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| - | [[Image:1h2g.gif|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_1h2g| PDB=1h2g | SCENE= }} | | {{STRUCTURE_1h2g| PDB=1h2g | SCENE= }} |
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| - | '''ALTERED SUBSTRATE SPECIFICITY MUTANT OF PENICILLIN ACYLASE'''
| + | ===ALTERED SUBSTRATE SPECIFICITY MUTANT OF PENICILLIN ACYLASE=== |
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| - | ==Overview==
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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 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.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_12511194}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 12511194 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_12511194}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Hydrolase]] | | [[Category: Hydrolase]] |
| | [[Category: Zymogen]] | | [[Category: Zymogen]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 18:20:30 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 1 06:30:50 2008'' |
Revision as of 03:30, 1 July 2008
Template:STRUCTURE 1h2g
ALTERED SUBSTRATE SPECIFICITY MUTANT OF PENICILLIN ACYLASE
Template:ABSTRACT PUBMED 12511194
About this Structure
1H2G is a Protein complex structure of sequences from Escherichia coli. 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 Tue Jul 1 06:30:50 2008
