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The electronic properties of the active-sites of the structurally unrelated serine peptidases, alpha-chymotrypsin and subtilisin, have been expressed in the form of three-dimensional electrostatic potential maps derived from integrals calculated at the quantum chemistry level. As a consequence of the asymmetrical distribution of the secondary structures that occur within a 7 A sphere around the serine of the catalytic triad, the active sites are highly polarized entities and exhibit large dipole moments. One part of the active sites generates a nucleophilic suction-pump. Its isocontour at -10 kcal mol-1 defines an impressive, negatively-charged volume which bears a narrow channel in the immediate vicinity of the active-site serine 195 in alpha-chymotrypsin or 221 in subtilisin. In native alpha-chymotrypsin, there is a perfect complementation between this nucleophilic suction-pump and the positively-charged electrophilic hole that is generated by the backbone NH of Ser 195 and Gly 193. In subtilisin, generation of the complementing electrophilic hole requires binding of a carbonyl donor ligand and may be achieved by rotation of the side-chain amide of Asn 155 towards the backbone NH of Ser 221. Small variations in the atomic co-ordinates of alpha-chymotrypsin used for the calculations, the presence of water molecules in its active site and the occurrence of point mutations in the amino acid sequence of subtilisin have little effects on the shape and characteristics of the electrostatic potential.

Summary Comparison of the predicted amino acid sequence of the cell-cycle RodA protein with the National Research Foundation protein sequence database shows that the 370-amino-acid RodA, a protein that is essential for wall elongation in Escherichia coli and maintenance of the rod shape of the cell, is highly analogous, in terms of primary structure, with the Bacillus subtilis SpoVE protein involved in stage V of sporulation.

Quantum dynamics in a valley ridge inflection (VRI) point region is analyzed in the case of the Diels-Alder endo-dimerization of cyclopentadiene pointed out recently by [Caramella et al., J. Am. Chem. Soc. 124, 1130 (2002)]. The VRI point is located along the reaction path connecting the bispericyclic symmetrical transition structure put in evidence by Caramella et al. and the transition state of the Cope rearrangement. Dynamics is carried out by using constrained Hamiltonian methodology. The active coordinates are the first formed C–C bond length and the difference between the two other C–C bond lengths which achieve the dimerization as 4+2 or 2+4 adducts. A two-dimensional (2D) minimum-energy surface have been computed at the Becke 3 Lee–Yong–Parr∕6‐31G* level. The energy landscape can be classified as an uphill ridge-pitchfork VRI bifurcation according to a recent classification of bifurcation events [W. Quapp, J. Mol. Struct. 695–696, 95 (2004)]. Dynamics does not describe the thermal reaction but concerns wave packets which could be prepared by pulse reagents, i.e., by coherent control. We analyze how the shape and initial location on the ground potential-energy surface are linked to the synchronous or asynchronous mechanism of the final step after the first transition state. We use a one-dimensional model of optimum control theory to check the feasibility of such a coherent preparation. The wave-packet evolution in the VRI domain is well explained by semiclassical predictions even with the negative curvature of the unstable ridge. Finally, a crude model of dissipation has been introduced to test the stability of the 2D predictions.

Dans l'arsenal de la chimiotherapie antibacterienne, les antibiotiques de la famille de la penicilline ou β-lactamines sont les plus largement utilises, en raison de leur activite elevee et de leur absence presque totale d'effets secondaires. Les bacteries ont cependant developpe des mecanismes de resistance efficaces reposant sur differents facteurs genetiquement independants: une diminution de la sensibilite des cibles, des DD-peptidases ancrees a la face externe de la membrane cytoplasmique, la production d'enzymes qui catalysent la destruction de l'antibiotique et, chez les bacteries a Gram - et les mycobacteries, la presence d'une barriere de permeabilite qui ralentit la diffusion de l'antibiotique vers ses cibles. Des mecanismes actifs d'expulsion de l'antibiotique peuvent aussi se rencontrer. Les diverses strategies imaginees pour contrer cette resistance entrainent des reponses bacteriennes d'une remarquable diversite. Une lutte rationnelle contre les especes pathogenes demande une comprehension de ces phenomenes qui integre la microbiologie, la genetique moleculaire, l'enzymologie, l'analyse structurale des proteines impliquees et l'etude des reactions qu'elles catalysent par les methodes de la chimie theorique.

