Tetrahedral Intermediate Research Articles

Proximity effects in nucleophilic addition reactions to medium-bridged twisted lactams: Remarkably stable tetrahedral intermediates

Proximity effects in nucleophilic addition reactions to medium-bridged twisted lactams: Remarkably stable tetrahedral intermediates

Theoretical study on the aminolysis of ester catalyzed by TBD: Hydrogen bonding or covalent bonding of the catalyst?

In this work, 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-catalyzed aminolysis reaction of methyl acetate with methylamine has been investigated at the B3LYP/6-311++G(d,p) and MP2/6-311++G(d,p) levels of theory. In our calculations, we take into account three possible pathways for the reaction. Path 1 is a ‘full concerted’ mechanism via a S N2-like O → N acyl-transfer transition state through the hydrogen bonding of TBD; Path 2 is a stepwise mechanism involving the tetrahedral intermediates through the hydrogen bonding of TBD; and Path 3 is also a stepwise mechanism including the nucleophilic attack of TBD upon ester with covalently bonding of TBD. Results indicate that Path 2 is the likely mechanism and that the hydrogen bonding of TBD has an important role in the aminolysis of ester. The role of the TBD catalyst is to facilitate the proton transfer and accelerate the reaction.

Disparity in productive binding mode of the slow-reacting enantiomer determines the novel catalytic behavior of Candida antarctica lipase B

In the context of specifying the origin of enzyme enantioselectivity, the present study explores the lipase enantioselectivity towards secondary alcohols of similar structure from the perspective of substrate binding. By carrying out molecular mechanics minimization as well as molecular dynamics simulation on tetrahedral reaction intermediates which are used as a model of transition state, we identify an unconventional productive binding mode (PBM)—M/H permutation type for Candida antarctica lipase B (CALB). The in silico results also indicate that different PBMs of the slow-reacting enantiomer do exist in one lipase even when there is little structural differences between substrates, e.g. compounds with Ph or CH 2CH 2Ph group display the M/H permutation type PBM while molecules with CH 2Ph show the M/L permutation type PBM. By relating the PBMs of substrates to the experimentally determined E-values obtained by Hoff et al. [16], we find that disparity in PBM of the slow-reacting enantiomer determines why E-values of substrates with CH 2Ph were lower than E-values of substrates with Ph or CH 2CH 2Ph group. The modeling results also suggest that the “pushed aside” effect of the F atom and Br atom accommodates the medium size substituent of the substrate better in the stereospecificity pocket of the enzyme.

X-ray Crystallographic Analysis of α-Ketoheterocycle Inhibitors Bound to a Humanized Variant of Fatty Acid Amide Hydrolase

Three cocrystal X-ray structures of the alpha-ketoheterocycle inhibitors 3-5 bound to a humanized variant of fatty acid amide hydrolase (FAAH) are disclosed and comparatively discussed alongside those of 1 (OL-135) and its isomer 2. These five X-ray structures systematically probe each of the three active site regions key to substrate or inhibitor binding: (1) the conformationally mobile acyl chain-binding pocket and membrane access channel responsible for fatty acid amide substrate and inhibitor acyl chain binding, (2) the atypical active site catalytic residues and surrounding oxyanion hole that covalently binds the core of the alpha-ketoheterocycle inhibitors captured as deprotonated hemiketals mimicking the tetrahedral intermediate of the enzyme-catalyzed reaction, and (3) the cytosolic port and its uniquely important imbedded ordered water molecules and a newly identified anion binding site. The detailed analysis of their key active site interactions and their implications on the interpretation of the available structure-activity relationships are discussed providing important insights for future design.

