Reaction mechanism of the acyl-enzyme formation in b-lactam hydrolysis by means of quantum chemical modeling J.R. Alvarez-Idaboy a,b , R. Gonza ´lez-Jonte a,b , A. Herna ´ndez-Laguna c , Y.G. Smeyers a, * a C.S.I.C, Instituto de Estructura de la Materia, Serrano 123, E-28006 Madrid, Spain b Universidad de La Habana, Laboratorio de Quı ´mica Computacional y Teo ´ria, Facultad de Quı ´mica, Havana 10400, Cuba c C.S.I.C, Estacio ´n Experimental del Zaidı ´n, Profesor Albareda 1, E-18008 Granada, Spain Abstract The acylation step of the b-lactam hydrolysis by a class A b-lactamase has been modeled by means of the quantum chemical PM3 method. A model of the catalytic site of the protein has been built using two segments of three and four amino acids, respectively, which include four of the most important residues of the enzyme. The mechanism is based on that proposed by Strynadka et al. in 1992 (Strynadka et al., Nature 359 (1992) 700–705). The results confirm that Lys-73 is expected to be unprotonated in the wild-type enzyme, and therefore acts as a general base in the acylation step of the reaction. Previous contradictory results are discussed, and a new proton transfer pathway from Lys-73 to b-lactam nitrogen is proposed.  2000 Elsevier Science B.V. All rights reserved. Keywords: b-Lactam antibiotics; PBP’s interaction with the b-lactam antibiotics; Quantum chemical calculations 1. Introduction b-Lactam antibiotics: penicillins and cephalos- porins, are clinically important drugs, which are used as effective agents against bacterial diseases. b-Lactam antibiotics inhibit bacterial cell wall synthesis, by forming an irreversible adduct with the necessary enzymes: peptidoglycan transpeptidases and d-alanyl-d-alanine carboxypeptidases [1–3] (protein binding penicillins: PBPs). However, b-lactam antibotics also selectively form an irreversible adduct with some bacterial enzymes, i.e. b-lactamases.The clinically useful drugs are destroyed by b-lactamases. Indeed b-lactamases present some similarities with peptidoglycan transpeptidases and d-alanyl-d-alanine carboxypeptidases. Class A b-lactamases are serine hydrolases (serine proteases) produced by both Gram-positive and Gram-negative bacteria. The active site of b-lactamases and their inter- actions with the targeted antibiotics and inhibitors have been the subject of many experimental and theoretical papers [4–42]. The essential experimental tools were X-ray diffraction and kinetic enzymatic analysis combined with directed site mutagenesis. By X-ray diffraction of 1.7 A ˚ resolution, Strynadka et al. [4] studied the crystal structure of the penicillin G molecular complex with a deacylation-defective mutant of class A (RTEM1) b-lactamase from Escherichia coli. These authors proposed a reaction mechanism, in which the Lys-73 amine group acts as a general base, transferring a proton from Ser-70 to the b-lactam nitrogen using Ser-130 as an intermediate. In class C b-lactamases, Lys-67 would play the role of Lys-73. This reaction is not generally accepted in Journal of Molecular Structure (Theochem) 504 (2000) 13–28 0166-1280/00/$ - see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0166-1280(00)00351-1 www.elsevier.nl/locate/theochem * Corresponding author. E-mail address: smeyers@iem.cfmac.csic.es (Y.G. Smeyers).