Váli Z, Kilár F, Lakatos S, Venyaminov S A, Závodszky P
Biochim Biophys Acta. 1980 Sep 9;615(1):34-47. doi: 10.1016/0005-2744(80)90006-6.
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.
通过亲和色谱法从嗜热栖热菌这种嗜热微生物中分离并纯化出了一种热稳定的L-丙氨酸脱氢酶。用沉降平衡法测定，该酶的分子量为290000，十二烷基硫酸钠凝胶电泳结果表明其由六个分子量相同的亚基组成。已对该酶在活性的pH和底物浓度依赖性、底物特异性、D-丙氨酸和D-半胱氨酸的抑制作用以及氨基酸组成等方面进行了表征。所获得的参数与嗜温微生物枯草芽孢杆菌的L-丙氨酸脱氢酶所报道的参数非常相似（吉田，A.和弗里斯，E.（1965年）《生物化学与生物物理学学报》96，248 - 262）。嗜热栖热菌酶的热稳定性远高于枯草芽孢杆菌酶。测定了嗜热酶和嗜温酶的丙氨酸脱氨反应以及热失活反应的活化自由能（ΔG）、活化焓（ΔH）和活化熵（ΔS）值。催化反应所获得的值实际上是相等的。然而，在为酶失活测定的这些参数方面，这两种酶有显著差异，这表明确保嗜热栖热菌酶耐热性的因素不会影响酶的活性。
