title: The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase.
abstract: Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of beta-lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active-site phenylalanines. Mutation of alphaF146, betaF24 and betaF57 to Tyr, Trp, Ala or Leu yielded mutants that were still capable of hydrolysing the chromogenic substrate 2-nitro-5-[(phenylacetyl)amino]-benzoic acid. Mutations on positions alphaF146 and betaF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40-fold decrease for alphaF146Y and alphaF146W to a threefold increase for alphaF146L and betaF24A, using 6-aminopenicillanic acid as the nucleophile. Further analysis of the betaF24A mutant showed that it had specificity constants (kcat/Km) for p-hydroxyphenylglycine methyl ester and phenylglycine methyl ester that were similar to the wild-type values, whereas the specificity constants for p-hydroxyphenylglycine amide and phenylglycine amide had decreased 10-fold, due to a decreased kcat value. A low amidase activity was also observed for the semisynthetic penicillins amoxicillin and ampicillin and the cephalosporins cefadroxil and cephalexin, for which the kcat values were fivefold to 10-fold lower than the wild-type values. The reduced specificity for the product and the high initial transferase/hydrolase ratio of betaF24A resulted in high yields in acyl transfer reactions.
authors: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, the Netherlands.
authors: Wynand B L
authors: WB
authors: Alkema
authors: Anne-Jan
authors: AJ
authors: Dijkhuis
authors: Erik
authors: E
authors: De Vries
authors: Dick B
authors: DB
authors: Janssen
mesh_terms: D062365:Aminobenzoates / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D007769:Lactams; D016297:Mutagenesis, Site-Directed; D009579:Nitrobenzoates / Q000378:metabolism; D010399:Penicillin Amidase / Q000737:chemistry* / Q000378:metabolism; D010648:Phenylacetates / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D062365:Aminobenzoates; D000900:Anti-Bacterial Agents; D007769:Lactams; D009579:Nitrobenzoates; D010648:Phenylacetates; D010649:Phenylalanine; C008641:6-nitro-3-phenylacetamidobenzoic acid; D010399:Penicillin Amidase; C025136:phenylacetic acid