title: Two nucleophilic mutants of the Micromonospora viridifaciens sialidase operate with retention of configuration by two different mechanisms.
abstract: 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 Brønsted 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.
authors.affiliation: Department of Chemistry, Simon Fraser University, University Drive, Burnaby, BC, Canada.
authors.forename: Jacqueline N
authors.initials: JN
authors.lastname: Watson
authors.forename: Simon
authors.initials: S
authors.lastname: Newstead
authors.forename: Arun A
authors.initials: AA
authors.lastname: Narine
authors.forename: Garry
authors.initials: G
authors.lastname: Taylor
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bennet
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D007700:Kinetics; D008847:Micromonospora / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D015394:Molecular Structure; D016296:Mutagenesis; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D014867:Water / Q000737:chemistry
chemical_list: D014867:Water; D009439:Neuraminidase