Acylation Step Research Articles

Enantiopure building blocks for the synthesis of 3-methyl-2-alkanols. Diastereoselective methylmetal addition to a chiral 2-methylaldehyde followed by lipase catalysed esterification

The racemic synthetic building block (2 R*,3 R*)-3-methyl-4-(phenylsulfanyl)butan-2-ol (2 R*,3 R*)- 2 was obtained in a high diastereomeric ratio [95:5, (2 R*,3 R*)/(2 R*,3 S*)-ratio] by Lewis acid catalysed dimethylzinc addition to racemic 2-methyl-3-(phenylsulfanyl)propanal ( rac- 1). Two consecutive acylations with vinyl acetate catalysed by Chirazyme L-2 (immobilised Candida antarctica lipase B, CAL-B) led to preferential esterification of three of the four stereoisomers leaving (2 S,3 S)-3-methyl-4-(phenylsulfanyl)butan-2-ol (2 S,3 S)- 2 of 98:2 dr and 98% ee. The stereoisomerically impure acetate of (2 R,3 R)-3-methyl-4-(phenylsulfanyl)butan-2-ol (2 R,3 R)- 2, obtained in the first CAL-B-catalysed acylation step, was hydrolysed and reesterified using CAL-A (immobilised Novozyme SP 525) as the catalyst, which left (2 R,3 R)-3-methyl-4-(phenylsulfanyl)butan-2-ol (2 R,3 R)- 2 of 98:2 dr and 99% ee as the remaining substrate. The individual enantiomers of 2-methyl-3-(phenylsulfanyl)propanal 1 were prepared from readily available ( S)- and ( R)-3-hydroxy-2-methylpropanoic acid methyl ester and reacted with dimethylzinc to give both enantiomers of (2 R*,3 R*)-3-methyl-4-(phenylsulfanyl)butan-2-ol (2 R, 3 R)- or (2 S,3 S)- 2 of both high dr and ee. These products were purified by lipase catalysed acylation to give the enantiomerically and diastereomerically highly pure enantiomers (>99.5:0.5 dr, >99.9% ee). Pure (2 S,3 S)-3-methyl-4-(phenylsulfanyl)butan-2-ol (2 S,3 S)- 2 was transformed into a potential pheromone precursor isolated from some pine sawflies of the genus Gilpinia, (2 S,3 R)-3-methylpentadecan-2-ol in 54% yield over eight steps.

The Importance of a Critical Protonation State and the Fate of the Catalytic Steps in Class A β-Lactamases and Penicillin-binding Proteins

Beta-lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to beta-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 beta-lactamase from Escherichia coli. These techniques included perturbation of the pK(a) of Lys-73 by the study of the gamma-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK(a) of Lys-73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A beta-lactamases has actually raised the pK(a) of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine beta-lactamases and penicillin-binding proteins.

Molecular cloning and characterization of a taxadienol acetyl transferase cDNA from Taxus x media

A full-length cDNA encoding taxadienol acetyl transferase (designated as TmTAT), that catalyzes the first acylation step in Taxol biosynthesis, was cloned from young leaves of Taxus x media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTAT was 1459 bp and contained a 1317-bp open reading frame (ORF) encoding a protein of 439 amino acids. The deduced protein had a calculated molecular weight of about 49 kDa and an isoelectric point (p I) of 5.3, similar to acetyl transferases from Taxus cuspidata and Taxus chinensis. Sequence comparison analysis revealed that the TmTAT had high similarity with other members of plant transferase family. Phylogenetic tree analysis showed that TmTAT had close relationship with acetyl transferases from T. chinenisis and T. cuspidata. Tissue expression pattern analysis revealed that TmTAT expressed strongly in leaves, weak in stems and no expression could be detected in fruits, indicating that TmTAT was a tissue-specific gene.

Conversion of alpha-haloaldehydes into acylating agents by an internal redox reaction catalyzed by nucleophilic carbenes.

Reactivity umpolung allows us to consider nontraditional bond disconnections. We report herein that treatment of an alpha-haloaldehyde with a nucleophile in the presence of catalytic amounts of nucleophilic carbenes results in an internal redox reaction giving rise to a dehalogenated acylating agent as an intermediate by a new reaction manifold. A brief illustration of the scope of this reaction is presented along with evidence supporting the direct intervention of the carbene in the acylation step.

The ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class A penicillin-binding protein.

A soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli 14C-labeled lipid II precursor. As a DD- peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k2/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.

