Salt effects on β-glucosidase: pH-profile narrowing

Abstract

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl − salts) the effectiveness follows the series: Cu +2, Fe +2 > Zn +2 > Li + > Ca +2 > Mg +2 > Cs + > NH 4 + > Rb + > K + > Na + and for anions (Na + salts) the series is: I − > ClO 4 − > −SCN > Br − ≈ NO 3 − > Cl − ≈ −OAc > F − ≈ SO 4 − 2 . The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k cat/ K m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic p K as and not to an effect on the pH-independent second-order rate constant, ( k cat/ K m) lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent p K a1 increases (from 3.7 to 4.9) and the apparent p K a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent ( k cat/ K m) lim (= 9 × 10 4 M − 1 s − 1 ) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the p K as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced p K a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a p K a of 3.7 and dissociated from the group with a p K a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two p K as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, ( k cat/ K m) lim, of the monoprotonated species. For other enzymes which may show such salt-induced p K a shifts, this provides a convenient test for the role of reverse protonation.