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

• Published in: Protein engineering, design and selection Vol. 17; no. 5; pp. 473 - 480
• Main Authors: Alkema, Wynand B.L., Hensgens, Charles M.H., Snijder, Harm J., Keizer, Evelien, Dijkstra, Bauke W., Janssen, Dick B.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-05-2004; Oxford Publishing Limited (England)
• Subjects: 3D structure; Catalytic Domain - genetics; Catalytic Domain - physiology; Crystallization; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Kinetics; ligand binding; Ligands; mutants; Mutation; penicillin acylase; Penicillin Amidase - chemistry; Penicillin Amidase - genetics; Penicillin Amidase - metabolism; Phenylacetates - metabolism; Protein Binding; Protein Conformation; 3D structure; kinetics; mutants; ligand binding; penicillin acylase
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzh057

Penicillin acylase catalyses the condensation of Cα-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-methylphenylacetic 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.