Oxygen-18 Exchange Studies of Energy Coupling in E1E2 Type ATPases

多种生命过程依赖于选择性运输 离子穿过细胞膜的浓度 梯度离心 尽管这一现象很重要， 最基本的问题的分子机制 主动运输仍未得到答复。 的目的 拟议的研究是了解离子运输是如何耦合到 催化生物储存的能量释放 分子（ATP）的酶，形成一个 磷酸酶中间体（E1 E2型）。 当前理论 是ATP和无机磷酸盐磷酸化 (P1)是由不同构象的酶催化的 这个想法将通过研究氧-18交换来验证， 当酶被磷酸化时被催化。 的 无机磷酸盐的出现和消失率 含有0到4个氧原子， 通过质谱或核磁共振。 的 同位素交换的模式取决于 反应 已经发现， 由质子传输的离子之一的浓度， 钾-ATP酶（H+）增加。 因此 构象状态与机制之间的关系可以 通过测量同位素交换来灵敏地探测 格局 催化钠钾的酶 交通也将被研究，因为有丰富的 关于Na+和K+如何影响构象的信息 酵素 在这些研究中使用的主要模型， 胃质子，钾-ATP酶，是重要的 生理上和理论上都是如此。

A variety of life processes depend on the selective transport of ions across cell membranes against a concentration gradient. Despite the importance of this phenomenon, the most fundamental questions about the molecular mechanism of active transport remain unanswered. The objective of the proposed research is to learn how ion transport is coupled to catalysis of energy release from the biological storage molecule (ATP) by the class of enzyme that forms a phosphoenzyme intermediate (E1E2-type). The current theory is that phosphorylation by ATP and by inorganic phosphate (P1) are catalyzed by different conformations of the enzyme. This idea will be tested by studying oxygen-18 exchange, which is catalyzed when the enzyme is phosphorylated. The rate of appearance and disappearance of inorganic phosphate containing from zero to four oxygen-18 atoms can be measured by mass spectrometry or nuclear magnetic resonance. The pattern of isotope exchange depends upon the mehcanism of the reaction. It has been found to change dramatically when the concentration of one of the ions transported by the proton, potassium-ATPase (H+) is increased. Therefore, the relationship between conformational state and mechanism can be sensitively probed by measuring the isotope exchange pattern. The enzyme that catalyzes sodium, potassium transport will also be studied, because there is a wealth of information on how Na+ and K+ affect the conformation of the enzyme. The primary model to be used in these studies, the gastric proton, potassium-ATPase, is important physiologically as well as theoretically.