KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

(1)本文介绍了一种采用高效液相色谱技术的改进的纯化方法。 建立了一种纯化Mg（II）-ATP依赖性蛋白激酶的方法 骨骼肌。 纯化的酶由38和31 KDa亚基组成， 其分别对应于催化和调节子单元。 一 失活酶的激活机制涉及一种瞬时的 激酶Fa催化的调节子亚基的磷酸化。 几 支持磷酸化的证据表明， 亚基转化为其活性形式包括观察到， 通过限制蛋白水解的调节剂不能激活失活的（天然的） 酶，除非它已首先磷酸化或硫代磷酸化。 支持缓慢异构化的证据，这种异构化导致活性酶 它的非活性形式被证明。 II型调节亚基（R） cAMP依赖性蛋白激酶是一种有效的抑制剂， 磷酸酶。 用曲线拟合法估计解离常数 R.磷酸酶、R. I型催化片段和Fa.磷酸酶的常数 配合物 结果表明，R结合的催化亚基。 在 此外，使用cAMP依赖性的循环级联的计算机模拟， 蛋白激酶和上述磷酸酶作为转化酶揭示， 构成了一个高效的监管机制。 (2)循环级联与简单变构调控的比较研究 表明简单的变构控制不能提供相同的调节作用， 效率的循环级联系统所表现出的。 (3)提出了一种描述界面反应的简单方法 动力学 这一分析揭示了在哪些条件下， 可以预期的维度，以提高整体效率或 双分子反应的反应速率。 (4)用亚片段-1研究骨骼肌肌动球蛋白ATP酶循环 代替肌球蛋白揭示了一个六态模型，它由六个 肌球蛋白结合的反应中间体，含有ATP形式的磷酸盐 或ADP.Pi，是描述AT酶所需的最简单的动力学模型 周期

(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.