easily detected experimentally by UV spectroscopy. Interestingly, the same keto-deoxyribonucleotide is also a proposed intermediate of the normal substrate pathway, but by some unknown reason, it does not dissociate from the active site and does not inactivate the enzyme. Therefore, if the currently accepted mechanism for substrate reduction is correct, there must be some specific reason that makes such a reactive intermediate behave differently, not dissociating from the active site during substrate reduction. In this article, we propose to validate the current substrate mechanism by showing that the keto-deoxyribonucleotide dissociates from the active site only in the case of the inhibitors, and therefore, it corresponds to a viable intermediate in the substrate mechanism. Furthermore, we answer unexplained experimental observations that concern the predomination of the normal reduction mechanism over the abnormal ketone formation in the FdNDP and the release of F(-), either in the normal or in the abnormal turnover. For that purpose, we have investigated the interaction between the enzyme and this keto-deoxyribonucleotide generated from the normal substrate and from two widely studied representative inhibitors. A model containing 140 atoms was used to represent the desired structures. The results allowed us to conclude that the solvation free energy of the 2'-substituents, its influence inside the active site, and the charge transfer mechanism from a protein side chain to solution are the thermodynamic driving forces for the intermediate dissociation and subsequent RNR inhibition. Such charge transfer cannot be accomplished by the natural substrate, preventing its dissociation. These results elucidate a paradox which has been unexplained for more than 20 years and further validates both the proposed substrate and inhibition chemical mechanisms.     

Capreomycin and hygromycin B modulate the catalytic activity of the delta ribozyme in a manner that depends on the protonation and complexation with Cu2+ ions of these antibiotics

Catalytic RNA molecules (ribozymes) have often been used for the testing of interactions of antibiotics with ribonucleic acids. We showed that the impact of capreomycin and hygromycin B on delta ribozyme catalysis might change dramatically, from stimulation to inhibition, depending on conditions. In order to evaluate possible mechanisms of modulation of the ribozyme catalytic activity we used our earlier data on species distribution for protonated forms of capreomycin and hygromycin B and their complexes with Cu(2+) ions at different pH values. We proposed that, upon inhibition, the protonated amino group of capreomycin was located in the ribozyme catalytic cleft interfering with binding catalytic Mg(2+). Such a mechanism was also supported by the results of ribozyme inhibition with capreomycin complexed with Cu(2+). The effects of stimulation of the delta ribozyme activity by capreomycin and hygromycin B were less pronounced than inhibition. Possibly, the amino functions of these antibiotics might be involved in a general acid-base catalysis performed by the ribozyme, acting as proton acceptors/donors.     

Catalytic oxidation and ammoxidation of propylene. Modelling studies on well-defined molybdenum complexes

Molybdenum (VI)-allyloxo and -allylamido complexes possessing ancillary oxo and imido ligands have been synthesised and their subsequent decomposition studied in the perspective of modelling the postulated surface intermediates of the catalytic oxidation and ammoxidation of propylene. In solution and under mild conditions, these complexes undergo two types of reactions corresponding effectively to certain elemental steps of the heterogeneous processes, i.e. oxidative dehydrogenation of the allyl groups and allyl migration to oxo and imido functions. The mechanisms and the relative rates of these reactions are compared with that proposed for the surface catalytic chemistry. The possibility of alternate reaction pathways is discussed such as for CN bond formation and the involvement of an allylideneamine intermediate in the catalytic cycle.     

A QM/MM study of the racemization of vinylglycolate catalyzed by mandelate racemase enzyme.

