Solvent isotope effects on α-glucosidase

Abstract

The solvent kinetic isotope effects (SKIE) on the yeast α-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl- d-glucopyranoside were measured at 25 °C. With p-nitrophenyl- d-glucopyranoside (pNPG), the dependence of k cat/ K m on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, DOD( k cat/ K m), of 1.9 (±0.3). The two p K as characterizing the pH profile were increased in D 2O. The shift in p K a2 of 0.6 units is typical of acids of comparable acidity (p K a=6.5), but the increase in p K a1 (=5.7) of 0.1 unit in going from H 2O to D 2O is unusually small. The initial velocities show substrate inhibition ( K is/ K m∼200) with a small solvent isotope effect on the inhibition constant [ DOD K is=1.1 (±0.2)]. The solvent equilibrium isotope effects on the K is for the competitive inhibitors d-glucose and α-methyl d-glucoside are somewhat higher [ DOD K i=1.5 (±0.1)]. Methyl glucoside is much less reactive than pNPG, with k cat 230 times lower and k cat/ K m 5×10 4 times lower. The solvent isotope effect on k cat for this substrate [=1.11 (±0. 02)] is lower than that for pNPG [=1.67 (±0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.