title: Chemical mechanism of beta-xylosidase from Trichoderma reesei QM 9414: pH-dependence of kinetic parameters.
abstract: The variation of kinetic parameters of beta-xylosidase from Trichoderma reesei QM 9414 with pH was used to elucidate the chemical mechanism of the p-nitrophenyl beta-D-xylopyranoside hydrolysis. The pH-dependence of V and V/K(m) showed that a group on the enzyme with a pK value of 3.20 must be unprotonated and a group with a pK value of 5.20 must be protonated for activity and both are involved in catalysis. Solvent-perturbation studies indicated that these groups are neutral acid type. Temperature dependence of kinetic parameters suggested the stickiness of the substrate at lower temperatures than the optimum and the calculated ionization enthalpies pointed to carboxyl groups as responsible for both pKs. Chemical modification with triethyloxonium tetrafluoroborate and protection with the substrate studies demonstrated essential carboxyl groups on the enzyme. Profiles of pK(i) for D-gluconic acid lactone indicated that a group with a pK value of 3.45 must be protonated for binding and it has been assigned to the carboxyl group of D-gluconic acid formed by lactone ring breakdown in solution.
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Gómez
authors.forename: P
authors.initials: P
authors.lastname: Isorna
authors.forename: M
authors.initials: M
authors.lastname: Rojo
authors.forename: P
authors.initials: P
authors.lastname: Estrada
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology; D005942:Gluconates / Q000494:pharmacology; D006027:Glycosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*; D014995:Xylosidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D005942:Gluconates; D006027:Glycosides; D007783:Lactones; C008646:4-nitrophenyl beta-D-xyloside; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C106477:glucono-1,4-lactone