Jault J M, Dou C, Grodsky N B, Matsui T, Yoshida M, Allison W S
Department of Chemistry and Biochemistry, School of Medicine, University of California at San Diego, La Jolla, California 92093-0601, USA.
J Biol Chem. 1996 Nov 15;271(46):28818-24. doi: 10.1074/jbc.271.46.28818.
The hydrolytic properties of the mutant alpha3(betaT165S)3gamma and wild-type alpha3beta3gamma subcomplexes of TF1 have been compared. Whereas the wild-type complex hydrolyzes 50 microM ATP in three kinetic phases, the mutant complex hydrolyzes 50 microM ATP with a linear rate. After incubation with a slight excess of ADP in the presence of Mg2+, the wild-type complex hydrolyzes 2 mM ATP with a long lag. In contrast, prior incubation of the mutant complex under these conditions does not affect the kinetics of ATP hydrolysis. The ATPase activity of the wild-type complex is stimulated 4-fold by 0. 1% lauryl dimethylamine oxide, whereas this concentration of lauryl dimethylamine oxide inhibits the mutant complex by 25%. Compared with the wild-type complex, the activity of the mutant complex is much less sensitive to turnover-dependent inhibition by azide. This comparison suggests that the mutant complex does not entrap substantial inhibitory MgADP in a catalytic site during turnover, which is supported by the following observations. ATP hydrolysis catalyzed by the wild-type complex is progressively inhibited by increasing concentrations of Mg2+ in the assay medium, whereas the mutant complex is insensitive to increasing concentrations of Mg2+. A Lineweaver-Burk plot constructed from rates of hydrolysis of 20-2000 microM ATP by the wild-type complex is biphasic, exhibiting apparent Km values of 30 microM and 470 microM with corresponding kcat values of 26 and 77 s-1. In contrast, a Lineweaver-Burk plot for the mutant complex is linear in this range of ATP concentration, displaying a Km of 133 microM and a kcat of 360 s-1.
已对TF1的突变体α3(βT165S)3γ和野生型α3β3γ亚复合物的水解特性进行了比较。野生型复合物在三个动力学阶段水解50μM ATP，而突变体复合物以线性速率水解50μM ATP。在Mg2+存在下与略微过量的ADP孵育后，野生型复合物水解2 mM ATP时存在较长的延迟期。相比之下，在此条件下预先孵育突变体复合物不会影响ATP水解的动力学。野生型复合物的ATP酶活性被0.1%的月桂基二甲基氧化胺刺激4倍，而该浓度的月桂基二甲基氧化胺会抑制突变体复合物25%。与野生型复合物相比，突变体复合物的活性对叠氮化物的周转依赖性抑制不太敏感。这种比较表明，突变体复合物在周转过程中不会在催化位点截留大量抑制性的MgADP，以下观察结果支持了这一点。野生型复合物催化的ATP水解在测定介质中会随着Mg2+浓度的增加而逐渐受到抑制，而突变体复合物对Mg2+浓度的增加不敏感。由野生型复合物水解20 - 2000μM ATP的速率构建的Lineweaver - Burk图是双相的，表观Km值分别为30μM和470μM，相应的kcat值分别为26和77 s-1。相比之下，突变体复合物在该ATP浓度范围内的Lineweaver - Burk图是线性的，显示Km为133μM，kcat为360 s-1。