Evidence of Kinetic Control of Ligand Binding and Staged Product Release in MurA (Enolpyruvyl UDP-GlcNAc Synthase)-Catalyzed Reactions,

MurA (enolpyruvyl UDP-GlcNAc synthase) catalyzes the first committed step in peptidoglycan biosynthesis. In this study, MurA-catalyzed breakdown of its tetrahedral intermediate (THI), with a k(cat)/K(M) of 520 M(-1) s(-1), was far slower than the normal reaction, and 3 x 10(5)-fold slower than the homologous enzyme, AroA, reacting with its THI. This provided kinetic evidence of slow binding and a conformationally constrained active site. The MurA cocrystal structure with UDP-N-acetylmuramic acid (UDP-MurNAc), a potent inhibitor, and phosphite revealed a new "staged" MurA conformation in which the Arg397 side chain tracked phosphite out of the catalytic site. The closed-to-staged transition involved breaking eight MurA.ligand ion pairs, and three intraprotein hydrogen bonds helping hold the active site loop closed. These were replaced with only two MurA.UDP-MurNAc ion pairs, two with phosphite, and seven new intraprotein ion pairs or hydrogen bonds. Cys115 appears to have an important role in forming the staged conformation. The staged conformation appears to be one step in a complex choreography of release of the product from MurA.

The Thiolase Reaction Mechanism: The Importance of Asn316 and His348 for Stabilizing the Enolate Intermediate of the Claisen Condensation

The biosynthetic thiolase catalyzes a Claisen condensation reaction between acetyl-CoA and the enzyme acetylated at Cys89. Two oxyanion holes facilitate this catalysis: oxyanion hole I stabilizes the enolate intermediate generated from acetyl-CoA, whereas oxyanion hole II stabilizes the tetrahedral intermediate of the acetylated enzyme. The latter intermediate is formed when the alpha-carbanion of acetyl-CoA enolate reacts with the carbonyl carbon of acetyl-Cys89, after which C-C bond formation is completed. Oxyanion hole II is made of two main chain peptide NH groups, whereas oxyanion hole I is formed by a water molecule (Wat82) and NE2(His348). Wat82 is anchored in the active site by an optimal set of hydrogen bonding interactions, including a hydrogen bond to ND2(Asn316). Here, the importance of Asn316 and His348 for catalysis has been studied; in particular, the properties of the N316D, N316A, N316H, H348A, and H348N variants have been determined. For the N316D variant, no activity could be detected. For each of the remaining variants, the k(cat)/K(m) value for the Claisen condensation catalysis is reduced by a factor of several hundred, whereas the thiolytic degradation catalysis is much less affected. The crystal structures of the variants show that the structural changes in the active site are minimal. Our studies confirm that oxyanion hole I is critically important for the condensation catalysis. Removing either one of the hydrogen bond donors causes the loss of at least 3.4 kcal/mol of transition state stabilization. It appears that in the thiolytic degradation direction, oxyanion hole I is not involved in stabilizing the transition state of its rate limiting step. However, His348 has a dual role in the catalytic cycle, contributing to oxyanion hole I and activating Cys89. The analysis of the hydrogen bonding interactions in the very polar catalytic cavity shows the importance of two conserved water molecules, Wat82 and Wat49, for the formation of oxyanion hole I and for influencing the reactivity of the catalytic base, Cys378, respectively. Cys89, Asn316, and His348 form the CNH-catalytic triad of the thiolase superfamily. Our findings are also discussed in the context of the importance of this triad for the catalytic mechanism of other enzymes of the thiolase superfamily.

Solvent assisted decomposition of the tetrahedral intermediate of the transesterification reaction to biodiesel production. A density functional study

B3LYP/6–31 + G(d) calculations were employed to investigate the mechanism of the transesterification reaction between a model monoglyceride and the methoxide and ethoxide anions. The gas-phase results reveal that both reactions have essentially the same activation energy (5.9 kcal mol −1) for decomposition of the key tetrahedral intermediate. Solvent effects were included by means of both microsolvation and the polarizable continuum solvation model CPCM. Both solvent approaches reduce the activation energy, however, only the microsolvation model is able to introduce some differentiation between methanol and ethanol, yielding a lower activation energy for decomposition of the tetrahedral intermediate in the reaction with methanol (1.1 kcal mol −1) than for the corresponding reaction with ethanol (2.8 kcal mol −1), in line with experimental evidences. Analysis of the individual energy components within the CPCM approach reveals that electrostatic interactions are the main contribution to stabilization of the transition state.

