1h2g
From Proteopedia
| Line 5: | Line 5: | ||
==Overview== | ==Overview== | ||
| - | 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 .. | + | 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 2 A, 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. |
==About this Structure== | ==About this Structure== | ||
| - | 1H2G is a | + | 1H2G is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with CA and EDO as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Penicillin_amidase Penicillin amidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.1.11 3.5.1.11] Structure known Active Site: CA1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1H2G OCA]. |
==Reference== | ==Reference== | ||
| Line 29: | Line 29: | ||
[[Category: zymogen]] | [[Category: zymogen]] | ||
| - | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 5 13:57:10 2007'' |
Revision as of 11:51, 5 November 2007
|
ALTERED SUBSTRATE SPECIFICITY MUTANT OF PENICILLIN ACYLASE
Overview
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 2 A, 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.
About this Structure
1H2G is a Protein complex structure of sequences from Escherichia coli with CA and EDO as ligands. Active as Penicillin amidase, with EC number 3.5.1.11 Structure known Active Site: CA1. Full crystallographic information is available from OCA.
Reference
Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding., Morillas M, McVey CE, Brannigan JA, Ladurner AG, Forney LJ, Virden R, Biochem J. 2003 Apr 1;371(Pt 1):143-50. PMID:12511194
Page seeded by OCA on Mon Nov 5 13:57:10 2007
