ISSN:
1573-4986
Keywords:
β-glycosidase
;
temperature dependence
;
kinetics
;
glucose
;
transglycosylation
;
(Thermus thermophilus)
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract A β-glycosidase of a thermophile, Thermus thermophilus, belonging to the glycoside hydrolase family 1, was cloned and overexpressed in Escherichia coli. The purified enzyme (Ttβgly) has a broad substrate specificity towards β-D-glucoside, β-D-galactoside and β-D-fucoside derivatives. The thermostability of Ttβgly 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. Ttβgly 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 cat and K m ) were calculated at different temperatures. Both k cat and K m increased with an increase in temperature, but up to 75[emsp4 ]°C the values of k cat increased much more rapidly than the values of K 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 Ttβgly. 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1007104030314
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