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 (kcat/Km as much as 116-fold) by the addition of divalent metal cations, Ca2+, Co2+, Fe2+, Mg2+, Mn2+ and Ni2+. Slow activation by Mg2+ was demonstrated (kon 0.013s−1mM−1, koff 0.008s−1) at pH 7.0 and 25°C. koff and kon values are independent of Mg2+ concentration, but koff and kon are slower in the presence of increasing levels of substrate 4-nitrophenyl-β-d-xylopyranoside. The kinetics strongly suggest that M2+ binds to the enzyme rapidly, forming E M2+, followed by slow isomerization to the activated enzyme, E∗ M2+. Moderately high values of kcat (7–30s−1) were found for M2+-activated RS223-BX acting on xylobiose (natural substrate) at pH 7.0 and 25°C. Certain M2+-activated RS223-BX exhibit the highest reported values of kcat/Km of any β-xylosidase acting on natural substrates: for example, at pH 7.0 and 25°C, xylobiose (Mn2+, 190s−1mM−1), xylotriose (Ca2+, 150s−1mM−1) and xylotetraose (Ca2+, 260s−1mM−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.