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

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 Cys^B71 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 2Å resolution, shows a flip of the side chain of Phe^B256 into the periphery of the catalytic centre, associated with loss of the π-stacking interactions between Phe^B256 and Phe^B71. Molecular modelling demonstrates that glutaryl-l-leucine may bind with the uncharged glutaryl group in the S₁ 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.