Solvent isotope effects on alpha-glucosidase.
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.
O'donnell
Anne H
AH
Department of Chemistry, Tulane University, New Orleans, LA 70118, USA.
Yao
Xiaojie
X
Byers
Larry D
LD
D002021:Buffers; D017666:Deuterium Oxide / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007554:Isotopes / Q000378:metabolism*; D007700:Kinetics; D008757:Methylglucosides / Q000378:metabolism / Q000494:pharmacology; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012997:Solvents / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism*
D002021:Buffers; D004791:Enzyme Inhibitors; D007554:Isotopes; D008757:Methylglucosides; D011522:Protons; D011994:Recombinant Proteins; D012997:Solvents; C027020:methylglucoside; D000520:alpha-Glucosidases; D005947:Glucose; D017666:Deuterium Oxide