Effect of Fractional Fluorination on the Properties of ATRP Surface-Initiated Poly(hydroxyethyl methacrylate) Films

Acylation of a surface-initiated poly(hydroxyethyl methacrylate) (PHEMA) film with a perfluoroalkyl acid chloride (C7F15COCl) results in a highly blocking film with fluorocarbon side groups that orient at the air−film interface to produce an ultralow-energy surface. The kinetics of the acylation step was monitored with reflectance-absorption infrared spectroscopy to reveal that the rate of fluorination depends on the chain length and solution concentration of the perfluorinated acid chloride as well as the solvent used. By control of the time of exposure to C7F15COCl, films with fractional conversion of the hydroxyl groups to fluorinated esters between 0 and 0.8 were produced. Increasing fractional conversion from 0 to ∼0.2 has a dramatic effect on surface wettability and electrochemical barrier properties as a densely packed, oriented fluorocarbon surface region is formed that greatly reduces water and ion transport. Additional fluorination has no effect on wettability but gradually increases film resistance while lowering film capacitance. These results suggest that structuring of fluorocarbon groups at the outermost surface of the film has a major impact on film properties and establish the minimum content of fluorocarbon groups required to achieve the structured interface via this grafting process.

Pre‐steady‐state kinetic studies of rat kidney γ‐glutamyl transpeptidase confirm its ping‐pong mechanism

AbstractThe enzyme γ‐glutamyl transpeptidase (GGT) is implicated in cellular detoxification, the biosynthesis of leukotrienes and control of the physiological concentration of glutathione. It also plays important roles in Parkinson's disease, diabetes, apoptosis inhibition and cancer drug resistance. It catalyses the breakdown of its in vivo substrate, glutathione, by cleaving the amide bond between the γ‐glutamyl and the cysteinylglycine moieties. Threonine is proposed to act as the nucleophile of GGT in the formation of the γ‐glutamyl acyl enzyme intermediate during the acylation step. The γ‐glutamyl moiety is then transferred to a primary amine acceptor substrate (an amino acid or dipeptide) or to a water molecule, to form a compound containing a new isopeptide bond or glutamate in the transamidation or hydrolysis reactions, respectively. In spite of the importance of the role of GGT in human physiology, there is a lack of information about the mechanisms of its catalytic reactions, and in particular the nature of the intermediate formed during the acylation step. In order to gain insight into the formation of the acyl enzyme intermediate, different D‐γ‐glutamylanilides substituted in the para position with electron‐withdrawing and electron‐donating groups were used as donor substrates under conditions where water served as acceptor substrate. A Hammett plot with a slope of zero was obtained for the steady‐state hydrolysis reaction for which deacylation is the rate‐ limiting step. To confirm the ping‐pong mechanism, pre‐steady‐state kinetics of this reaction were then performed with the donor substrate D‐γ‐glutamyl‐p‐nitroanilide, which liberates the chromophore p‐nitroaniline. Experiments using a stopped‐ flow spectrometer and a rapid mix–quench apparatus gave biphasic traces with a burst up to ∼65 ms, the amplitude of which corresponds well with the concentration of the enzyme. These burst kinetics were also observed in the presence of L‐methionine at concentrations ∼15‐fold below its KM value, where deacylation would still be rate limiting. These observations are consistent with the formation of an intermediate during the rapid acylation step and support the modified ping‐pong mechanism proposed for GGT‐ mediated hydrolysis and aminolysis. Copyright © 2004 John Wiley & Sons, Ltd.

Modeling of serine protease prototype reactions with the flexible effective fragment potential quantum mechanical/molecular mechanical method

A complete cycle of chemical transformations for the serine protease prototype reaction is modeled following calculations with the flexible effective fragment quantum mechanical/molecular mechanical (QM/MM) method. The initial molecular model is based on the crystal structure of the trypsin–bovine pancreatic trypsin inhibitor complex including all atoms of the enzyme within approximately 15–18 A of the oxygen center Oγ of the catalytic serine residue. Several selections of the QM/MM partitioning are considered. Fractions of the side chains of the residues from the catalytic triad (serine, histidine and aspartic acid) and a central part of a model substrate around the C–N bond to be cleaved are included into the QM subsystem. The remaining part, or the MM subsystem, is represented by flexible chains of small effective fragments, whose potentials explicitly contribute to the Hamiltonian of the QM part, but the corresponding fragment–fragment interactions are described by the MM force fields. The QM/MM boundaries are extended over the Cα–Cβ bonds of the peptides assigned to the QM subsystem in the enzyme, C–C and C–N bonds in model substrates. Multiple geometry optimizations have been performed by using the RHF/6-31G method in the QM part and OPLSAA or AMBER sets of MM parameters, resulting in a series of stationary points on the complex potential-energy surfaces. All structures generally accepted for the serine protease catalytic cycle have been located. Energies at the stationary points found have been recomputed at the MP2/6-31+G* level for the QM part in the protein environment. Structural changes along the reaction path are analyzed with special attention to hydrogen-bonding networks. In the case of a model substrate selected as a short peptide CH3(NHCO-CH2)2 – HN–CO–(CH2–NHCO)CH3 the computed energy profile for the acylation step shows too high activation energy barriers. The energetics of this rate-limiting step is considerably improved, if more realistic model for the substrate is considered, following the motifs of the ThrI11–GlyI12–ProI13-–CysI14–LysI15–AlaI16–ArgI17–IleI18–IleI19 sequence of the bovine pancreatic trypsin inhibitor.

