Lack of relevance of kinetic parameters for exocellular DD-peptidases to cephalosporin MICs

Abstract

MICs of a set of cephalosporins against a variety of gram-positive and gram-negative pathogens showed no strong correlations with the rate at which these inhibitors acylate or are deacylated by P-lactam-sensitive DD-peptidases excreted by Streptomyces sp. strain R61 and Actinomadura sp. strain R39.Detailed biochemical investigations of the 3-lactamsensitive enzymes with D-alanyl-D-alanine transpeptidase and carboxypeptidase activities date back over 10 years and have been comprehensively reviewed (16-23). The R61 and R39 DD-peptidases, which are excreted by Streptomyces and Actinomadura spp., respectively, have been advanced as models for membrane-bound, P-lactam-sensitive proteins.The model DD-peptidases catalyze transpeptidation and hydrolysis reactions of peptides like Ac2-L-Lys-D-Ala-D-Ala which are analogs of the natural pentapeptide substrates of the enzymes that regulate peptidoglycan biosynthesis. In addition, the model enzymes are inhibited to varying degrees by a variety of P-lactams, including penicillins, cephalosporins, penems, carbapenems, and monobactams (17,22).There is accumulating evidence that the DD-peptidases bind covalently to ,-lactam antibiotics, and possibly also the substrate analogs, through a serine in the active site (17,22). This is a feature which they share in common with membrane-bound penicillin-binding proteins (PBPs) and with many ,B-lactamases (49). The DD-peptidases have been assumed to interact with ,B-lactams via the kinetic modelwhere K is the dissociation constant of the reversible complex, k2 is the first-order rate constant for covalent Even the very early research (18) with the DD-peptidases showed that cephalosporin C, a clinically unimportant compound, was one of the most potent, long-acting inhibitors, whereas ampicillin, a widely used therapeutic agent, was relatively ineffective in those studies. Recently, it was concluded that the exocellular enzymes are not the killing targets in the R61 and R39 strains (17). The sensitivity profiles of the purified enzymes to an assortment of ,B-lactam antibiotics were not related to those of the organisms that generated the enzymes.Prior to the latest publications (17, 22), attempts to make in-depth, quantitative comparisons of biochemical and microbiological data have been thwarted by lack of sufficient kinetic data on a structurally related set of ,3-lactam antibiotics. Structure-activity relationships are most likely to be discernible and meaningful when closely related chemical structures are compared. The longest series of compounds for which there are now in vitro kinetic data are A3-cephalosporins. There are 17 in this group. However, of these, only six are clinically useful ones, the rest being experimental compounds about which much less is known microbiologically.It is the purpose of this paper to make use of available biochemical and microbiological data to determine if relationships between them exist. That none are found calls attention to the need to study enzymes more relevant to the mode of action of ,B-lact...