Mueller D M, Indyk V, McGill L
Chicago Medical School, Department of Biological Chemistry, IL 60064.
Eur J Biochem. 1994 Jun 15;222(3):991-9. doi: 10.1111/j.1432-1033.1994.tb18950.x.
Unisite ATPase kinetic constants were measured for wild-type yeast Saccharomyces cerevisiae F1-ATPase and F1-ATPase with the Thr197-->Ser mutation in the beta subunit. Under unisite conditions, the concentration of ATP is greater than that of the enzyme, ATP hydrolysis is slow and the affinity of the enzyme for ATP and ADP is high. The Thr197-->Ser mutation in the yeast F1-ATPase increases the specific activity of ATP hydrolysis threefold and makes the enzyme much less sensitive to azide and oxyanions [Mueller, D. M. (1989) J. Biol. Chem. 264, 16552-16556]. A unifying hypothesis is that the affinity of F1-ATPase for ADP is altered by azide, oxyanions and the Thr197-->Ser mutation. To address this hypothesis, kinetic and thermodynamic constants were measured for the wild-type and mutant enzymes in the absence and presence of azide and oxyanions. The results indicate that sulfite and azide do not significantly alter unisite thermodynamic binding constants of either enzyme for ADP at the catalytic site. The mutation Thr197-->Ser has little effect on the binding constant for ADP, or on other unisite kinetic constants of the enzyme, in the presence or absence of azide or oxyanions. However, the binding of ADP to the enzyme was affected by oxyanions and the Thr197-->Ser mutation as measured by determining the KiADP values for multisite ATPase activity (saturating ATP). The Ki for ADP on ATPase activity was measured for the wild-type and mutant enzymes in the presence and absence of sulfite under multisite conditions. Sulfite increases the KiADP values for ATP hydrolysis under multisite conditions approximately threefold for the wild-type and mutant enzymes and the Thr197-->Ser mutation increases KiADP ninefold. The effect of sulfite on KiADP is additive to the effect of the Thr197-->Ser mutation, suggesting that these are distinct effects. These results indicate that the effects of azide, oxyanions, and the Thr197-->Ser mutation on the biochemistry of F1-ATPase are limited primarily to multisite conditions. Both sulfite and the Thr197-->Ser mutation decrease the affinity of the enzyme for ADP, as measured by the increase in the Ki values. Furthermore, the mechanisms of activation by sulfite and the Thr197-->Ser mutations are different. This difference occurs despite their common biochemical consequences on the apparent affinity for ADP.
测定了野生型酿酒酵母F1 - ATP酶以及β亚基中苏氨酸197突变为丝氨酸的F1 - ATP酶的单位点ATP酶动力学常数。在单位点条件下，ATP的浓度高于酶的浓度，ATP水解缓慢，且酶对ATP和ADP的亲和力较高。酵母F1 - ATP酶中的苏氨酸197突变为丝氨酸会使ATP水解的比活性提高三倍，并使该酶对叠氮化物和含氧阴离子的敏感性大大降低[穆勒，D. M.（1989年）《生物化学杂志》264卷，16552 - 16556页]。一个统一的假说是，叠氮化物、含氧阴离子以及苏氨酸197突变为丝氨酸会改变F1 - ATP酶对ADP的亲和力。为了验证这一假说，测定了野生型和突变型酶在不存在和存在叠氮化物及含氧阴离子情况下的动力学和热力学常数。结果表明，亚硫酸盐和叠氮化物不会显著改变两种酶在催化位点对ADP的单位点热力学结合常数。在存在或不存在叠氮化物或含氧阴离子的情况下，苏氨酸197突变为丝氨酸对ADP的结合常数或该酶的其他单位点动力学常数影响很小。然而，通过测定多位点ATP酶活性（ATP饱和）的KiADP值发现，含氧阴离子和苏氨酸197突变为丝氨酸会影响ADP与酶的结合。在多位点条件下，测定了野生型和突变型酶在不存在和存在亚硫酸盐情况下ATP酶活性对ADP的Ki。亚硫酸盐使野生型和突变型酶在多位点条件下ATP水解的KiADP值大约提高三倍，而苏氨酸197突变为丝氨酸使KiADP提高九倍。亚硫酸盐对KiADP的影响与苏氨酸197突变为丝氨酸的影响具有加和性，这表明它们是不同的效应。这些结果表明，叠氮化物、含氧阴离子以及苏氨酸197突变为丝氨酸对F1 - ATP酶生物化学性质的影响主要限于多位点条件。通过Ki值的增加可以看出，亚硫酸盐和苏氨酸197突变为丝氨酸都会降低酶对ADP的亲和力。此外，亚硫酸盐和苏氨酸197突变为丝氨酸的激活机制不同。尽管它们对ADP的表观亲和力有共同的生化影响，但这种差异仍然存在。