The experimentally postulated mechanism for the interconversion between (S)-vinylglycolate and (R)-vinylglycolate catalyzed by mandelate racemase enzyme consists of a two-step quite symmetric process through a dianionic enolic intermediate that is formed after the abstraction of the alpha-proton of vinylglycolate by a basic enzymatic residue and is then reprotonated by another residue. The challenging problem behind this reaction is how the enzyme manages to stabilize such an intermediate, that is, how it lowers enough the high pK(a) of the alpha-proton for the reaction to take place. The QM/MM simulations performed in this paper indicate that catalysis is based on the stabilization of the negative charge developed on the substrate along the reaction. We have identified three different reaction mechanisms starting from different quasi-degenerate structures of the substrate-enzyme complex. In two of them the stabilizing role is done by means of a catalytic proton transfer that avoids the formation of a dianionic intermediate, and they involve six steps instead of the two experimentally proposed. On the contrary, the third mechanism passes through a dianionic species stabilized by the concerted approach of a protonated enzymatic residue during the proton abstraction. The potential energy barriers theoretically found along these mechanisms are qualitatively in good agreement with the experimental free energy barriers determined for racemization of vinylglycolate and mandelate. The theoretical study of the effect of the mutation of Glu317 by Gln317 in the kinetics of the reaction reveals the important role in the catalysis of the hydrogen bond formed by Glu317 in the native enzyme, as only one of the mechanisms, the slower one, is able to produce the racemization in the active site of the mutant. However, we have found that this hydrogen bond is not an LBHB within our model.     

Molecular orbital studies of enzyme mechanisms. II. Catalytic oxidation of alcohols by liver alcohol dehydrogenase

Semiempirical (AM1) molecular orbital theory has been used to investigate the oxidation of alcohols at the active site of liver alcohol dehydrogenase (LADH). The model active site consists of a zinc dication coordinated to two methyl-mercaptans (Cys-46, Cys-176), an imidazole (His-67), and a water. An imidazole (His-51) hydrogen bonded to a hydroxy-acetate (Ser-48) forms the remote base. AM1 calculations that address the two distinct steps in the catalytic mechanism of ethanol oxidation by LADH are reported. These two steps are: (1) the deprotonation of ethanol by imidazole (His-51) via hydrogen-bonded hydroxy-acetate (Ser-48), creating a proton relay system; and (2) the rate-limiting hydride transfer step from ethanol C1 to nicotinamide adenine dinucleotide (NAD⁺), leading to product formation. Detailed calculations have been used to resolve the unsolved problems of mechanisms that have been suggested on the basis of kinetic data and crystal structures of several LADH complexes. We investigated two possible mechanisms for the deprotonation of ethanol, by zinc-bound OH^? and by direct deprotonation of zinc-bound ethanol by imidazole via hydroxyacetate (Ser-48). Our calculations show that there is no need for LADH to activate a water molecule at the active site as in many other zinc enzymes. This result agrees with experimental evidence. Our calculations also indicate that substrates are bound in an inner-sphere-pentacoordinated complex to the active site zincion. In this case, spectroscopic investigations agree with our results but crystallographic data do not. The highest activation energy is found for the hydride transfer, in agreement with the experiment. Finally, we proposed an alternative mechanism for the mode of action of LADH based upon our results. © 1993 John Wiley & Sons, Inc.     

Theoretical study of the human DNA repair protein HOGG1 activity

We have examined the role of the catalytic lysine (Lys 249) in breaking the glycosidic bond of 8-oxoguanine in the enzyme human 8-oxoguanine DNA glycosylase. Until quite recently it has been assumed that this lysine acts as a nucleophile in an S(N)2 type of reaction after being activated through a donation of a proton to a strictly conserved aspartate, also located in the active site. However, evidence from crystallographic, as well as biochemical studies, questions this assumption mainly because the lysine is not ideally positioned for such an attack. In addition, the catalytic activity is preserved even after that aspartate is mutated to a residue not accepting protons, but still keeping the interactions in the active site. In this study, we have investigated several different reaction mechanisms to discover plausible ways where the lysine could assist in breaking the glycosidic bond. We use hybrid density functional theory to characterize both associative and dissociative pathways. We find that the smallest energetical barrier involves an S(N)1 type of mechanism where the lysine electrostatically stabilizes the dissociating base and then donates a proton with a very small barrier and then finally attacks the sugar ring to create the covalently bound protein-DNA intermediate complex. The S(N)2 mechanism also has a lower barrier than the "spontaneous" bond breaking but considerably above that of the S(N)1 reaction. However, in current conditions, the reactants placed in a conformation posed for an S(N)2 reaction is substantially more stable than if posed for the S(N)1 reaction, indicating that the active site has to bind stronger to the latter in order to achieve a full catalytic effect. An analysis of the polarization of the transition states shows that the polarization is largest for the S(N)1 reaction, indicating that this path will gain most by being placed in a prepolarized active site. These findings give further support to the hypothesis that a dissociative mechanism may be the preferred mode of action for this type of enzymes.     

