D-Alanine carboxypeptidases of bacillus stearothermophilus: Solubilisation of particulate enzyme and mechanism of action of penicillin

Abstract

Two D-alanine carboxypeptidases have been found to be present in protoplast membranes from Bacillus stearothermophilus. D-Alanine carboxypeptidase I removes the terminal D-alanine residue from the substrate UDP-MurNAc- L-Ala- D-Glu- meso-Dap- D-[ 14C]Ala- D-[ 14C]Ala; (MurNAc, N-acetylmuramyl; Dap, α, ε-diaminopimelic acid); D-alanine carboxypeptidase II removes the penultimate D-alanine residue. Both enzymes have been obtained in a soluble form by treatment of protoplast membranes with butan-1-ol at 20 °C. The properties of D-alanine carboxypeptidase I in the membrane-bound state have been studied. The enzyme is inactivated by penicillin G but the inactivation can be reversed by treatment with β-lactamase. Inhibition of enzyme by penicillin G shows hyperbolic competitive kinetics. Penicillin G does not appear to act as a substrate analogue and compete for the entire active site of the enzyme. Under certain conditions the penicillin sensitivity of D-alanine carboxypeptidase I is lost without concomitant loss of catalytic activity. It is suggested that the enzyme has a penicillin-binding site that is separate from the substrate-binding site, a suggestion which is strongly supported by kinetic evidence.