Inactivation of BacterialDD-Peptidase by â-Sultams †

N-Acyl-â-sultams are time-dependent, irreversible active site-directed inhibitors of Streptomyces R61 DD-peptidase. The rate of inactivation is first order with respect to â-sultam concentration, and the second-order rate constants show a dependence on pH similar to that for the hydrolysis of a substrate. Inactivation is due to the formation of a stable 1:1 enzyme-inhibitor complex as a result of the active site serine being sulfonylated by the â-sultam as shown by ESI-MS analysis and by X-ray crystallography. A striking feature of the sulfonyl enzyme is that the inhibitor is not bound to the oxyanion hole but interacts extensively with the “roof” of the active site where the Arg 285 is located. D-Alanyl-D-alanine carboxypeptidase/transpeptidases [DDpeptidases and also known as penicillin-binding proteins, or PBPs (1)] are enzymes produced by bacteria that catalyze the final steps of their cell wall biosynthesis (1, 2). Bacteria, with a very few exceptions, have a cross-linked cell wall, the formation of which involves a transpeptidation reaction that is catalyzed by bacterial transpeptidases/PBPs. The PBPs have been classified into two groups: the low molecular mass (LMM) PBPs and the high molecular mass (HMM) PBPs (3). The low molecular mass group includes â-lactamases that catalyze the hydrolysis of â-lactam antibiotics, the D-AlaD-Ala carboxypeptidases, and endopeptidases. The HMM PBPs catalyze the transpeptidation reaction that cross-links the glycan strands of the cell wall (3, 4). The DD-peptidases are the inhibition targets of â-lactam antibiotics, such as penicillin and cephalosporins. These enzymes have an active site serine residue that is acylated by the D-Ala-D-Ala natural peptide substrate as an intermediate stage of the acyl transfer reaction (5) (Scheme 1). The â-lactam antibiotics act as mechanism-based inhibitors for the PBPs by acting as substrates for nucleophilic attack by the active site serine causing C-N bond fission and ring opening to form stable acyl-enzyme intermediates that are then not susceptible to further nucleophilic attack by exogenous amines or water (Scheme 2). Deacylation is very slow because the leaving amino group remains covalently attached to the acyl-enzyme and presumably blocks nucleophilic attack by the amino group of the acceptor or by water (6). Bacterial resistance to â-lactam antibiotics has emerged primarily through two mechanisms: the production of â-lactam degrading enzymes, the â-lactamases, and the accumulation of mutations in their HMM PBP targets which render them less susceptible to inhibition by â-lactam antibiotics. The most important defense mechanism is the production of â-lactamases [thought to be evolutionary descendants of DD-peptidases (3-7)]. Some of the â-lactamases are serine enzymes and also catalyze the hydrolysis of â-lactams through the formation of an acyl-enzyme but, in this case, to give a short-lived intermediate that is quickly hydrolyzed before the â-lactams can reach their transpeptidase or DD-peptidase targets. The DD-peptidase of Streptomyces R61 is secreted in the extracellular medium as a soluble protein and catalyzes the cleavage of the C-terminal D-alanine of simple synthetic peptides. In the presence of acceptor compounds having a suitably located amino group, they also perform transpeptidation reactions (Scheme 1) (8, 9). The specificity of the enzymes for the acceptors reflects the structure of the bacteria’s peptidoglycan cross-bridges, and for Streptomyces R61 DD-peptidase, dipeptides, such as Gly-L-Ala, are good acceptors. The R61 enzyme is a 37.5 kDa enzyme (EC 3.4.16.4) that has 349 residues and has been used extensively as a kinetic and structural model for PBPs (9, 10). The use of ester and thiolester substrates for the DD-peptidases † This research was supported by the University of Huddersfield. * To whom correspondence should be addressed: phone, +1484 472169; fax, +1484 472182; e-mail, m.i. page@hud.ac. uk. ‡ The University of Huddersfield. § Institut de Chimie B6. | University of Connecticut. Scheme 1 Scheme 2 7738 Biochemistry 2005, 44, 7738-7746 10.1021/bi050110o CCC: $30.25 © 2005 American Chemical Society Published on Web 05/07/2005 represented a significant advance in the study of their reactions. The study of thioesters as substrates can be followed by spectrophotometry, allowing a continuous monitoring of enzyme activity. Moreover, with the thioester substrates, deacylation is rate-limiting with several enzymes, so that the complete catalytic pathway could be more easily studied (11). In this study N-benzoyl-D-Ala-thiolactate (1) and N-benzoyl-D-Ala-thioglycolate (2) (see Chart 1) were synthesized and used as substrates to monitor the activity of