Interaction of the substrate radical and the 5'-deoxyadenosine-5'-methyl group in vitamin B(12) coenzyme-dependent ethanolamine deaminase

The distance and relative orientation of the C5' methyl group of 5'-deoxyadenosine and the substrate radical in vitamin B(12) coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The (S)-2-aminopropanol-generated substrate radical catalytic intermediate was prepared by cryotrapping steady-state mixtures of enzyme in which catalytically exchangeable hydrogen sites in the active site had been labeled by previous turnover on (2)H(4)-ethanolamine. Simulation of the time- and frequency-domain ESEEM requires two types of coupled (2)H. The strongly coupled (2)H has an effective dipole distance (r(eff)) of 2.2 A, and isotropic coupling constant (A(iso)) of -0.35 MHz. The weakly coupled (2)H has r(eff) = 3.8 A and A(iso) = 0 MHz. The best (2)H ESEEM time- and frequency-domain simulations are achieved with a model in which the hyperfine couplings arise from one strongly coupled hydrogen site and two equivalent weakly coupled hydrogen sites located on the C5' methyl group of 5'-deoxyadenosine. This model indicates that the unpaired electron on C1 of the substrate radical and C5' are separated by 3.2 A and are thus at closest contact. The close proximity of C1 and C5' indicates that C5' of the 5'-deoxyadenosyl moiety directly mediates radical migration between cobalt in cobalamin and the substrate/product site over a distance of 5-7 A in the active site of ethanolamine deaminase.     

Synthesis and inhibition properties of conformational probes for the mutase-catalyzed UDP-galactopyranose/furanose interconversion

UDP-galactose mutase is a flavoenzyme that catalyzes the isomerization of UDP-galactopyranose into UDP-galactofuranose, a key step in the biosynthesis of important bacterial oligosaccharides. Several mechanisms for this unique ring-contraction have been proposed, one of them involving a putative 1,4-anhydrogalactopyranose as an intermediate in the reaction. The purpose of this study was to probe the mutase binding site with conformationally restricted analogues of its substrate. Thus, we describe the straightforward synthesis of two C-glycosidic UDP-galactose derivatives: analogue 1, presenting a galactose moiety locked in a bicyclic (1,4)B boat conformation, and UDP-C-Galf 2, where the galactose residue is locked in the conformation of the mutase substrate. The two molecules were found to be inhibitors of UDP-galactose mutase at levels depending on the redox state of the enzyme. Strong inhibition of the native enzyme, but a low one of the reduced mutase, were observed with UDP-C-Galf 2, whereas 1 displayed intermediate inhibition levels under both native and reducing conditions. These data provide evidence of a significant conformational difference of the mutase binding pocket in the reduced enzyme and in the native one, the enzyme switching from a low Galf-affinity state (reduced enzyme) to a very strong one (native enzyme). It is remarkable that the mutase binds the boat-locked analogue 1 with similar affinities in both its conformational states. These results support a mechanism involving the formation of 1,4-anhydrogalactopyranose as a low-energy intermediate. An alternative explanation would be that the distortion of the galactose moiety during the cycle contraction transiently brings the carbohydrate into a conformation close to a (1,4)B boat.     

