Molecular Modeling of Henry ± Michaelis and Acyl±Enzyme Complexes between Imipenem and Enterobacter cloacae P99 b-Lactamase by Cristina Fenollar-Ferrer, Josefa Donoso*, Juan Frau, and Francisco Mun Äoz Instituto Universitario de Investigacio  n en Ciencias de la Salud (IUNICS), Departamento de Química, Universidad de las Islas Baleares, Ctra. de Vallemossa km 7.5, 07122 Palma de Mallorca, Spain (e-mail: josefa.donoso@uib.es) We report a molecular-mechanics (AMBER*) study on the Henry±Michaelis complex and the corresponding acyl ± enzyme adduct formed between imipenem (1), a transient inhibitor of b-lactamases, and Enterobactercloacae P99, a class C-b-lactamase. We have examined the influence of the structural configuration of the functional groups in the substrate on their three-dimensional (3D) arrangement at the active site, which was compared with those adopted by typical penicillins and cephalosporins. Our results confirm that the carboxy group of the antibiotic plays a prominent role in the binding of the substrate to the active site, and that it activates Ser 64 through interaction with the phenolic OH group of Tyr 150 . The binding of imipenem to E.cloacae P99 increases the distance between Tyr 150 and Ser 64 due to the presence of a hydrophobic Me group in the ( R)-1- hydroxyethyl substituent at C(6). This, together with the 3D arrangement of its carboxy group, leads to an interaction with the active site in a manner that hinders H  exchange between the nucleophile in Ser 64 and its basic activator, the phenolic group of Tyr 150 . 1. Introduction. ± b-Lactams are still our main general defense against bacterial infections. Nevertheless, their effectiveness is being seriously threatened by expression of b-lactamases, the most-common cause of bacteria resistence to b-lactam antibiotics [1±3]. b-Lactamases (E.C. 3.5.2.6) are widespread bacterial enzymes that effectively cleave and inactivate b-lactam antibiotics by catalyzing the hydrolysis of the amido group of the b-lactam ring. They can be split into four different classes (A ± D) according to the homology of their primary amino acid sequence [4] . Except for class-B b-lactamases, which belong to metalo-enzymes, these compounds have serine in their active sites, participating in the formation of acyl ± enzyme intermediates with the b- lactam [2]. These serine enzymes, together with penicillin-binding proteins (PBPs), form the so-called penicilloyl serine transferase superfamily [5]. In recent years, several clarifying articles have been published on b-lactamases, PBPs, their kinships and evolutive relationships [6 ± 10]. Class-C b-lactamases are the second-most-common class of b-lactamase hydro- lyzing enzymes. They are most commonly chromosomally encoded in Gram-negative bacteria, and are inducible [6]. Class-C b-lactamases hydrolyze cephalosporins very efficiently. Hence, redepression of the chromosomal genes leads to class-C enzyme production and to resistance to most cephalosporins, including extended-spectrum cephalosporins, cefepime, cefpirome, and a-metoxy-b-lactams such as cefoxitin [11] [12]. The discovery of plasmid-encoded class-C b-lactamases in several Gram- negative species [13 ± 15] has given rise to concern about the usefulness of b-lactam CHEMISTRY & BIODIVERSITY ± Vol. 2 (2005) 645  2005 Verlag Helvetica Chimica Acta AG, Zürich