The pKaof the General Acid/Base Carboxyl Group of a Glycosidase Cycles during Catalysis: A13C-NMR Study ofBacillus circulansXylanase

Abstract

The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl−enzyme intermediate. In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water. The dual role of Glu172 places specific demands upon its ionization states and hence pK_a values. ¹³C-NMR titrations of xylanase, labeled with [δ-¹³C]glutamic acid, have revealed pK_a values of 4.6 and 6.7 for Glu78 and Glu172, respectively. These agree well with the apparent pK_a values obtained from a study of the pH dependence of k_cat/K_m and demonstrate that, at the enzyme's pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated. Remarkably, the pK_a for Glu172 drops to 4.2 in a trapped covalent glycosyl−enzyme intermediate, formed by reaction with 2‘,4‘-dinitrophenyl 2-deoxy-2-fluoro-β-xylobioside [Miao et al. (1994) Biochemistry 33, 7027−7032]. A similar pK_a is measured for Glu172 when a glutamine is present at position 78. This large decrease in pK_a of ∼2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. Such “pK_a cycling” during catalysis is likely to be a common phenomenon in glycosidases.