Paclitaxel production by plant-cell-culture technology.

Plant cells catalyze multiple-step reactions of secondary metabolite biosynthesis, and selectively synthesize chiral compounds with polycyclic structures. Taking advantage of this characteristic, we studied the production of the anticancer drug paclitaxel, which is currently produced in limited supply. Callus culture investigations indicate that woody plant medium supplemented with 10(-5) mol L(-1) 1-naphthylacetic acid and without the NH4+ -type ion is the best condition for growth of the callus. The accumulation of paclitaxel and related taxanes in Taxus plants is thought to be a biological response to specific external stimuli. Several signal transducers were screened; taxane biosynthesis was strongly promoted by methyl jasmonate (MeJA) and silver thiosulfate (STS) as an anti-ethylene compound. Of ten taxane-type diterpenoids isolated from T. baccata suspension-cultured cells treated with MeJA, five have a phenylisoserine side-chain at the C-13 position of the taxane skeleton. Time-course analysis revealed two regulatory steps in taxane biosynthesis: the taxane-ring formation step and the acylation step of the C-13 position. Methyl jasmonate promoted the formation of the taxane-ring. The production of paclitaxel reached a maximum level of 295 mg L(-1) in a large-scale culture of T. x media cells using a two-stage process.

Novel mannitol based non-ionic surfactants from biocatalysis

Two new mannitol ester based non-ionic surfactants were synthesised using immobilised lipases. The synthesis started from mannitol derivatives having two of the six hydroxyls protected by an isopropylidene group. In the acylation step five different immobilised lipases were compared, for efficiency of substrate conversion. The commercial immobilised lipase from Candida antarctica B (Novozym 435) gave the highest conversion in the shortest time for both substrates. Selective hydrolysis of the isopropylidene group yielded 1- O-lauroyl- d-mannitol and 1,6-di- O-lauroyl- d-mannitol. Characterisation of the products was performed by 13 C NMR. In the enzymatic step it has been thought that it is mainly the primary hydroxyl groups that are esterified. However, as detected from 13 C NMR, also other isomers were found. The formation of these isomers may be due to the non-specificity of the lipase, and to the acyl migration phenomenon.

Homogeneous time resolved fluorescence assay to measure histamine release.

Histamine is a biogenic amine synthesized by the enzymatic decarboxylation of histidine. Implication of histamine in allergy is well described but histamine is also found in some specific neurones, functions as a neurotransmitter and regulates sleep/wake cycles, hormonal secretion, cardiovascular control and thermo-regulation. We have developed a TR-FRET histamine assay, based on the competition between sample histamine and allophycocyanine (XL665) labelled histamine for binding to a Europium cryptate (EuK) labelled antibody. As histamine is a small monoamine molecule, high affinity antibodies have been raised against carrier protein conjugated histamine. Therefore, sample histamine needs to be derivatized in the same way as the conjugated histamine, so that the antibody will have a similar affinity for both molecules. This acylation step is performed directly in wells and does not need to be done in separate vials, making handling easier for large numbers of samples. The incubation takes place at room temperature for 3 hours. The assay covers a measurement range of 1.56 to 400 nM and shows an analytical sensitivity of 1.3nM. We have shown that miniaturization of sample and reagents volumes down to 20 micro l does not alter these performances. This histamine release assay provides a particularly well adapted procedure for HTS and secondary screening compared to current heterogeneous methods.

Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases.

