title: Activation of a GH43 β-xylosidase by divalent metal cations: slow binding of divalent metal and high substrate specificity.
abstract: RS223-BX of glycoside hydrolase family 43 is a β-d-xylosidase that is strongly activated (k(cat)/K(m) as much as 116-fold) by the addition of divalent metal cations, Ca(2+), Co(2+), Fe(2+), Mg(2+), Mn(2+) and Ni(2+). Slow activation by Mg(2+) was demonstrated (k(on) 0.013 s(-1) mM(-1), k(off) 0.008 s(-1)) at pH 7.0 and 25 °C. k(off) and k(on) values are independent of Mg(2+) concentration, but k(off) and k(on) are slower in the presence of increasing levels of substrate 4-nitrophenyl-β-D-xylopyranoside. The kinetics strongly suggest that M(2+) binds to the enzyme rapidly, forming E M(2+), followed by slow isomerization to the activated enzyme, E* M(2+). Moderately high values of kcat (7-30 s(-1)) were found for M(2+)-activated RS223-BX acting on xylobiose (natural substrate) at pH 7.0 and 25 °C. Certain M(2+)-activated RS223-BX exhibit the highest reported values of k(cat)/K(m) of any β-xylosidase acting on natural substrates: for example, at pH 7.0 and 25°C, xylobiose (Mn(2+), 190 s(-1) mM(-1)), xylotriose (Ca(2+), 150 s(-1) mM(-1)) and xylotetraose (Ca(2+), 260 s(-1) mM(-1)). There is potential for the enzyme to add value to industrial saccharification operations at low substrate and high d-glucose and high d-xylose concentrations.
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: Charles C
authors.initials: CC
authors.lastname: Lee
authors.forename: Kurt
authors.initials: K
authors.lastname: Wagschal
authors.forename: Jay D
authors.initials: JD
authors.lastname: Braker
mesh_terms: D055162:Biocatalysis; D050260:Carbohydrate Metabolism; D002413:Cations, Divalent / Q000378:metabolism* / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology*; D013379:Substrate Specificity; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D008670:Metals; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase