1wcq: Difference between revisions

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MUTAGENESIS OF THE NUCLEOPHILIC TYROSINE IN A BACTERIAL SIALIDASE TO PHENYLALANINE.

OverviewOverview

Mutants of the Micromonospora viridifaciens sialidase, Y370E and Y370F, are catalytically active retaining enzymes that operate by different, mechanisms. Previous substitutions with smaller amino acids, including, Y370D, yielded inverting sialidases. At least one water molecule can fit, into the active-site cavity of this mutant and act as a nucleophile from, the face opposite the leaving group (Biochemistry 2003, 42, 12 682). Thus, addition of a CH(2) unit (Asp versus Glu) changes the mechanism from, inversion back to retention of configuration. Based on Bronsted beta(lg), values, it is proposed that the Y370E mutant reacts by a, double-displacement mechanism (beta(lg) on k(cat)/K(m) -0.36+/-0.04) with, Glu370 acting as the nucleophile. However, the Y370F mutant (beta(lg) on, k(cat)/K(m) -0.79+/-0.12) reacts via a dissociative transition state. The, crystal structure of the Y370F mutant complexed with, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid shows no significant, active-site perturbation relative to the wild-type enzyme.

About this StructureAbout this Structure

1WCQ is a Single protein structure of sequence from Micromonospora viridifaciens with NA, DAN and GOL as ligands. Active as Exo-alpha-sialidase, with EC number 3.2.1.18 Structure known Active Site: AC1. Full crystallographic information is available from OCA.

ReferenceReference

Two nucleophilic mutants of the Micromonospora viridifaciens sialidase operate with retention of configuration by two different mechanisms., Watson JN, Newstead S, Narine AA, Taylor G, Bennet AJ, Chembiochem. 2005 Nov;6(11):1999-2004. PMID:16206228

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