Kinetic study of a thermostable b-glycosidase of Thermus thermophilus. Effects of temperature and glucose on hydrolysis and transglycosylation reactions

A b-glycosidase of a thermophile, Thermus thermophilus, belonging to the glycoside hydrolase family 1, was cloned and overexpressed in Escherichia coli. The purified enzyme (Ttbgly) has a broad substrate specificity towards b-D-glucoside, b-D-galactoside and b-D-fucoside derivatives. The thermostabi...

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Bibliographic Details
Published in:Glycoconjugate journal 2000-06, Vol.17 (6), p.377-383
Main Authors: Fourage, Laurent, Dion, Michel, Colas, Bernard
Format: Article
Language:English
Subjects:
Escherichia coli
Thermus thermophilus
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Summary:A b-glycosidase of a thermophile, Thermus thermophilus, belonging to the glycoside hydrolase family 1, was cloned and overexpressed in Escherichia coli. The purified enzyme (Ttbgly) has a broad substrate specificity towards b-D-glucoside, b-D-galactoside and b-D-fucoside derivatives. The thermostability of Ttbgly was exploited to study its kinetic properties within the range 25-80[emsp4 ]C. Whatever the temperature, except around 60[emsp4 ]C, the enzyme displayed non-Michaelian kinetic behavior. Ttbgly was inhibited by high concentrations of substrate below 60[emsp4 ]C and was activated by high concentrations of substrate above 60[emsp4 ]C. The apparent kinetic parameters (k sub(cat) and K sub(m)) were calculated at different temperatures. Both k sub(cat) and K sub(m) increased with an increase in temperature, but up to 75[emsp4 ]C the values of k sub(cat) increased much more rapidly than the values of K sub(m). The observed kinetics might be due to a combination of factors including inhibition by excess substrate and stimulation due to transglycosylation reactions. Our results show that the substrate could act not only as a glycosyl donor but also as a glycosyl acceptor. In addition, when the glucose was added to reaction mixtures, inhibition or activation was observed depending on both substrate concentration and temperature. A reaction model is proposed to explain the kinetic behavior of Ttbgly. The scheme integrates the inhibition observed at high concentrations of substrate and the activation due to transglycosylation reactions implicating the existence of a transfer subsite.
ISSN:0282-0080
1573-4986
DOI:10.1023/A:1007104030314