Glycopeptide transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reactions.

Abstract

In 1929, Fleming discovered penicillins and observed that this group of substances kills gram-positive bacteria more effectively than gram-negative bacteria (thus implying some important physiological difference between the two groups of organisms). I Subsequent knowledge of the bacterial cell wall made it possible to establish both on physiological and chemical grounds that penicillins are specific and highly selective inhibitors of the biosynthesis of cell walls, both in gram-positive and in gram-negative bacteria.2-6 The reason for the relative insensitivity of gramnegative bacteria to most penicillins has remained obscure. More recent knowledge of the structure and biosynthesis of bacterial cell walls has suggested that, in a terminal reaction in cell wall synthesis, linear glycopeptide strands are cross-linked in a transpeptidation, accompanied by release of the terminal D-alanine of the pentapeptide precursor, with formationi of a two- or threedimensional network. Direct chemical analyses of cell walls prepared from cells treated with penicillinll 8 and isotopic studies of wall biosynthesis9' 10 suggested that penicillin was interfering with this hypothetical g]ycopeptide cross-linking reaction. In the presence of penicillin, nascent glycopeptide, an uncross-linked mlonomeric uniit of the wall, accumulated.8 Pulse-labeling experiments have established that this nascent unit is an immediate precursor of the final cross-linked glycopeptide; penicillin completely inhibited its integration into the iietwork.10 Moreover, molecular models of penicillin resembled the acyl-D-alanyl-D-alanine in the ]inear glycopeptide and it was possible to suggest a molecular mlechanism for the transpeptidation and its inhibition by penicillin.8 However, the actual reaction or reactions inhibited had not been demoonstrated. In this paper we wish to report that a cell-free enzymatic system has been obtained from ce]ls of Escherichia coli which catalyzes this cross-linking reactiorn. We call the enzyme which catalyzes this step glycopeptide transpeptidase, since it catalyzes a reaction in which the peptide chains of two linear glycopeptide strands are cross-linked by a transpeptidation, in which the terminal D-alanine residue of one of the strands is elimirnated; this peptide bond synthesis proceeds without any other source of energy and is reversible. The terminal D-alanine residue of the other strand is also removed by hydrolysis catalyzed by a D-alanine carboxypeptidase in the preparation (Fig. 1). Both of these reactions are inhibited by low levels of penici]lin G, other penicillins, and a cephalosporin. Materials and Methods.-Preparation of substrates: UDP-MtirNAc L-ala -D-glu H3-mesoDAP D-ala D-ala and UDP-MurNAc L-ala- D-glu meso-DAP C14-D-ala C14-D-ala were prepared enzymatically by sequential addition of amino acids to the appropriate uridine nucleotides. lOa UJDP-GlcNAc-C14 is the same preparation uised previously. Preparation of enzyme: Two particulate enzymes, obtained from cells of E. coli strain Y-1O or E. coli strain B, were employed. The cells were ground with alumina. The fraction of the dis