Kocabiyik S, Erduran I, Russel R J, Danson M J, Hough D W
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey.
Biochem Biophys Res Commun. 1996 Jul 5;224(1):224-8. doi: 10.1006/bbrc.1996.1011.
In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma citrate synthase is very similar to wild-type citrate synthase in its substrate and co-factor specificities, pH profile and thermal stability. The mutation, however, has decreased the enzyme activity. The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity.
在本研究中，我们将嗜热古菌嗜热栖热菌重组柠檬酸合酶中的丝氨酸-43替换为半胱氨酸，用于位点特异性标记，并研究了该突变对酶的生化特性和热稳定性的影响。使用Matrex Gel Red A亲和色谱法将野生型和突变型酶均纯化至均一状态。突变型嗜热栖热菌柠檬酸合酶在底物和辅因子特异性、pH曲线和热稳定性方面与野生型柠檬酸合酶非常相似。然而，该突变降低了酶活性。新引入的反应性巯基可以很容易地被5,5'-二硫代双(2-硝基苯甲酸)修饰并用4-氯-7-磺基苯并呋喃标记，而不会损失任何活性。
