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Thioglucoside Hydrolysis Catalyzed by β‐Glucosidase
Author(s) -
Shen Hong,
Byers Larry D.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a272-c
Subject(s) - chemistry , protonation , enzyme kinetics , deprotonation , hydrolysis , carboxylate , leaving group , acid dissociation constant , medicinal chemistry , stereochemistry , kinetic isotope effect , glycosyl , catalysis , organic chemistry , active site , aqueous solution , deuterium , quantum mechanics , ion , physics
Sweet almond β‐glucosidase (EC 3.2.1.21 ) is a retaining “family 1” exo ‐glycohydrolase. The activity of it depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. Here we report on the hydrolysis of thioglucosides and thiogalactosides catalyzed by β‐glucosidase. While the K m values for the S‐ and O‐ glycosides are similar, the k cat values are about 1,000‐times lower for the S ‐glycosides. Remarkably, the pH‐profile for k cat /K m for hydrolysis of p ‐nitrophenyl thioglucoside (pNPTG) shows the identical dependence on a deprotonated carboxylate (pK a = 4.5) and a protonated group (pK a = 6.7) as does the pH‐profile for hydrolysis of the corresponding O ‐glycoside. For the few glycohydrolases that have been shown to be able to also catalyze the hydrolysis of thioglycosides, the bell‐shaped pH‐profile usually shows no dependence on the ionization of the acidic group. While substitution of D 2 O for H 2 O results in the expected increase in the enzymic pK a s, there is no solvent isotope effect on the limiting (pH‐independent) second‐order rate constant (k cat /K m ) or on the limiting k cat value. These results suggest the reason that the glutamic acid residue must be protonated is not that it is acting as a general acid catalyst, but rather, that the unionized glutamic acid avoids electrostatic repulsion with the incipient thiolate. Subsequent proton transfer, from this acid to the thiolate, favors partitioning of the glycosyl‐enzyme intermediate towards products, over reversion to substrate.