Di Cera E, De Cristofaro R, Albright D J, Fenton J W
Department of Biochemistry, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry. 1991 Aug 13;30(32):7913-24. doi: 10.1021/bi00246a007.
The amidase activity of human alpha-thrombin has been studied at steady state in the pH range 6-10, as a function of NaCl concentration from 1 mM to 1 M and temperature from 10 to 40 degrees C. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over this pH range with a minimum around pH 7.5 in the presence of 0.1 M NaCl at 25 degrees C. The catalytic constant, kcat, also has a bell-shaped pH dependence with multiple inflection points that are more evident at low NaCl concentrations and a maximum around pH 8.2 in the presence of 0.1 M NaCl at 25 degrees C. A detailed analysis of the results in terms of a general linkage scheme has allowed a thorough characterization of the linkage between proton and substrate binding and its dependence on NaCl concentration, as well as the relevant entropic and enthalpic contributions to binding and catalytic events. Formulation of detailed partition functions for each enzyme intermediate involved in the catalytic cycle suggests that (at least) three groups are responsible for the control of thrombin amidase activity as a function of pH. One group is to be identified with the active site His, due to its pK values in the free enzyme and the adduct and its enthalpy of ionization. The effect of NaCl concentration on amidase activity seems to be extremely specific. Comparative steady-state measurements carried out in the presence of NaCl, NaBr, NaI, KCl, and MgCl2 show that human alpha-thrombin is capable of discriminating among different cations and anions. This suggests that small ions participate as allosteric effectors in the regulation of thrombin activity. The linkage with NaCl is strongly pH dependent and increases with decreasing pH. The present results provide information on the basic aspects of human alpha-thrombin activity and regulation and enable a rigorous thermodynamic approach to other important regulatory interactions in human alpha-thrombin and its structurally perturbed derivatives.
已在pH值6 - 10范围内对人α-凝血酶的酰胺酶活性进行了稳态研究，该活性是NaCl浓度（从1 mM至1 M）和温度（从10至40摄氏度）的函数。米氏常数Km在此pH范围内呈现钟形依赖性，在25摄氏度下0.1 M NaCl存在时，在pH 7.5左右达到最小值。催化常数kcat也具有钟形pH依赖性，有多个拐点，在低NaCl浓度下更明显，在25摄氏度下0.1 M NaCl存在时，在pH 8.2左右达到最大值。根据通用连锁方案对结果进行的详细分析，已全面表征了质子与底物结合之间的连锁及其对NaCl浓度的依赖性，以及对结合和催化事件的相关熵和焓贡献。为催化循环中涉及的每个酶中间体制定详细的分配函数表明，（至少）有三组因素负责控制凝血酶酰胺酶活性随pH的变化。由于其在游离酶、加合物中的pK值及其电离焓，其中一组与活性位点His相关。NaCl浓度对酰胺酶活性的影响似乎极具特异性。在NaCl、NaBr、NaI、KCl和MgCl2存在下进行的比较稳态测量表明，人α-凝血酶能够区分不同的阳离子和阴离子。这表明小离子作为变构效应剂参与凝血酶活性的调节。与NaCl的连锁强烈依赖于pH，并随pH降低而增加。目前的结果提供了关于人α-凝血酶活性和调节基本方面的信息，并使得能够对人α-凝血酶及其结构扰动衍生物中的其他重要调节相互作用采用严格的热力学方法。