Theoretical Studies on the Binding Mode and Reaction Mechanism of TLP Hydrolase kpHIUH

In this work, we have investigated the binding conformations of the substrate in the active site of 5-HIU hydrolase kpHIUH and its catalytic hydrolysis mechanism. Docking calculations revealed that the substrate adopts a conformation in the active site with its molecular plane laying parallel to the binding interface of the protein dimer of kpHIUH, in which His7 and His92 are located adjacent to the hydrolysis site C6 and have hydrogen bond interactions with the lytic water. Based on this binding conformation, density functional theory calculations indicated that the optimal catalytic mechanism consists of two stages: (1) the lytic water molecule is deprotonated by His92 and carries out nucleophilic attack on C6=O of 5-HIU, resulting in an oxyanion intermediate; (2) by accepting a proton transferred from His92, C6–N5 bond is cleaved to completes the catalytic cycle. The roles of His7, His92, Ser108 and Arg49 in the catalytic reaction were revealed and discussed in detail.     

Role of carbocation's flexibility in sesquiterpene biosynthesis: computational study of the formation mechanism of terrecyclene

Two mechanisms have been proposed in the literature to explain the formation of the skeleton of terrecyclic acid from farnesyl diphosphate. Both mechanisms satisfy the experimental data obtained using isotopic labeling, but computational results at the mPW1B95/6-31+G(d,p) level of theory allow the differentiation between them. While one of the mechanisms is basically a carbocation cascade, the other one requires several steps that imply high energetic demands. Specifically, there is a [1,3] hydride shift that requires approximately 100 kcal/mol making this mechanisms unlikely. The other mechanism is more plausible, and it suggests the participation of two secondary carbocation as intermediates, but these were not observed as minimums on the potential energy surface analyzed; they only appear as a point near the transition state in the intrinsic reaction coordinate. Both mechanisms proposed a [1,3] hydride shift, but in the less likely mechanism, the rigidity of the intermediate that undergoes the hydride shift greatly increases the energy of the corresponding transition state.     

Catalytic reaction mechanism of homogentisate dioxygenase: a hybrid DFT study

Human homogentisate dioxygenase is an Fe(II)-dependent enzyme responsible for aromatic ring cleavage. The mechanism of its catalytic reaction has been investigated with the hybrid density functional method B3LYP. A relatively big model of the active site was first used to determine the substrate binding mode. It was found that binding of the substrate dianion with a vacant position trans to Glu341 is most favorable. The model was then truncated to include only the most relevant parts of the active-site residues involved in iron coordination and substrate binding. Thus, methylimidazole was used to model His292, His335, His365, and His371, while propionate modeled Glu341. The computational results suggest that the catalytic reaction of homogentisate dioxygenases involves three major chemical steps: formation of the peroxo intermediate, homolytic cleavage of the O-O bond leading to an arene oxide radical, and finally, cleavage of the six-membered ring. Calculated barriers for alternative reaction paths are markedly higher than for the proposed mechanism, and thus the computational results successfully explain the product specificity of the enzyme. Interestingly, the results indicate that the type of ring scission, intra or extra with respect to the substituents coordinating to iron, is controlled by the barrier heights for the decay of the arene oxide radical intermediate.     

A quantum chemical study of the reaction mechanism of acetyl-coenzyme a synthase

Recent experimental and theoretical studies have focused on the mechanism of the A-cluster active site of acetyl-CoA synthase that produces acetyl-CoA from a methyl group, carbon monoxide, and CoA. Several proposals have been made concerning the redox states of the (Ni-Ni) bimetallic center and the iron-sulfur cluster connected to one of the metals. Using hybrid density functional theory, we have investigated putative intermediate states from the catalytic cycle. Among our conclusions are the following: (i) the zerovalent state proposed for the proximal metal is unlikely if the charge on the iron-sulfur cluster is +2; (ii) a mononuclear mechanism in which both CO and CH(3) bind the proximal nickel is favored over the binuclear mechanism in which CO and CH(3) bind the proximal and distal nickel ions, respectively; (iii) the formation of a disulfide bond in the active site could provide the two electrons necessary for the reaction but only if methylation occurs simultaneously; and (iv) the crystallographic closed form of the active site needs to open to accommodate ligands in the equatorial site.