title: Beta-lactamase-catalyzed aminolysis of depsipeptides: peptide inhibition and a new kinetic mechanism.
abstract: The aminolysis of the depsipeptide m-[[(phenylacetyl)glycyl]oxy]benzoic acid (1) by D-phenylalanine, catalyzed by the beta-lactamase of Enterobacter cloacae P99, is inhibited by the product of the reaction, (phenylacetyl)glycyl-D-phenylalanine (2), by the peptide analogue of 1, m-[(phenylacetyl)-glycinamido]benzoic acid (3), and by (3-dansylamidophenyl)boronic acid. Analysis of the steady-state kinetics of the effect of 2 and 3 on the reaction indicated that both a competitive binding mode and a noncompetitive binding mode existed for each peptide. Thus, there probably are two distinct binding sites (sites 1 and 2) that 2 and 3, and by implication 1, are able to simultaneously occupy on the enzyme surface. Given this information, it was possible to devise a new kinetic mechanism for the aminolysis reaction which yielded the experimentally observed empirical rate equation [Pazhanisamy, S., Govardhan, C. P., & Pratt, R. F. (1989) Biochemistry (first of three papers in this issue)] but did not involve initial binding of D-phenylalanine to the free enzyme, which has been shown not to occur [Pazhanisamy, S., & Pratt, R. F. (1989) Biochemistry (second of three papers in this issue)]. The mechanism requires two different 1:1 enzyme/1 complexes, only one of which leads to the hydrolysis and aminolysis reactions (1 in site 1), and a 1:2 enzyme/1 complex (1 in both sites), which leads only to hydrolysis. The dansyl boronate inhibits by binding competitively with 1 in site 1. It is suggested that this scheme also applies to the analogous transpeptidase reactions of small model peptides catalyzed by the bacterial cell wall DD-peptidases, where similar steady-state kinetics have been observed.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, Wesleyan University, Middletown, Connecticut 06457.
authors.forename: S
authors.initials: S
authors.lastname: Pazhanisamy
authors.forename: R F
authors.initials: RF
authors.lastname: Pratt
mesh_terms: D001665:Binding Sites; D004754:Enterobacter / Q000201:enzymology*; D004755:Enterobacteriaceae / Q000201:enzymology*; D005998:Glycine / Q000378:metabolism / Q000031:analogs & derivatives; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010455:Peptides / Q000494:pharmacology; D010648:Phenylacetates / Q000378:metabolism*; D011485:Protein Binding; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D010455:Peptides; D010648:Phenylacetates; D065093:beta-Lactamase Inhibitors; C061747:3-(((phenylacetyl)glycyl)oxy)benzoic acid; D001618:beta-Lactamases; D005998:Glycine