Covalent posttranslational protein modifications by eukaryotic transglutaminases proceed by a kinetic pathway of acylation and deacylation. Ammonia is released as the acylenzyme is formed, whereas the cross-linked product is released later in the deacylation step. Superposition of the active sites of transglutaminase type 2 (TG2) and the structurally related cysteine protease, papain, indicates that in the formation of tetrahedral intermediates, the backbone nitrogen of the catalytic Cys-277 and the N1 nitrogen of Trp-241 of TG2 could contribute to transition-state stabilization. The importance of this Trp-241 side chain was demonstrated by examining the kinetics of dansylcadaverine incorporation into a model peptide. Although substitution of the Trp-241 side chain with Ala or Gly had only a small effect on the Michaelis constant Km (1.5-fold increase), it caused a >300-fold lowering of the catalytic rate constant kcat. The wild-type and mutant TG2-catalyzed release of ammonia showed kinetics similar to the kinetics for the formation of cross-linked product, indicating that transition-state stabilization in the acylation step was rate-limiting. In papain, a Gln residue is at the position of TG2-Trp-241. The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower kcat than wild-type enzyme suggest evolutionary specialization in the use of the indole group. This notion is further supported by the observation that transition-state-stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease.

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Brønsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.

Identification of Glu166 as the general base in the acylation reaction of class A beta-lactamases through QM/MM modeling.

Bacterial class A beta-lactamases are responsible for the most known resistance against beta-lactam antibiotics. With the continuing rise in antibiotic resistance, improved knowledge of the mechanisms of action of these enzymes is needed in the development of effective therapeutic agents and strategies. The mechanism of the deacylation step in class A beta-lactamases is well accepted. In contrast, the mechanism of the acylation step has been uncertain, with several conflicting proposals put forward. We have modeled the acylation step in a class A beta-lactamase, using a combined quantum mechanics/molecular mechanics approach. The results provide an atomic level description of the reaction and show that Glu166 acts as the general base in the reaction, deprotonating Ser70 via an intervening water molecule. Ser70 acts as the nucleophile for attack on the lactam ring in a concerted reaction. The results do not rule out the importance of Lys73 in catalysis, in agreement with experimental data.

Incorporation of unprotected heterocyclic side chains into peptoid oligomers via solid-phase submonomer synthesis.

Peptoids (N-substituted glycines) are an important class of biomimetic oligomers that have made a significant impact in the areas of combinatorial drug discovery, gene therapy, drug delivery, and biopolymer folding in recent years. Sequence-specific peptoid oligomers are easily assembled from primary amines by the solid-phase submonomer method. However, most amines that contain heterocyclic nitrogens in the side chain do not incorporate efficiently. We present here a straightforward revision of the submonomer method that allows efficient incorporation of unprotected imidazoles, pyridines, pyrazines, indoles, and quinolines into oligomers as long as 15 monomers in length. This improved method uses chloroacetic acid instead of bromoacetic acid in the acylation step of the monomer addition cycle, and allows for the incorporation of new side chains that should enable the synthesis of peptoids with entirely new properties.

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system: effects of water on kinetic parameters

The influence of water on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates to the enzyme while removing reaction products. In this system, interactions between the enzyme and nonreacting molecules are avoided, since no solvent is present, and it is thus more easy to assess the role of water. To this end, alcohol inhibition constant, substrates dissociation constants as well as acylation rate constant and ratio of acylation to deacylation rate constants have been determined as a function of water activity ( a w). Data obtained highlight that n-propanol inhibition constant and dissociation constant of methyl propionate are a lot affected by a w variations whereas water has no significant effect on the catalytic acylation step nor on the ratio of acylation to deacylation rate constants. These results suggest the water-independent character of the transition step.

Acylation is rate-limiting in glycosylasparaginase-catalyzed hydrolysis of N4-(4'-substituted phenyl)-L-asparagines.

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond between N-acetyl-D-glucosamine and L-asparagine in the catabolism of glycoproteins. The mechanism has been proposed to resemble that of serine proteases involving an acylation step where a nucleophilic attack by a catalytic Thr residue on the carbonyl carbon of the N-glycosylic bond gives rise to a covalent beta-aspartyl-enzyme intermediate, and a deacylation step to give the final products. The question posed in this study was: Is the acylation step the rate-limiting step in the hydrolysis reaction as in serine proteases? To answer this question a series of mostly new substituted anilides was synthesized and characterized, and their hydrolysis reactions catalyzed by glycosylasparaginase from human amniotic fluid were studied. Five N4-(4'-substituted phenyl)-L-asparagine compounds were synthesized and characterized: 4'-hydrogen, 4'-ethyl, 4'-bromo, 4'-nitro, and 4'-methoxy. Each of these anilides was a substrate for the enzyme. Hammett plots of the kinetic parameters showed that acylation is the rate-limiting step in the reaction and that upon binding the electron distribution of the substrate is perturbed toward the transition state. This is the first direct evidence that acylation is the rate-limiting step in the enzyme-catalyzed reaction. A Brønsted plot indicates a small, negative charge (-0.25) on the nitrogen atom of the leaving group anilines containing electron-withdrawing groups, and a small, positive charge (0.43) on the nitrogen atom of the leaving group anilines containing electron-donating groups. The free energy (incremental) change of binding (delta deltaGb) in the enzyme-substrate transition state complexes shows that substitution of a substituted phenyl group for the pyranosyl group in the natural substrate results in an overall loss of binding energy equivalent to a weak hydrogen bond, the magnitude of which is dependent on the substituent group. The data are consistent with a mechanism for glycosylasparaginase involving rapid formation of a tetrahedral structure upon substrate binding, and a rate-limiting breakdown of the tetrahedral structure to a covalent beta-aspartyl-enzyme intermediate that is dependent on the electronic properties of the substituent group and on the degree of protonation of the leaving group in the transition state by a general acid.

Stereoselective Hydrolysis of Quaternary Quinuclidinium Benzoates Catalyzed by Butyrylcholinesterase

AbstractFour chiral, quaternary, N‐methyl and N‐benzyl derivatives of (R)‐ and (S)‐quinuclidin‐3‐yl benzoates were synthesized and studied as substrates of horse serum butyrylcholinesterase (BChE). The kcat for the substrates decreased in the order (R)‐N‐methyl > (R)‐N‐benzyl (2.3‐fold slower) >> (S)‐N‐methyl (70.5‐fold slower reaction), while for the (S)‐N‐benzyl ester inhibition of the enzyme was observed. The kinetics of inhibition (Ka = 3.3 μM) indicated that binding to the catalytic site of BChE occurred. From the ratio of the kcat/KM values of both enantiomers an enantiomeric excess of 95% was calculated for N‐methyl derivatives. Thus, BChE is suitable as a biocatalyst for the resolution of racemic quaternary quinuclidinium esters. In order to explain the experimental data, combined quantum chemical (HF/3−21G*) and semiempirical (PM3) calculations within the ONIOM scheme of the stable species in the acylation step were performed. Geometry optimizations were carried out for all benzoate esters for an assumed active site model of BChE. It was confirmed that hydrolysis is affected to an appreciable extent by a proper geometrical orientation of substrates at the choline subsite. The energies of the optimized systems were in good agreement with the experimental data. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

A combinatorial biocatalysis approach to an array of cholic acid derivatives.

A 39-member library of bile acid derivatives was prepared starting from 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid methyl ester using a combinatorial biocatalytic approach. A regioselective oxidation step, catalyzed by hydroxysteroid dehydrogenases, followed by an acylation step with a series of different acyl donors catalyzed by Candida antarctica lipase B, led to the modification of the bile acid scaffold. Each member of the library was obtained in high purity and good yield.

PKa, MM, and QM studies of mechanisms of beta-lactamases and penicillin-binding proteins: acylation step.

The acylation step of the catalytic mechanism of beta-lactamases and penicillin-binding proteins (PBPs) has been studied with various approaches. The methods applied range from molecular dynamics (MD) simulations to multiple titration calculations using the Poisson-Boltzmann approach to quantum mechanical (QM) methods. The mechanism of class A beta-lactamases was investigated in the greatest detail. Most approaches support the critical role of Glu-166 and hydrolytic water in the acylation step of the enzymatic catalysis in class A beta-lactamases. The details of the catalytic mechanism have been revealed by the QM approach, which clearly pointed out the critical role of Glu-166 acting as a general base in the acylation step with preferred substrates. Lys-73 shuffles a proton abstracted by Glu-166 O(epsilon ) to the beta-lactam nitrogen through Ser-130 hydroxyl. This proton is transferred from O(gamma) of the catalytic Ser-70 through the bridging hydrolytic water to Glu-166 O(epsilon ). Then the hydrogen is simultaneously passed through S(N)2 inversion mechanism at Lys-73 N(zeta) to Ser-130 O(gamma), which loses its proton to the beta-lactam nitrogen. The protonation of beta-lactam nitrogen proceeds with an immediate ring opening and collapse of the first tetrahedral species into an acyl-enzyme intermediate. However, the studies that considered the effect of solvation lower the barrier for the pathway, which utilizes Lys-73 as a general base, thus creating a possibility of multiple mechanisms for the acylation step in the class A beta-lactamases. These findings help explain the exceptional efficiency of these enzymes. They emphasize an important role of Glu-166, Lys-73, and Ser-130 for enzymatic catalysis and shed light on details of the acylation step of class A beta-lactamase mechanism. The acylation step for class C beta-lactamases and six classes of PBPs were also considered with continuum solvent models and MD simulations.