title: Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase.
abstract: Penicillin acylase catalyses the condensation of Calpha-substituted phenylacetic acids with beta-lactam nucleophiles, producing semi-synthetic beta-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for Calpha-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, alphaF146Y, betaF24A and alphaF146Y/betaF24A, which all had a 2- to 10-fold higher affinity for Calpha-substituted compounds than wild-type enzyme. In addition, betaF24A 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 alphaF146Y for (R)-alpha-methylphenylacetic acid can be explained by van der Waals interactions between alphaY146:OH and the Calpha-substituent. The betaF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-alpha-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 betaF24A structure with other open conformations of penicillin acylase showed that betaF24 has a fixed position, whereas alphaF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, The Netherlands.
authors.forename: Wynand B L
authors.initials: WB
authors.lastname: Alkema
authors.forename: Charles M H
authors.initials: CM
authors.lastname: Hensgens
authors.forename: Harm J
authors.initials: HJ
authors.lastname: Snijder
authors.forename: Evelien
authors.initials: E
authors.lastname: Keizer
authors.forename: Bauke W
authors.initials: BW
authors.lastname: Dijkstra
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
mesh_terms: D020134:Catalytic Domain / Q000235:genetics* / Q000502:physiology; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010648:Phenylacetates / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D008024:Ligands; D010648:Phenylacetates; D010399:Penicillin Amidase; C025136:phenylacetic acid