Catalysis by β-glucosidase A3of Aspergillus wentii

Abstract

(1)α-Deuterium kinetic isotope effects on V_max. for hydrolysis of both β-D-glucopyranosylpyridinium ions and aryl β-D-glucopyranosides are in the range k_H/k_D 1.08–1.14, indicating that bond-breaking limits the rate of hydrolysis of both sets of substrates. The variation of k_cat with aglycone acidity, expressed by log k_cat=A+β_lgpK_a, is governed by a β_lg value of –0.96 ± 0.19 for the N-glycosides and –0.05 ± 0.05 for the O-glycosides. The latter, low value of |β_lg| is evidence for extensive proton donation to the oxygen at the transition state, even for the departure of acidic aglycones. The dependence of rate on protonation of a group of pK_a < 6 required by this idea is indeed observed. (2) Comparison of log k_cat values with spontaneous hydrolysis rates indicates that nucleophilic and non-covalent interactions accelerate C–N bond cleavage in the ES complex by a factor of 10^{(8 + 0.3[pk_a]_N)} for glucosyl pyridinium salts where [pK_a]_N is the pK_a of the pyridine. On the assumption that these interactions are similar for O-glucosides, proton donation to the aglycone oxygen atom can be estimated to contribute a rate-enhancement of 10^{(0.1 + 0.7[pK_a]_O)}, where [pK_a]_O is that of the free phenol. (3)D-Glucono-δ-lactone and 5-amino-5-deoxy-D-gluconolactam are bound 10^2.8 and 10^2.2 times, respectively, more tightly than β-D-glucopyranose, because of their analogy to a transition state in which α-deuterium kinetic isotope effects have shown the anomeric carbon atom to have substantial sp² character. (4) Cationic inhibitors are bound 10^2.5–10^3.5 times more tightly than directly comparable neutral ones, if the positive charge resides on the equatorial substituent at C-1, but protonated 2-amino-2-deoxyglucose is bound no more tightly than glucose. This indicates the presence of a negative charge in the El complex near C-1 on the α face of the pyranose ring (an aspartate residue previously identified by covalent labelling) which is partly compensated by a positive charge near C-2.