The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase

Wynand B L Alkema, Anne-Jan Dijkhuis, Erik De Vries, Dick B Janssen

Research output: Contribution to journalArticleAcademicpeer-review

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.

Original languageEnglish
Pages (from-to)2093-2100
JournalEuropean journal of biochemistry
Volume269
Issue number8
DOIs
Publication statusPublished - 9 Apr 2002
Externally publishedYes

Keywords

  • aminobenzoates/metabolism
  • anti-bacterial agents/chemistry
  • binding sites
  • escherichia coli/enzymology
  • kinetics
  • lactams
  • mutagenesis, site-directed/methods
  • nitrobenzoates/metabolism
  • penicillin amidase/chemistry
  • phenylacetates/metabolism
  • phenylalanine/genetics
  • substrate specificity

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