1gm8: Difference between revisions

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.

1GM8 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 SOX 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=1GM8 OCA].  

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