含有半胱氨酸-255-丝氨酸和半胱氨酸-255-甲硫氨酸替代的假单胞菌sp.101甲酸脱氢酶突变体的催化特性和稳定性
Catalytic properties and stability of a Pseudomonas sp.101 formate dehydrogenase mutants containing Cys-255-Ser and Cys-255-Met replacements.
作者信息
Tishkov V I, Galkin A G, Marchenko G N, Egorova O A, Sheluho D V, Kulakova L B, Dementieva L A, Egorov A M
机构信息
Department of Chemical Enzymology, Chemistry Faculty, Moscow State University, Russia.
出版信息
Biochem Biophys Res Commun. 1993 Apr 30;192(2):976-81. doi: 10.1006/bbrc.1993.1511.
Two mutants of bacterial formate dehydrogenase from Pseudomonas sp.101 (EC 1.2.1.2, FDH)-C255S (FDH-S) and C255M (FDH-M), were obtained and its properties were studied. Both mutations provided the high resistance to inactivation by Hg2+. Slow inactivation of mutants by DTNB reveals the presence in FDH molecule of another essential cysteine residue. Specific activities of FDH, FDH-S and FDH-M were 16, 16 and 9.5 U/mg of protein, respectively. Km on formate was 7.5, 7.5 and 20 mM and Km on NAD(+)-0.1, 0.3 and 0.6 mM for FDH, FDH-S and FDH-M, respectively. Mutations of Cys255 on Ser or Met resulted in increasing of enzyme stability at 25 degrees C and decreasing of thermostability (above 45 degrees C). Data obtained show that Cys255 is unique residue for providing both enzyme thermostability and catalytically optimal binding of coenzyme.
获得了来自假单胞菌属sp.101的细菌甲酸脱氢酶（EC 1.2.1.2，FDH）的两个突变体——C255S（FDH-S）和C255M（FDH-M），并对其性质进行了研究。这两个突变体对Hg2+介导的失活均具有高度抗性。DTNB对突变体的缓慢失活表明FDH分子中存在另一个必需的半胱氨酸残基。FDH、FDH-S和FDH-M的比活性分别为16、16和9.5 U/mg蛋白质。FDH、FDH-S和FDH-M对甲酸的Km分别为7.5、7.5和20 mM，对NAD(+)的Km分别为0.1、0.3和0.6 mM。将半胱氨酸255突变为丝氨酸或甲硫氨酸会导致酶在25℃时稳定性增加，而热稳定性降低（45℃以上）。所得数据表明，半胱氨酸255是提供酶热稳定性和辅酶催化最佳结合的独特残基。
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