Identification of the Acid/Base Catalyst in Agrobacterium faecalis .beta.-Glucosidase by Kinetic Analysis of Mutants

Abstract

The catalytic mechanism of the retaining beta-glucosidase (Abg) from Agrobacterium faecalis involves a double-displacement process in which an alpha-glucosyl-enzyme intermediate is formed with general acid catalytic assistance and then hydrolyzed with general base assistance. Glu170 was identified as an important residue, possibly the acid/base catalyst, on the basis of sequence alignments. This glutamate is conserved in almost all enzymes in family 1 of glycoside hydrolases. Detailed pre-steady-state and steady-state kinetic analyses of the mutant E170G suggested very strongly that Glu170 is the acid/base catalyst. First, kcat values were invariant with pH over the range of 5.0-9.0. Secondly, rates of formation of the glycosyl-enzyme, calculated from kcat/Km and k2, were similar to those of wild-type enzyme for substrates not requiring protonic assistance but dramatically reduced for those needing acid catalysis. Thirdly, addition of azide as a competitive nucleophile increased kcat values 100-300-fold for substrates whose rate-limiting step is deglycosylation, yielding beta-glucosyl azide, but had no effect on the wild-type enzyme. Other anionic nucleophiles had similar, but less dramatic effects. Previous results [Gebler, J.C., et al. (1995) 34, 14547-14553] had indicated that Tyr298F is important for catalysis. The kinetic consequences of the mutations in the double mutant E170G-Y298F are additive, resulting in a 10(6)-fold reduction in kcat values and allowing the accumulation of a stable (t1/2 > 7 h) glucosyl-enzyme intermediate. Thus, Glu170 and Tyr298 function independently, and a possible role for Tyr298 in modulating the pKa of the catalytic nucleophile is proposed.

Cloning and Comparison of Third β-Glucoside Utilization (bglEFIA) Operon with Two Operons ofPectobacterium carotovorumsubsp.carotovorumLY34

“…Agrobacterium faecalis, 35,36) site-directed mutagenesis was performed on BglA (Table 2). Based on the results, we propose that Glu 178 and Glu 378 are catalytic sites in BglA.…”

Structural Determinants of Substrate Specificity in Family 1 β-Glucosidases

“…The roles of the 2 catalytic glutamates (8,9) included in the TFNEP and Y(I/V)TENG peptide motifs of ␤-glucosidases as the general acid/base catalyst and the nucleophile, respectively (10), are now well understood. These residues are 5.5-6 Å apart within the active-site pocket on opposite sides of the glycosidic bond (8,11) and are required in the two steps of the substrate hydrolysis that results in retention of the anomeric conformation of C-1 at the point of cleavage.…”

The role of residues R97 and Y331 in modulating the pH optimum of an insect β‐glycosidase of family 1

“…These glutamic acids are very close inside the active site (about 4.5 Å apart) [2], and during the reaction the first glutamic acid acts as proton donor, and the second acts as a nucleophile. The catalytic nucleophile pK a is around 5.0 and the catalytic proton donor pK a is around 7.0 [3][4][5][6][7].A plot of b-glycosidase activity vs. pH presents a bell shape, indicating that in the pH optimum the catalytic nucleophile is deprotonated and the catalytic proton donor is protonated. Hence the branch of the curve below the pH optimum is determined mainly by the ionization of the catalytic nucleophile, whereas the catalytic ionization of the proton donor determines the branch above the pH optimum.…”