A case for reverse protonation: identification of Glu160 as an acid/base catalyst in Thermoanaerobacterium saccharolyticum beta-xylosidase and detailed kinetic analysis of a site-directed mutant.

The catalytic mechanism of the family 39 Thermoanaerobacterium saccharolyticum beta-xylosidase (XynB) involves a two-step double-displacement mechanism in which a covalent alpha-xylosyl-enzyme intermediate is formed with assistance from a general acid and then hydrolyzed with assistance from a general base. Incubation of recombinant XynB with the newly synthesized active site-directed inhibitor, N-bromoacetyl-beta-D-xylopyranosylamine, resulted in rapid, time-dependent inactivation of the enzyme (k(i)/K(i) = 4.3 x 10(-4) s(-1)mM(- 1)). Protection from inactivation using xylose or benzyl 1-thio-beta-xyloside suggested that the inactivation was active site-directed. Mass spectrometric analysis indicated that incubation of the enzyme with the inactivator resulted in the stoichiometric formation of a new enzyme species bearing the label. Comparative mapping of peptic digests of both the labeled and unlabeled enzyme by HPLC coupled to an electrospray ionization mass spectrometer permitted the identification of a labeled peptide. Sequencing of this peptide by tandem mass spectrometry identified Glu160 within the sequence (157)IWNEPNL(164) as the site of attachment of the N-acetyl-beta-D-xylopyranosylamine moiety. Kinetic analysis of the Glu160Ala mutant strongly suggests that this residue is involved in acid/base catalysis as follows. First, a significant difference in the dependence of k(cat)/K(m) on pH as compared to that seen for the wild-type enzyme was found, as expected for a residue that is involved in acid/base catalysis. The changes, however, were not as simple as those seen in other cases. Second, a dramatic decrease (up to 10(5)-fold) in the catalytic efficiency (k(cat)/K(m)) of the enzyme with a substrate requiring protonic assistance is observed upon such mutation. In contrast, the catalytic efficiency of the enzyme with substrates bearing a good leaving group, not requiring acid catalysis, is only moderately impaired relative to that of the wild-type enzyme (8-fold). Surprisingly, however, the glycosylation step was rate-limiting for all but the most reactive substrates. Last, the addition of azide as a competitive nucleophile resulted in the formation of a beta-xylosyl azide product and increased the k(cat) and K(m) values up to 8-fold while k(cat)/K(m) remained relatively unchanged. Such kinetic behavior is consistent with azide acting competitively with water as a nucleophile in the second step of the enzyme catalyzed reaction involving breakdown of the xylosyl-enzyme intermediate. Together, these results provide strong evidence for a role of Glu160 in acid/base catalysis but suggest that it may be partnered by a second carboxylic acid residue and that the enzyme may function through using acid catalysis involving reverse protonation of active site residues.