Kinetics and mechanism of the reactions of O‐aryl S‐(4‐nitrophenyl) dithiocarbonates with anilines in aqueous ethanol

AbstractThe reactions of O‐(4‐methylphenyl) S‐(4‐nitrophenyl) dithiocarbonate and O‐(4‐chlorophenyl) S‐(4‐nitrophenyl) dithiocarbonate with a series of anilines are subjected to a kinetic investigation in 44 wt% ethanol–water, at 25.0 °C and an ionic strength of 0.2 M. The reactions are followed spectrophotometrically at 420 nm (appearance of 4‐nitrobenzenethiolate anion). Under excess amine, pseudo‐first‐order rate coefficients (kobs) are found. For the reactions of both substrates with anilines, plots of kobs versus free amine concentration at constant pH are nonlinear upwards, according to a second‐order polynomial equation. This kinetic behavior is in agreement with a stepwise mechanism consisting of two tetrahedral intermediates, one zwitterionic (T±) and the other anionic (T−), with a kinetically significant proton transfer from T± to an aniline to yield T−. The rate equation was derived from the proposed mechanism. By nonlinear least‐squares fitting of the rate equation to the experimental data, values of the rate micro‐coefficients involved in both steps were determined. Copyright © 2009 John Wiley & Sons, Ltd.

Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state

The perhydrolysis reaction in hydrolases is an important example of catalytic promiscuity and has many potential industrial applications. The mechanisms of perhydrolase activity of a subtilisin Carlsberg mutant and of an aryl-esterase mutant have been investigated using classical molecular dynamics simulations of the second tetrahedral intermediate (TI) state. The simulations demonstrated that hydrogen bonding between the second TI of the perhydrolysis reaction is possible in the mutants but not wild type. The stabilization by hydrogen bonds was specific for the perhydrolysis intermediate and either no hydrogen bonding or only weakened hydrogen bonding to the second TI state of the hydrolysis reaction was observed. Furthermore, a significant hindrance to the formation of the catalytically important hydrogen bond between His64 and Ser221 in the catalytic triad by competing hydrogen bonds was found for the subtilisin mutant but not wild type enzyme in case of the hydrolysis intermediate. The opposite was observed in case of the perhydrolysis intermediate. The result offers a qualitative explanation for the overall reduced hydrolysis activity of the subtilisin mutant. In addition, the simulations also explain qualitatively the perhydrolysis activity of the enzyme variants and may be helpful for designing enzyme mutants with further improved perhydrolysis activity.

Inhibition of human folylpolyglutamate synthetase by diastereomeric phosphinic acid mimics of the tetrahedral intermediate

Phosphorus-containing pseudopeptides, racemic at the C-terminal α-carbon, are potent mechanism-based inhibitors of folylpolyglutamate synthetase (FPGS). They are mimics of the tetrahedral intermediate postulated to form during FPGS-catalyzed biosynthesis of poly(γ- l-glutamates). In the present paper, the FPGS inhibitory activity of each diastereomer coupled to three heterocycles is reported. The high R f pseudopeptide containing the 5,10-dideazatetrahydropteroyl (DDAH 4Pte) heterocycle is most potent ( K is = 1.7 nM). While the heterocyclic portion affects absolute FPGS inhibitory potency, the high R f species is more potent in each pair containing the same heterocycle. This species presumably has the same stereochemistry as the natural folate polyglutamate, i.e., ( l-Glu-γ- l-Glu). Unexpectedly, the low R f (presumed l-Glu-γ- d-Glu) species are only slightly less potent (<30-fold) than their diastereomers. Further study of this phenomenon comparing l-Glu-γ- l-Glu and l-Glu-γ- d-Glu dipeptide-containing FPGS substrates shows that <1% contamination of commercial d-Glu precursors by l-Glu may give misleading information if l-Glu-γ- l-Glu substrates have low K m values.

Annotating Enzymes of Uncertain Function: The Deacylation of d-Amino Acids by Members of the Amidohydrolase Superfamily,

The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k(cat)/K(m) = 5.8 x 10(6) M(-1) s(-1)), N-acetyl-d-glutamate (k(cat)/K(m) = 5.2 x 10(6) M(-1) s(-1)), and l-methionine-d-glutamate (k(cat)/K(m) = 3.4 x 10(5) M(-1) s(-1)). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k(cat)/K(m) = 3.2 x 10(4) M(-1) s(-1)), N-acetyl-d-tryptophan (k(cat)/K(m) = 4.1 x 10(4) M(-1) s(-1)), and l-tyrosine-d-leucine (k(cat)/K(m) = 1.5 x 10(4) M(-1) s(-1)). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the alpha-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional approximately 250 sequences identified as members of this group suggest that there are no simple motifs that allow prediction of substrate specificity for most of these unknowns, highlighting the challenges for computational annotation of some groups of homologous proteins.

Recent Advances in Our Mechanistic Understanding of SNV Reactions

Nucleophilic vinylic substitution (S(N)V), in which a leaving group such as halogen is replaced by a carbon, oxygen, nitrogen, sulfur, or other nucleophile, is an important synthetic tool. It generates compounds with a carbon- or heteroatom-substituted carbon-carbon double bond, such as vinyl ethers, enamines, a variety of heterocyclic systems, and intermediates to pharmaceutically important compounds. The S(N)V reaction has many mechanistic variants, which depend on the substituents, nucleophile, leaving group, and solvent, among other factors. Among these mechanisms, the "addition-elimination" S(N)V route is the most important to synthetic chemists. S(N)V reactions are involved in several biological processes, notably (i) in the inactivation of proteases, (ii) in intermediates of herbicide metabolism, and (iii) in the formation of mutagenic intermediates by reaction of glutathione with the environmental pollutant trichloroethylene. A variant involving a tetrahedral intermediate was found in the enzymatic transfer of an enolpyruvyl group of phosphoenolpyruvate. The main S(N)V mechanism was previously analyzed in terms of a variable transition state with perpendicular nucleophilic attack. Electron-withdrawing groups Y and Y' in the beta position adjacent to the C(alpha) reaction site increase the nucleophilic attack rate; the retention of stereochemistry was mostly ascribed to formation of carbanionic intermediate 1, in which internal rotation is slower than nucleofuge expulsion (k2). As predicted, poor nucleofuges and high activation led to partial or complete stereoconvergence, and an intramolecular element effect in polyhaloethylenes gave competition ratios, kF/kBr < 1. Evidence for a zwitterionic intermediate comes from amine-catalyzed substitutions with amines. The mechanistic spectrum investigated is wide in terms of rate constants, electron-withdrawing groups, nucleophiles, leaving groups, and solvents. However, the two extremes, that is, the very slightly activated systems where in-plane invertive substitution is feasible and conversely the highly activated systems carrying poor nucleofuges where the intermediate may be observable and kinetics examined, remained almost unexplored for a long time. In this Account, we describe the progress during the last two decades in these areas. Computations on low-reactivity systems showed that the in-plane invertive single-step nucleophilic sigma attack can have a lower barrier than the pi-perpendicular retentive attack. A kBr/kCl > 1 could be deduced for the H2C=CHX (X = Cl, Br) system. Several inverted substitution-cyclizations or inverted ring openings were observed. Alkenyl iodonium salts with superb nucleofuges, showed in-plane substitutions by various nucleophiles. In parallel, we demonstrated that several highly activated systems carrying poor nucleofuges enabled a direct detection of the intermediate 1 when attacked by strong nucleophiles. Poor correlation between the equilibrium constants K1(RS) for RS- attack and pKa(CH2YY') indicates large nucleofuge steric effects (SPr > SMe > OMe >> H). Rate and equilibrium constants for RS- attack as a function of YY' also correlate poorly owing to differences in intrinsic barriers caused by different resonance effects of YY'. The expulsion of either the nucleofuge (k2) or the nucleophile (k(-1)) from 1 was analyzed with respect to several factors. Challenges still remain, including acquiring experimental data for unactivated systems and observing an intermediate carrying a good nucleofuge.

