Steady state kinetic studies of purified yeast plasma membrane proton-translocating ATPase.
Steady state kinetic studies of purified yeast plasma membrane proton-translocating ATPase.
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DOI：
10.1016/s0021-9258(17)38473-9
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发表时间：
1986-05
期刊：
影响因子：
--
通讯作者：
J. Koland;G. Hammes
中科院分区：
文献类型：
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作者：
J. Koland;G. Hammes
对面包酵母质膜H+- atp酶进行纯化，用磷脂酰丝氨酸进行重组。在较宽的Mg2+和ATP浓度范围内研究了H+-ATP酶催化ATP水解的稳态动力学。而MgATP被底物水解，过量浓度的Mg2+或ATP均有抑制作用。在固定浓度的Mg2+下，ATP水解的稳态初始速度与MgATP的浓度呈s型关系，而不是双曲线关系。这排除了仅涉及Mg2+激活和抑制以及ATP竞争性抑制的机制。对这些结果的两种解释是：1)酶具有多个协同作用的催化位点；或者2)酶可以以多种构象状态存在，通过平行途径催化MgATP水解。两种机制的速率定律是相同的，因此不能根据动力学数据来区分这两种机制。这些数据很好地符合这些机制的速率定律，包括Mg2+和ATP的竞争性抑制以及Mg2+的独立抑制位点。
The plasma membrane H+-ATPase from bakers' yeast was purified and reconstituted with phosphatidylserine. The steady state kinetics of ATP hydrolysis catalyzed by the H+-ATPase were studied over a wide range of Mg2+ and ATP concentrations. Whereas MgATP was the substrate hydrolyzed, excess concentrations of either Mg2+ or ATP were inhibitory. The dependence of the steady state initial velocity of ATP hydrolysis on the concentration of MgATP at a fixed concentration of Mg2+ was sigmoidal rather than hyperbolic. This precluded mechanisms involving only activation and inhibition by Mg2+ and competitive inhibition by ATP. Two alternative interpretations of these results are: 1) the enzyme possesses multiple catalytic sites which interact cooperatively; or 2) the enzyme can exist in multiple conformational states which catalyze MgATP hydrolysis by parallel pathways. The rate laws for both mechanisms are identical so that the two mechanisms cannot be distinguished on the basis of the kinetic data. The data are well fit by the rate law for these mechanisms with the inclusion of competitive inhibition by Mg2+ and ATP and an independent inhibition site for Mg2+.