Reference: O'donnell AH, et al. (2004) Solvent isotope effects on alpha-glucosidase. Biochim Biophys Acta 1703(1):63-7

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Abstract


The solvent kinetic isotope effects (SKIE) on the yeast alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 degrees 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 pK(a)s characterizing the pH profile were increased in D(2)O. The shift in pK(a2) of 0.6 units is typical of acids of comparable acidity (pK(a)=6.5), but the increase in pK(a1) (=5.7) of 0.1 unit in going from H(2)O to D(2)O is unusually small. The initial velocities show substrate inhibition (K(is)/K(m) approximately 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 alpha-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 x 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.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't | Comparative Study
Authors
O'donnell AH, Yao X, Byers LD
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