Crystallographic Insights into the Autocatalytic Assembly Mechanism of a Bacteriophage Tail Spike

The tailed bacteriophage phi29 has 12 "appendages" (gene product 12, gp12) attached to its neck region that participate in host cell recognition and entry. In the cell, monomeric gp12 undergoes proteolytic processing that releases the C-terminal domain during assembly into trimers. We report here crystal structures of the protein before and after catalytic processing and show that the C-terminal domain of gp12 is an "autochaperone" that aids trimerization. We also show that autocleavage of the C-terminal domain is a posttrimerization event that is followed by a unique ATP-dependent release. The posttranslationally modified N-terminal part has three domains that function to attach the appendages to the phage, digest the cell wall teichoic acids, and bind irreversibly to the host, respectively. Structural and sequence comparisons suggest that some eukaryotic and bacterial viruses as well as bacterial adhesins might have a similar maturation mechanism as is performed by phi29 gp12 for Bacillus subtilis.

The neutral hydrolysis of methyl acetate — Part 2. Is there a tetrahedral intermediate?

A computational strategy that reproduces the experimental rates of hydration of formaldehyde, acetaldehyde, acetone, and cyclohexanone and the rates of acetic acid and 2-hydroxypyridine-catalyzed hydration of acetone has been extended to the results of the neutral hydrolysis of methyl acetate reported in Part 1. Calculations have been performed for one-step and two-step mechanisms, with cooperative assistance from one to three additional water molecules in the presence and absence of the acetic acid product. The calculations predict that, for the neutral reaction, a one-step mechanism will be favoured if tetrahedral intermediates have a short lifetime and do not interconvert prior to breakdown (case A), and a two-step mechanism will be operative if tetrahedral intermediates are allowed to interconvert prior to breakdown (case B). The experimental results are consistent with the predictions of case A. In the presence of acetic acid, case A predicts that the acid will contribute only 1.6% to the overall rate, a negligible acceleration over the noncatalytic process, and case B predicts general acid catalysis to be an order of magnitude greater than the experimental result. It is concluded that the neutral hydrolysis of methyl acetate is mainly a cooperative one-step process, and that general acid catalysis by the acetic acid product does not occur.

ONIOM Study of the Mechanism of the Enzymatic Hydrolysis of Biodegradable Plastics

Abstract A cutinase-like enzyme (CLE), which is purified experimentally from the yeast Cryptococcus sp. strain S-2, has been recently found to degrade biodegradable plastics very efficiently. In this study, we theoretically examine the mechanism of hydrolysis of biodegradable plastics by the CLE by means of the ONIOM method. We optimize all the intermediates and the transition states involved in the considered enzymatic reaction and determine the energy surface of the entire catalytic cycle. The calculations show that the amino acid residues inside the pocket of the active site, Thr17 and Gln86, which stabilize the tetrahedral intermediates, and Gly115 in addition to Ser85, His180, and Asp165, which compose the catalytic triad, significantly contribute to the catalytic reaction. This is similar to the case of serine protease reported to date. Moreover, we have newly found that the energy barrier of the catalytic cycle is significantly lowered by the electronic effect of the OH group in the side-chain of Thr17 and bound water. The calculated potential energy surface of the reaction shows that the cleavage of the ester bond is a rate-determining step.

A quantum mechanics study on the reaction mechanism of chalcone formation from p-coumaroyl-CoA and malonyl-CoA catalyzed by chalcone synthase

Chalcone synthase catalyzes formation of phenylpropanoid chalcone from one p-coumaroyl-coenzyme A (CoA) and three malonyl-CoA molecules. In order to elucidate structural and energetic features of the reaction mechanism, we performed the quantum mechanics calculations and obtained the following results. In loading step, only a tetrahedral intermediate is located without transition state (TS). Our results indicate that His303 acts as a H31 donor, but not a hydrogen bond donor, to stabilize the intermediate formation. In decarboxylation step, the reaction proceeds via a TS and is sensitive to the environment. In elongation step, a tetrahedral TS is located. All of the results above support the reaction mechanism and further complement the proposal of Noel JP et al.

