Active-site-serine D-alanyl-D-alanine-cleaving-peptidase-catalysed acyl-transfer reactions. Procedures for studying the penicillin-binding proteins of bacterial plasma membranes

• Published in: Biochemical journal 1986-04, Vol.235 (1), p.159-165
• Main Authors: Ghuysen, J M, Frère, J M, Leyh-Bouille, M, Nguyen-Distèche, M, Coyette, J
• Format: Article
• Language: English
• Subjects: active sites; Acylation; Anti-Bacterial Agents - metabolism; bacteria; Bacterial Proteins; Binding Sites; Biochemistry, biophysics & molecular biology; Biochimie, biophysique & biologie moléculaire; Carboxypeptidases - metabolism; Carrier Proteins - metabolism; Cell Membrane - metabolism; Hexosyltransferases; Kinetics; Lactams; Life sciences; Microbiologie; Microbiology; Muramoylpentapeptide Carboxypeptidase - metabolism; penicillin-binding protein; Penicillin-Binding Proteins; Penicillins - metabolism; Peptidyl Transferases; plasma membranes; proteinase; Sciences du vivant; Serine-Type D-Ala-D-Ala Carboxypeptidase; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0264-6021; 1470-8728; 1470-8728
• DOI: 10.1042/bj2350159

Under certain conditions, the values of the parameters that govern the interactions between the active-site-serine D-alanyl-D-alanine-cleaving peptidases and both carbonyl-donor substrates and beta-lactam suicide substrates can be determined on the basis of the amounts of (serine ester-linked) acyl-protein formed during the reactions. Expressing the 'affinity' of a beta-lactam compound for a DD-peptidase in terms of second-order rate constant of enzyme acylation and first-order rate constant of acyl-enzyme breakdown rests upon specific features of the interaction (at a given temperature) and permits study of structure-activity relationships, analysis of the mechanism of intrinsic resistance and use of a 'specificity index' to define the capacity of a beta-lactam compound of discriminating between various sensitive enzymes. From knowledge of the first-order rate constant of acyl-enzyme breakdown and the given time of incubation, the beta-lactam compound concentrations that are necessary to achieve given extents of DD-peptidase inactivation can be converted into the second-order rate constant of enzyme acylation. The principles thus developed can be applied to the study of the multiple penicillin-binding proteins that occur in the plasma membranes of bacteria.