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

Eur J Biochem. 2002 Apr;269(8):2093-100. doi: 10.1046/j.1432-1033.2002.02857.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminobenzoates / metabolism
  • Anti-Bacterial Agents / chemistry
  • Anti-Bacterial Agents / metabolism
  • Binding Sites
  • Escherichia coli / enzymology
  • Kinetics
  • Lactams
  • Mutagenesis, Site-Directed
  • Nitrobenzoates / metabolism
  • Penicillin Amidase / chemistry*
  • Penicillin Amidase / metabolism
  • Phenylacetates / metabolism
  • Phenylalanine / genetics
  • Substrate Specificity

Substances

  • Aminobenzoates
  • Anti-Bacterial Agents
  • Lactams
  • Nitrobenzoates
  • Phenylacetates
  • Phenylalanine
  • 6-nitro-3-phenylacetamidobenzoic acid
  • Penicillin Amidase
  • phenylacetic acid