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

A beta -glycosidase of a thermophile, Thermus thermophilus, belonging to th e glycoside hydrolase family 1, was cloned and overexpressed in Escherichia coli. The purified enzyme (Tt beta gly) has a broad substrate specificity towards beta -D-glucoside, beta -D-galactoside and beta -D-fucoside derivat ives. The thermostability of Tt beta gly was exploited to study its kinetic properties within the range 25-80 degreesC. Whatever the temperature, exce pt around 60 degreesC, the enzyme displayed non-Michaelian kinetic behavior . Tt beta gly was inhibited by high concentrations of substrate below 60 de greesC and was activated by high concentrations of substrate above 60 degre esC. The apparent kinetic parameters (k(cat) and K-m) were calculated at di fferent temperatures. Both k(c)at and K-m increased with an increase in tem perature, but up to 75 degreesC the values of k(cat) increased much more ra pidly than the values of K-m. The observed kinetics might be due to a combi nation of factors including inhibition by excess substrate and stimulation due to transglycosylation reactions. Our results show that the substrate co uld act not only as a glycosyl donor but also as a glycosyl acceptor. In ad dition, when the glucose was added to reaction mixtures, inhibition or acti vation was observed depending on both substrate concentration and temperatu re. A reaction model is proposed to explain the kinetic behavior of Tt beta gly. The scheme integrates the inhibition observed at high concentrations of substrate and the activation due to transglycosylation reactions implica ting the existence of a transfer subsite.