Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 444, Issue 1, Pages 66-75Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2005.09.013
Keywords
beta-glucosidase; transglucosidic reactions; hydrolytic reactions; kinetics; tryptophan
Categories
Ask authors/readers for more resources
Trp-262 of the Aspergillus niger family 3 beta-glucosidase is shown in this report to be a key residue for determining the ratio of this enzyme's hydrolytic and transglucosidic activities. TLC showed that when cellobiose was both the substrate and the acceptor, beta-glucosidases with substitutions (Phe, Ala, Leu, and Cys) for Trp-262 formed very high amounts of transglucosidic adducts. When pNPGlc was the substrate and the acceptor of the substituted beta-glucosidases, only transglucosidic adducts and pNP were produced. Little or no Glc could be detected, indicating that the reactions occurring were mainly transglucosidic. GLC studies with cellobiose quantitatively showed that one Glc was transferred for each free Glc produced. Since this is the maximum level of transglucosidation possible, this again showed that the reaction is predominantly transglucosidic. Analyses of the K-m and K-i values of cello-oligosaccharides of increasing length, of the Ki values of Glc and of the transglucosidic activity at low acceptor concentration, showed that substitution for Trp-262 causes poor binding at the binding site for the non-reducing Glc of the substrate while the affinity for other Glc units is only minimally affected. The acceptor sites become saturated with substrate (acceptor) at the concentrations needed for glucosidic bond cleavage and thus only transglucosidic reactions occur. In addition, the data indicate that substitution for Trp-262 causes the rate of the hydrolysis step (k(3)) to be small. (c) 2005 Elsevier Inc. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available