Interactions between beta-Lactam Antibiotics and Isolated Membranes of Streptococcus faecalis ATCC 9790

Abstract

The DD-carboxypeptidase -exchange membrane-bound enzyme in Streptococcus faecalis ATCC 9790 reacts with p-lactam antibiotics to form complexes with rather long half-lives. Depending upon the antibiotic, the second-order rate constants for complex formation range from 0.75 -560 M-' s-l (at 37 "C and in water) and the first-order rate constants for complex breakdown range from 1.3 to 26 x lo-' s-' (at 37 "C and in 5 mM phosphate buffer pH 7.5). There are about 30 pmol of DD-carboxypeptidase -exchange enzyme per mg of membrane protein. The degradation products arising from benzylpenicillin are phenylacetylglycine and probably N-formyl-D-penicillamine. Isolated membranes also contain other penicillin binding sites (about 70 pmol/mg membrane protein). That part of benzylpenicillin which reacts with at least some of these latter sites is slowly degraded into penicilloic acid. Normal functioning of the DD-carboxypeptidase -exchange membrane-bound enzyme is important, if not essential, for cell growth. With the P-lactam antibiotics tested, inhibition of cell growth is mainly related to the rates of formation of the inactive enzyme-antibiotic complexes. The relationship, however, is not a direct one probably due to the competitive effect exerted by the other penicillin binding sites. Isolated membranes of Streptococcus faecalisATTC 9790 contain at least two activities that may constitute or at least be part of the peptide crosslinking enzyme system involved in cell wall peptidoglycan synthesis [l]: (a) a transfer activity that is revealed by the standard exchange reaction ACZ-L-LYS- alanine and which occurs at pH 10 and at pH 6. Previous studies [I] suggested that both hydrolysis and exchange reactions were catalysed by the same enzyme, or at least by two very closely related enzymes. They failed to prove, however, whether or not the above simple exchange reaction was a model of the transpeptidation reaction through which the nascent peptidoglycan undergoes peptide crosslinking. Experiments were therefore designed in order to Abbreviations. I D~o , concentration of antibiotic which inhibits the enzyme activity by 50%; [I],,,, minimal antibiotic concentration which prevented growth aftcr 18 h of incubation a1 37 C (stationary phase cultures).Enzyme. DD-Carboxypeptidase-exchange enzyme (EC 3.4.12.6).establish whether the hydrolysis and/or the exchange reaction, as they were revealed by the above standard reactions, were physiologically important and for this purpose, the nature and the mechanism of the interactions between the isolated membranes and several p-lactam antibiotics were investigated. MATERIALS AND METHODS Plasma MembranesThe techniques used to prepare the plasma membranes were those previously described [l, 21. Membrane suspensions were in water at 20 mg protein/ml. In the exchange reaction carried out in 50mM carbonate buffer pH 3 0, the K, (app) values for AcZ-L-Lys-D-Ala-D-Ala at infinite concentration of D-alanine and for D-alanine at infinite concentration of Ac2-L-Lys-~-Ala-~-Ala were virtually identica...

The Penicillin‐Binding Proteins in Streptococcus faecalis ATCC 9790

“…Saturation curves indicated that maximal binding of [14C]benzylpenicillin to growing cells (using the conditions described in Materials and Methods) and to isolated membranes (as described in [2] …”

“…linkage of the J-lactam ring [1,2]. Spontaneous breakdown (at pH 7.5) of the complexes formed between [14C]benzylpenicillin and the isolated membrane (thus involving all the penicillin-binding proteins present) also causes regeneration of the membrane-bound DD-carboxypeptidase activity [2].…”

“…Spontaneous breakdown (at pH 7.5) of the complexes formed between [14C]benzylpenicillin and the isolated membrane (thus involving all the penicillin-binding proteins present) also causes regeneration of the membrane-bound DD-carboxypeptidase activity [2]. At about 90 enzyme recovery, 39% of the total radioactivity is released in the form of phenylacetylglycine, 29 remains bound to the membranes, 3 0 x is released as a soluble material which is immobile on paper electrophoresis at pH 6.5, and 8 y( as benzylpenicilloate.…”