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Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding

Morillas, M, McVey, C E, Brannigan, J A, Ladurner, A G, Forney, L J and Virden, R (2003) Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding. Biochemical Journal. pp. 143-150. ISSN 1470-8728

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Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k(cat)/K-m values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K-s. Values of k(cat/)K(m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k(cat)/K-m for the CySB71 mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 X resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S-1 subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity.

Item Type: Article
Copyright, Publisher and Additional Information: © 2003 Biochemical Society. Mounted on the Internet with permission from the Biochemical Society 2003.
Institution: The University of York
Academic Units: The University of York > Chemistry (York)
Depositing User: Sherpa Assistant
Date Deposited: 24 Jun 2005
Last Modified: 05 Apr 2016 22:14
Status: Published
Refereed: Yes
URI: http://eprints.whiterose.ac.uk/id/eprint/463

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