1gm7: Difference between revisions
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==Overview== | ==Overview== | ||
The crystal structure of penicillin G acylase from Escherichia coli has | The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base. | ||
==About this Structure== | ==About this Structure== | ||
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[[Category: Penicillin amidase]] | [[Category: Penicillin amidase]] | ||
[[Category: Protein complex]] | [[Category: Protein complex]] | ||
[[Category: Brannigan, J | [[Category: Brannigan, J A.]] | ||
[[Category: Dodson, G | [[Category: Dodson, G G.]] | ||
[[Category: Mcvey, C | [[Category: Mcvey, C E.]] | ||
[[Category: Walsh, M | [[Category: Walsh, M A.]] | ||
[[Category: Wilson, K | [[Category: Wilson, K S.]] | ||
[[Category: CA]] | [[Category: CA]] | ||
[[Category: EDO]] | [[Category: EDO]] | ||
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[[Category: hydrolase]] | [[Category: hydrolase]] | ||
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Revision as of 13:51, 21 February 2008
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CRYSTAL STRUCTURES OF PENICILLIN ACYLASE ENZYME-SUBSTRATE COMPLEXES: STRUCTURAL INSIGHTS INTO THE CATALYTIC MECHANISM
OverviewOverview
The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base.
About this StructureAbout this Structure
1GM7 is a Protein complex structure of sequences from Escherichia coli with , and as ligands. Active as Penicillin amidase, with EC number 3.5.1.11 Known structural/functional Site: . Full crystallographic information is available from OCA.
ReferenceReference
Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism., McVey CE, Walsh MA, Dodson GG, Wilson KS, Brannigan JA, J Mol Biol. 2001 Oct 12;313(1):139-50. PMID:11601852
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