PhzA/B Catalyzes the Formation of the Tricycle in Phenazine Biosynthesis

Phenazines are redox-active bacterial secondary metabolites that participate in important biological processes such as the generation of toxic reactive oxygen species and the reduction of environmental iron. Their biosynthesis from chorismic acid depends on enzymes encoded by the phz operon, but many details of the pathway remain unclear. It previously was shown that phenazine biosynthesis involves the symmetrical head-to-tail double condensation of two identical amino-cyclohexenone molecules to a tricyclic phenazine precursor. While this key step can proceed spontaneously in vitro, we show here that it is catalyzed by PhzA/B, a small dimeric protein of the Delta(5)-3-ketosteroid isomerase/nuclear transport factor 2 family, and we reason that this catalysis is required in vivo. Crystal structures in complex with analogues of the substrate and product suggest that PhzA/B accelerates double imine formation by orienting two substrate molecules and by neutralizing the negative charge of tetrahedral intermediates through protonation. HPLC-coupled NMR reveals that the condensation product rearranges further, which is probably important to prevent back-hydrolysis, and may also be catalyzed within the active site of PhzA/B. The rearranged tricyclic product subsequently undergoes oxidative decarboxylation in a metal-independent reaction involving molecular oxygen. This conversion does not seem to require enzymatic catalysis, explaining why phenazine-1-carboxylic acid is a major product even in strains that use phenazine-1,6-dicarboxylic acid as a precursor of strain-specific phenazine derivatives.

Crystal Structure of Human Plasma Platelet-activating Factor Acetylhydrolase: STRUCTURAL IMPLICATION TO LIPOPROTEIN BINDING AND CATALYSIS

Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A2. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5 angstroms. It has a classic lipase alpha/beta-hydrolase fold, and it contains a catalytic triad of Ser273, His351, and Asp296. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser273. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase.

MurD ligase from E. coli: Tetrahedral intermediate formation study by hybrid quantum mechanical/molecular mechanical replica path method

MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase), a three-domain bacterial protein, catalyses a highly specific incorporation of D-glutamate to the cytoplasmic intermediate UDP-N-acetyl-muramoyl-L-alanine (UMA) utilizing ATP hydrolysis to ADP and P(i). This reaction is part of a biosynthetic path yielding bacterial peptidoglycan. On the basis of structural studies of MurD complexes, a stepwise catalytic mechanism was proposed that commences with a formation of the acyl-phosphate intermediate, followed by a nucleophilic attack of D-glutamate that, through the formation of a tetrahedral reaction intermediate and subsequent phosphate dissociation, affords the final product, UDP-N-acetyl-muramoyl-L-alanine-D-glutamate (UMAG). A hybrid quantum mechanical/molecular mechanical (QM/MM) molecular modeling approach was utilized, combining the B3LYP QM level of theory with empirical force field simulations to evaluate three possible reaction pathways leading to tetrahedral intermediate formation. Geometries of the starting structures based on crystallographic experimental data and tetrahedral intermediates were carefully examined together with a role of crucial amino acids and water molecules. The replica path method was used to generate the reaction pathways between the starting structures and the corresponding tetrahedral reaction intermediates, offering direct comparisons with a sequential kinetic mechanism and the available structural data for this enzyme. The acquired knowledge represents new and valuable information to assist in the ongoing efforts leading toward novel inhibitors of MurD as potential antibacterial drugs.

Kinetics and mechanism of the anilinolysis of S‐aryl N‐arylthiocarbamates in acetonitrile

AbstractThe aminolysis reactions of S‐aryl N‐arylthiocarbamates (YC6H4NHC(O)SC6H4Z, 1) with anilines in acetonitrile are studied. The reaction rates are more influenced by the nucleophilicity of the nucleophile than the nucleofugality of the leaving group, but the change in the effective charge from reactants to the TS for formation of the tetrahedral intermediate is slightly greater in the leaving group (βZ from−0.07 to −0.14) than in the nucleophile (βX = 0.04–0.12). The magnitude of the Brönsted coefficients are in the range of values that are consistent for a stepwise mechanism with rate‐limiting formation of the zwitterionic tetrahedral intermediate. Signs of cross‐interaction constants, ρXY (&gt;0), ρXZ (&gt;0) and ρYZ (&lt;0), are all consistent with a stepwise mechanism. It is concluded that the change of the amine from benzylamines to anilines causes a shift of the aminolysis mechanism from a concerted to a stepwise process. Copyright © 2008 John Wiley &amp; Sons, Ltd.