Characterization of a Chromogenic Substrate and Acyl Acceptor Design:Transpeptidation Reactions of a Specific Substrate Catalyzed by the Streptomyces R61 DD-Peptidase

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 (kappa_(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.