title: Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism β-xylosidases.
abstract: We obtained Cx1 from a commercial supplier, whose catalog listed it as a β-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein sequencing indicates Cx1 is similar to but not identical to β-xylosidases of GH52, including Q09LZ0, that have retaining mechanisms. Q09LZ0 β-xylosidase had been characterized biochemically in kinetic reactions that contained Tris. We overproduced Q09LZ0 and demonstrated that Tris is a competitive inhibitor of the β-xylosidase. Also, the previous work used grossly incorrect extinction coefficients for product 4-nitrophenol. We redetermined kinetic parameters using reactions that omitted Tris and using correct extinction coefficients for 4-nitrophenol. Cx1 and Q09LZ0 β-xylosidases were thus shown to possess similar kinetic properties when acting on 4-nitrophenyl-β-d-xylopyranoside and xylobiose. kcat pH profiles of Cx1 and Q09LZ0 acting on 4-nitrophenyl-β-d-xylopyranoside and xylobiose have patterns containing two rate increases with increasing acidity, not reported before for glycoside hydrolases. The dexylosylation step of 4-nitrophenyl-β-d-xylopyranoside hydrolysis mediated by Q09LZ0 is not rate determining for kcat(4NPX).
authors.affiliation: USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL 61604, USA. Douglas.Jordan@ars.usda.gov
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Jordan
authors.forename: Karl E
authors.initials: KE
authors.lastname: Vermillion
authors.forename: Arabela A
authors.initials: AA
authors.lastname: Grigorescu
authors.forename: Jay D
authors.initials: JD
authors.lastname: Braker
mesh_terms: D000595:Amino Acid Sequence; D004789:Enzyme Activation; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000145:classification*
chemical_list: D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase
keywords: Bioethanol; GH43; GH52; Retaining mechanism; k(cat) pH profile