ENERGETICS OF RIBONUCLEASE-A CATALYSIS .1. PH, IONIC-STRENGTH, AND SOLVENT ISOTOPE DEPENDENCE OF THE HYDROLYSIS OF CYTIDINE CYCLIC 2',3'-PHOSPHATE
ENERGETICS OF RIBONUCLEASE-A CATALYSIS .1. PH, IONIC-STRENGTH, AND SOLVENT ISOTOPE DEPENDENCE OF THE HYDROLYSIS OF CYTIDINE CYCLIC 2',3'-PHOSPHATE
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DOI：
10.1021/bi00291a011
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发表时间：
1983-01-01
期刊：
影响因子：
2.9
通讯作者：
BILTONEN, RL
中科院分区：
文献类型：
--
作者：
EFTINK, MR;BILTONEN, RL
主要通过使用流动微量热法来监测动力学，研究了 RNase A 催化的 2'',3''-cCMP 水解稳态动力学的 pH、离子强度和溶剂氘同位素依赖性。通过假设酶的 His-12 和 -119 以及第三个电离基团（假定位于底物的嘧啶环上）的参与来分析 Michaelis-Menten 参数的 pH 依赖性，以确定与 pH 无关的速率常数 kc 和 Km。报道的 pH 分析，连同现有的 NMR 数据和化学修饰研究，允许将 His-12 和 -119 的功能角色分别指定为一般酸和一般碱催化残基的功能。在高 pH 值下，表观 Km 值增加至 1。高 pH 条件下酶和底物之间的亲和力下降表明底物主要通过与活性位点组氨酸残基（尤其是 His-12）的静电相互作用与酶结合。与底物的核苷部分明显不存在相互作用可能是由于底物以其糖苷键以顺式构象存在，因此不能与酶的结合袋最佳地相互作用。这将导致酶-底物复合物相对不稳定，然后可以在过渡态形成后缓解。 RNase 活性的离子强度依赖性主要是其对组氨酸残基的 pKa 的影响以及随之而来的 Km 值变化的结果。
The pH, ionic strength and solvent deuterium isotope dependence of the steady-state kinetics of the RNase A-catalyzed hydrolysis of 2'',3''-cCMP was investigated by using, primarily, the technique of flow microcalorimetry to monitor the kinetics. The pH dependence of the Michaelis-Menten parameters was analyzed by assuming the participation of His-12 and -119 of the enzyme and a third ionizing group, postulated to be on the pyrimidine ring of the substrate, to determine the pH-independent rate constant, kc, and Km. The reported pH analysis, together with existing NMR data and chemical modification studies, allows an assignment of the functional roles of His-12 and -119 as being those of general acid and general base catalytic residues, respectively. At high pH, the apparent Km value increases to unity. This drop in affinity between the enzyme and the substrate at high pH indicates that the substrate binds to the enzyme primarily through an electrostatic interaction with the active-site histidine residues, particularly His-12. The apparent absence of an interction with the riboside portion of the substrate may be due to the fact that the substrate exists in a syn conformation about its glycosidic bond and thus cannot interact optimally with the enzyme''s binding pocket. This will result in a relative destabilization of the enzyme-substrate complex, which can then be relieved upon the formation of the transition state. The ionic strength dependence of RNase activity is primarily a result of its effect on the pKa of the histidine residues and a concomitant change in the value of Km.