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

• DOI: 10.1046/j.1432-1033.2002.02857.x
• Publication Date: 2003-03-11T18:41:57Z
• Authors: Wynand B. L. Alkema; Anne‐Jan Dijkhuis; Erik de Vries; Dick B. Janssen
• Keywords: AMPICILLIN; Lactams; substrate specificity; Phenylalanine; transferase/hydrolase ratio; Substrate Specificity; 03 medical and health sciences; BINDING; Escherichia coli; Aminobenzoates; Phenylacetates; penicillin acylase; LIGATION; 0303 health sciences; Binding Sites; beta-lactam antibiotics; NUCLEOPHILE; Anti-Bacterial Agents; MODEL; Kinetics; ESCHERICHIA-COLI; Nitrobenzoates; Mutagenesis, Site-Directed; BETA-LACTAM ANTIBIOTICS; Penicillin Amidase; site-directed mutagenesis

Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of β‐lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active‐site phenylalanines. Mutation of αF146, βF24 and βF57 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 αF146 and βF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40‐fold decrease for αF146Y and αF146W to a threefold increase for αF146L and βF24A, using 6‐aminopenicillanic acid as the nucleophile. Further analysis of the βF24A 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 βF24A resulted in high yields in acyl transfer reactions.