The conditions of CN bond breakage by the serine peptidases have been analysed. A two-way table has been generated where either formamide or protonated formamide serves as minimal model of the scissile CN bond and where either methanol or the couple CH 3 O − H + serves as minimal model of the attacking nucleophile. An addition-elimination reaction is proposed which links the enzyme acylation and deacylation steps.

Several types of active-site-directed inactivators (inhibitors) of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase were tested. (i) Among the heavy-atom-containing compounds examined, K2Pt(C2O4)2 inactivates the enzyme with a second-order rate constant of about 6 X 10(-2)M-1 X S-1 and has only one binding site located close to the Zn2+ cofactor within the enzyme active site. (ii) Several compounds possessing both a C-terminal carboxylate function and, at the other end of the molecule, a thiol, hydroxamate or carboxylate function were also examined. 3-Mercaptopropionate (racemic) and 3-mercaptoisobutyrate (L-isomer) inhibit the enzyme competitively with a Ki value of 5 X 10 X 10(-9)M. (iii) Classical beta-lactam compounds have a very weak inhibitory potency. Depending on the structure of the compounds, enzyme inhibition may be competitive (and binding occurs to the active site) or non-competitive (and binding causes disruption of the protein crystal lattice). (iv) 6-beta-Iodopenicillanate inactivates the enzyme in a complex way. At high beta-lactam concentrations, the pseudo-first-order rate constant of enzyme inactivation has a limit value of 7 X 10(-4)S-1 X 6-beta-Iodopenicillanate binds to the active site just in front of the Zn2+ cofactor and superimposes histidine-190, suggesting that permanent enzyme inactivation is by reaction with this latter residue.

The electrostatic interaction energy between methanol and the dyad water-imidazole has been computed numerically at three levels of approximation from 3D grids of the charge density of one partner and the electrostatic potential of the other. The minimum positions and energy values thus obtained compare well with those calculated analytically. The numerical procedure is especially interesting for the prediction of the stable conformers.

Bacteria possess proteases that are specific for the peptide bonds between D-alanine residues, one of which has a free α-carboxyl group. These D-alanyl-D-alanine peptidases catalyse carboxypeptidation and transpeptidation reactions involved in bacterial cell wall metabolism1,2, and are inactivated by β-lactam antibiotics. We have now elucidated the structure, at 2.5 A resolution, of the penicillin-resistant Zn2+-containing D-alanyl-D-alanine peptidase of Streptomyces albus (Zn2+ G peptidase)3,4. The enzyme is shown to consist of two globular domains, connected by a single link. The N-terminal domain has three α-helices, and the C-terminal domain has three α-helices and five β-strands. The Zn2+ ion is ligated by three histidine residues, and located in a cleft in the C-terminal domain. The mechanism of action of the enzyme may be related to that of other carboxypeptidases, which also contain functional Zn2+ ions.

Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-directed-mutagenesis experiments.

Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between beta-halogenopenicillanates and some class A beta-lactamases. This suggested the stabilization of a highly charged intermediate by solvation. Those data could be interpreted on the basis of a reaction pathway where an episulphonium ion was transiently formed. The various mechanisms proposed for explaining the formation of the dihydrothiazine chromophore are discussed.

PURPOSE: Cholesterol is a major lipid constituent of biological membranes which could be included in cyclodextrin (CD) cavities. Solubilization and cell extraction of cholesterol have been previously performed in order to study its interaction with beta-CD and methylated beta-derivatives notably. The present work aims at confirming the formation of inclusion complexes between these CDs and cholesterol in order to understand their solubilization and cell extraction capacities. METHODS: In this context, liquid-state NMR spectroscopy (1H NMR studies and ROESY experiments) as well as theoretical studies (molecular modeling) have been performed. RESULTS: Rather than preferential conformations, the spectroscopic studies showed us the possible interactions between cholesterol and dimethyl-beta-CD, trimethyl-beta-CD, randomly methylated beta-CD or Crysmeb®. Weak interactions were detected using the latter one, confirming the advantage of the low substitution to decrease membrane loss of integrity and cytotoxicity. Molecular modeling studies should be used to determine which stoichiometry and conformations are energically more favorable. The semi-empirical AM1 level was used to investigate both 1:1 and 1:2 complexes whereas 1:1 complexes were also studied using minimal or double zeta basis sets. Four conformations for each 1:2 complexes have been envisaged and studied for the methylated CDs. CONCLUSIONS: These studies allowed us to confirm the interactions between cholesterol and beta-CDs especially the methylated derivatives.