Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration.
Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration.
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DOI：
10.1021/bi00525a016
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发表时间：
1981-10
期刊：
影响因子：
2.9
通讯作者：
B. Springs;K. Welsh;B. Cooperman
中科院分区：
文献类型：
--
作者：
B. Springs;K. Welsh;B. Cooperman
我们已经开发了两种方法来定量测量无机焦磷酸盐（PPi）在10(3)- 10(4)摩尔过量无机磷酸盐（Pi）的存在，并使用它们来测量酵母菌无机焦磷酸酶和Pi的溶液中酶结合焦磷酸盐（EPPi）的形成程度。我们还测量了酶催化的H2O-磷酸氧交换速率。我们发现这两个过程对Mg2+和Pi浓度的依赖基本相同，从而为Janson等人（1979）最近提出的氧交换通过epi形成进行的建议提供了重要的证实。我们的结果与epi形成需要每个活性位点三个Mg2+的模型一致，但与每个活性位点只需要两个Mg2+的模型不一致，并允许制定无机焦磷酸酶催化PPi- Pi平衡的总体方案，以及该方案中的平衡和速率常数的评估。本研究的主要结果和结论如下：(a)酶结合产物PPi与酶结合产物2Pi平衡时的平衡常数为4.8；(b) PPi水解后，第一个释放的Pi含有来自溶剂水的氧；(c)酶水解PPi的步骤和两种产物Pi的释放都部分地决定了酶催化PPi水解的总体速率；(d)酶上PPi的生成速率决定了H2O—Pi氧交换；(e) PPi与酶的分离非常缓慢，是Pi- PPi交换的速率决定步骤（Cohn, 1958; Janson et al., 1979）。这也解释了MgPPi复合物与酶结合的解离常数的计算结果大大低于酶催化MgPPi水解的测量Km。
We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi--Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) the steps for PPi hydrolysis on the enzyme and for release of both product Pi's are all partially rate determining in overall enzyme-catalyzed PPi hydrolysis; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi--PPi exchange (Cohn, 1958; Janson et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.