title: Transpeptidation reactions of a specific substrate catalyzed by the streptomyces R61 DD-peptidase: characterization of a chromogenic substrate and acyl acceptor design.
abstract: 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.
authors.affiliation: Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
authors.forename: Ish
authors.initials: I
authors.lastname: Kumar
authors.forename: R F
authors.initials: RF
authors.lastname: Pratt
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D002863:Chromogenic Compounds / Q000138:chemical synthesis*; D004151:Dipeptides / Q000138:chemical synthesis; D004952:Esters; D018716:Molecular Mimicry; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D002863:Chromogenic Compounds; D004151:Dipeptides; D004952:Esters; D009842:Oligopeptides; D013438:Sulfhydryl Compounds; C493614:glycyl-L-alpha-amino-epsilon-pimelyl-D-alanyl-D-alanine; C061038:glycyl-alanyl-phenylalanine; C029660:glycylphenylalanine; C015905:glycylleucine; C065992:N-glycylalanine; D010458:Peptidyl Transferases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase