1k5q
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PENICILLIN ACYLASE, MUTANT COMPLEXED WITH PAA
OverviewOverview
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.
About this StructureAbout this Structure
1K5Q is a Protein complex structure of sequences from Escherichia coli with CA and PAC as ligands. Active as Penicillin amidase, with EC number 3.5.1.11 Full crystallographic information is available from OCA.
ReferenceReference
Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase., Alkema WB, Hensgens CM, Snijder HJ, Keizer E, Dijkstra BW, Janssen DB, Protein Eng Des Sel. 2004 May;17(5):473-80. Epub 2004 Jul 14. PMID:15254299
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