KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

生化反应的动力学、调控和机制

• 批准号: 4694455
• 负责人: P B CHOCK
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/4694455
• 关键词: 3'5' cyclic nucleotide phosphodiesterase; Escherichia coli; adenosinetriphosphatase; alkaline phosphatase; allosteric site; bioenergetics; chemical reaction; electron spin resonance spectroscopy; enzyme feedback; enzyme model; enzyme substrate complex; fluorescence polarization; glutamate ammonia ligase; light scattering; nuclear magnetic resonance spectroscopy; phosphorylation; stop flow technique

(1) An improved purification pocedure using the HPLC technique has been developed to purify the Mg(II)-ATP-dependent protein kinase from rabbit skeletal muscle. The purified enzyme consists of 38 and 31 KDa subunits, which correspond to the catalytic and modulator subunit, respectively. A mechanism for the activation of the inactive enzyme involves a transient phosphorylation of the modulator subunit catalyzed by kinase Fa. A few lines of evidence in support of the phosphorylation induces the catalytic subunit to its active form includes the observation that removal of the modulator by limiting proteolysis fails to activate the inactive (native) enzyme, unless it has been first phosphorylated or thiophosphorylated. Evidence supporting the slow isomerization which leads the active enzyme to its inactive form was demonstrated. The regulatory subunit (R) of type II cAMP-dependent protein kinase is an effective inhibitor for the phosphatase. A curve fitting method was used to estimate the dissociation constants for R.phosphatase, R.type I catalytic fragment and Fa.phosphatase complexes. The results suggest that R binds to the catalytic subunit. In addition, computer simulation of a cyclic cascade using the cAMP-dependent protein kinase and the above phosphatase as converter enzymes revealed that they constitute a highly efficient regulatory machanism. (2) A comparative study of cyclic cascade and simple allosteric control revealed that simple allosteric control cannot provide the same regulatory efficiency as those exhibited by the cyclic cascade systems. (3) A simple treatment was developed to describe interfacial reaction dynamics. This analysis revealed those conditions under which reduced dimensionality can be expected to enhance the overall efficiency or reaction rate of a bimolecular reaction. (4) A study of skeletal muscle actomyosin ATPase cycle using subfragment-1 in place of myosin revealed that a six-state model, which consists of six myosin bound reaction intermediates containing phosphate in the form of ATP or ADP.Pi, is the simplest kinetic model required to describe the ATase cycle.

（1）使用HPLC技术改进的纯化量已经 开发旨在纯化兔子的Mg（ii）-ATP依赖性蛋白激酶 骨骼肌。 纯化的酶由38和31 kDa亚基组成， 分别与催化和调节剂亚基相对应。 一个 激活非活性酶的机制涉及瞬态 由激酶FA催化的调节剂亚基的磷酸化。 几个 支持磷酸化的证据线诱导催化 其活性形式的亚基包括去除的观察 通过限制蛋白水解的调节剂无法激活非活性（天然） 酶，除非首先被磷酸化或硫磷酸化。 支持慢速异构化的证据，该异构化导致活性酶提升到 证明了它的不活动形式。 II型的调节亚基（R） cAMP依赖性蛋白激酶是一种有效的抑制剂 磷酸酶。 使用曲线拟合方法来估计解离 R.磷酸酶，R.Type I催化片段和FA。磷酸酶的常数 复合物。 结果表明R与催化亚基结合。 在 补充，使用CAMP依赖性的计算机模拟循环级联 蛋白激酶和上述磷酸酶作为转化酶表明， 它们构成了高效的监管机构。 （2）对循环级联和简单变构控制的比较研究 发现简单的变构控制不能提供相同的调节 效率如循环级联系统所表现出的效率。 （3）开发了一种简单的处理来描述界面反应 动力学。 该分析揭示了减少的条件 可以期望维度提高整体效率或 双分子反应的反应速率。 （4）使用Subsragment-1的骨骼肌肌动蛋白ATPase循环的研究 代替肌球蛋白表明，一个六态模型由六个模型组成 肌球蛋白结合反应中间体含有ATP的形式 或adp.pi，是描述ATAS​​E所需的最简单的动力学模型 循环。