THE PK(A) OF THE GENERAL ACID BASE CARBOXYL GROUP OF A GLYCOSIDASE CYCLES DURING CATALYSIS - A C-13-NMR STUDY OF BACILLUS CIRCULUNS XYLANASE/

The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme in termediate. 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. C-13-NMR titrations of xylanase, labeled with [delta-C-13]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 de pendence of k(cat)/K-m and demonstrate that, at the enzyme's pH optimu m of 5.7, the nucleophile Glu78 is deprotonated and the general acid G lu172 initially protonated. Remarkably, the pK(a) for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reac tion with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-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 dec rease in pK(a) of similar to 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appe ars to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. S uch ''pK(a) cycling'' during catalysis is likely to be a common phenom enon in glycosidases.