目的：采用定点突变方法探讨突变体对酵母Ndi1的NADH-泛醌氧化还原酶活性及泛醌结合部位的影响。方法定点突变方法将Ndi1的His残基（His193、His252、His301）突变为丙氨酸，转染大肠杆菌后表达蛋白，测定光谱特性，NADH-泛醌氧化还原酶活性和米氏常数Km及最大反应速度Vmax。结果各突变体蛋白H193A、H252A、H301A吸收光谱显示均在383nm和448nm处出现两个FAD峰与野生型一致；各突变体酶活性显示，突变体对UQ1的Vmax均明显减小，但仅H301A对UQ1的Km增加约3倍，对UQ亲和力明显减小。各突变体蛋白对NADH的Km未见明显增大，但Vmax均出现明显减小。结论酵母Ndi1中H301点突变可能是参与形成UQ结合位点所需的氨基酸残基之一，该位点突变影响了UQ与酶的结合及电子向UQ的传递。%Objective Using site-directed mutagenesis methods, we studyed the ubiquinone-binding site and enzyme activity of yeast single subunit NADH-ubiquinone oxidoreductase. Methods H193, H252, H301 of Ndi1 was mutated to alanine by site-directed mutagenesis methods. The recombinant plasmid pET-16b-Ndi1/mutant was transfromed into E. coli BL2l(DE3)pLysS for gene expression. Then the spectra of mutant enzymes and NADH-ubiquinone oxidoreductase activity were mesured. Results The 383 and 448 nm peaks derived from FAD were observed. The Vmax values decreased in H193A, H252A and H301.The Km value of UQ1 greatly increased in H301A by 3.0-fold only. The Km values of NADH was no significant changed in three mutants, but The Vmax values decreased all. Conclusions The highly conserved residue H301 may be involved in the formation of UQ-binding site in yeast single-subunit NADH-ubiquinone oxidoreductase.
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