Trp-262 is a key residue for the hydrolytic and transglucosidic reactivity of the Aspergillus niger family 3 β-glucosidase: Substitution results in enzymes with mainly transglucosidic activity

Heather F. Seidle, Kyle McKenzie, Ira Marten, Oded Shoseyov, Reuben E. Huber*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

Trp-262 of the Aspergillus niger family 3 β-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, β-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 β-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 Km and Ki 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 (k3) to be small.

Original languageAmerican English
Pages (from-to)66-75
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume444
Issue number1
DOIs
StatePublished - 1 Dec 2005

Bibliographical note

Funding Information:
This work was supported by funds from the Natural Science and Engineering Research Council of Canada (NSERC).

Keywords

  • Hydrolytic reactions
  • Kinetics
  • Transglucosidic reactions
  • Tryptophan
  • β-Glucosidase

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