L-alanine dehydrogenase from Thermus thermophilus.

A heat-stable L-alanine dehydrogenase was isolated and purified from the extremely thermophilic microorganism, Thermus thermophilus, by affinity chromatography. The enzyme has a molecular weight of 290 000, as determined by the sedimentation equilibrium method, and is composed of six subunits of identical molecular weight as concluded from sodium dodecyl sulphate gel electrophoresis. The enzyme has been characterized in terms of pH- and substrate concentration-dependence of activity, substrate specificity, inhibition by D-alanine and D-cysteine and amino acid composition. The parameters obtained are very similar to those reported for L-alanine dehydrogenase from the mesophilic microorganism, Bacillus subtilis (Yoshida, A. and Freese, E. (1965) Biochim. Biophys. Acta 96, 248--262). The thermal stability of the T. thermophilus enzyme is much greater than that of the B. subtilis enzyme. Activation free energy (delta G), activation enthalpy (delta H) and activation entropy (delta S) values were determined for both the alanine deamination and for the heat inactivation reactions of the thermophilic and mesophilic enzymes. The values obtained for the catalytic reaction were practically equal. However, the two enzymes differed significantly in these parameters determined for the enzyme inactivation, which indicates that the factors ensuring the thermoresistance of the enzyme from T. thermophilus do not affect enzyme activity.