Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase

Abstract

Penicillin acylase catalyses the condensation of Ca-substituted phenylacetic acids with β-lactam nucleophiles, producing semi-synthetic β-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for Cα-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, αF146Y, βF24A and αF146Y/βF24A, which all had a 2- to 10-fold higher affinity for Cα-substituted compounds than wild-type enzyme. In addition, βF24A had a 20-fold reduced affinity for phenylacetic acid. The molecular basis of the improved properties was investigated by X-ray crystallography. These studies showed that the higher affinity of αF146Y for (R)-α-methylphenyl acetic acid can be explained by van der Waals interactions between αY146:OH and the Cα-substituent. The βF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-α-methylphenylacetic acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the βF24A structure with other open conformations of penicillin acylase showed that βF24 has a fixed position, whereas αF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.