title: Identification of Tyr241 as a key catalytic base in the family 4 glycoside hydrolase BglT from Thermotoga maritima.
abstract: While the vast majority of glycosidases catalyze glycoside hydrolysis via oxocarbenium ion-like transition states and typically employ carboxylic acid residues as acid/base or nucleophile catalysts, two subfamilies of these enzymes (GH4 and GH109 in the CAZY classification) conduct hydrolysis via a redox-assisted mechanism involving anionic transition states. While good evidence of this mechanism has been obtained, the identities of the catalytic residues involved have not yet been confirmed. Mechanistic analyses of mutants of the 6-phospho-β-glucosidase from Thermotoga maritima (BglT), in which the active site tyrosine residue (Tyr 241) has been replaced with Phe and Ala, provide support for its role as a catalytic base. The pH dependence of k(cat) and k(cat)/K(m), particularly of the acidic limb corresponding to the base, is shifted relative to that of the wild-type enzyme. Kinetic isotope effects for hydrolysis of substrates deuterated at C1, C2, and C3 by the Tyr 241 mutants are strongly pH-dependent, with essentially full primary kinetic isotope effects being observed for the 2-deutero substrate at low pH with the Tyr241Ala mutant. This is consistent with a slowing of the deprotonation step upon removal of the base.
authors.affiliation: 2036 Main Mall, Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Vivian L Y
authors.initials: VL
authors.lastname: Yip
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D019943:Amino Acid Substitution; D002384:Catalysis; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
chemical_list: D009243:NAD; D014443:Tyrosine; D005959:Glucosidases; C066586:6-phospho-beta-glucosidase