Transpeptidation reactions of a specific substrate catalyzed by the Streptomyces R61 DD-peptidase: Characterization of a chromogenic substrate and acyl acceptor design

摘要

The Streptomyces R61 DD-peptidase, a functional model for penicillin-binding proteins, catalyzes the hydrolysis and aminolysis Of D-alanyl-D-alanine-terminating peptides by specific amines. In vivo, this reaction completes bacterial cell wall biosynthesis. For in vitro studies of this enzyme to date, various nonspecific acyl-donor substrates have been employed. Recently, however, a peptidoglycan-mimetic peptide substrate, glycyl-L-alpha-amino-epsilon-pimelyl-D-alanyl-D-alanine, has been described that is much more specific for this enzyme. In this paper, we describe the synthesis and kinetic characterization of an analogous thiolester substrate, 3-(N-glycyl-L-cysteinyl)-propanoyl-D-alanyl-D-thiolactate, that the enzyme hydrolyzes and aminolyzes very efficiently (k(cat)/K-m = 1.0 X 10(7) s(-1) M-1). Direct or indirect, by means of a thiol trap, spectrophotometric monitoring of the reactions of this substrate is readily achieved. Deacylation of the enzyme is rate-determining under substrate saturation conditions, and therefore the aminolysis reaction can be directly studied. The results show that D-amino acids and certain Gly-L-Xaa dipeptides and tripeptides may act as acyl acceptors at the active site of the enzyme. D-Phenylalanine and Gly-L-Phe were the most effective D-amino acid and dipeptide acceptors, respectively. On the basis of the dual specificity of the active site for acceptors (D-amino acids and Gly-L-Xaa peptides), "dual function" acceptors were designed and synthesized. Two of these, aminomalon-(N-ethyl)amide and aminomalon-(N-phenethyl)amide, were particularly effective. It did seem, however, that the observed rates of reaction of these very effective acceptors may be limited by some common, possibly physical, step. More extended, peptidoglycan-like, acceptors were found to be essentially unreactive. The reasons for this counterintuitive behavior are discussed.