DIRECT CALORIMETRIC ANALYSIS OF THE ENZYMATIC-ACTIVITY OF YEAST CYTOCHROME-C-OXIDASE
DIRECT CALORIMETRIC ANALYSIS OF THE ENZYMATIC-ACTIVITY OF YEAST CYTOCHROME-C-OXIDASE
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DOI：
10.1021/bi00098a030
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发表时间：
1991-08-27
期刊：
影响因子：
2.9
通讯作者：
FREIRE, E
中科院分区：
文献类型：
--
作者：
MORIN, PE;FREIRE, E
酵母细胞色素c氧化酶与其生物底物，亚铁细胞色素c，酶促反应的动力学和热力学参数已被测量，通过使用滴定微量热计直接监测产热率或吸收作为时间的函数。该技术允许在单个实验中确定各种条件下的反应的能量学和动力学。在不同电离度的缓冲系统中进行的实验允许确定反应过程中吸收或释放的质子的净数量。对于细胞色素c氧化酶，测定了反应的固有焓为bc-16.5 kcal/mol，每个氧化的亚铁细胞色素c分子消耗一个（0.96）质子。在10 mM磷酸钾（pH 7.40）和0.5 mM EDTA存在下，在0至200 mM KCl盐浓度范围内的活性测量显示电子转移酶活性对离子强度的双相依赖性，在50 mM KCl附近观察到峰值活性。离子强度的依赖性是相似的洗涤剂溶解和膜重构细胞色素c氧化酶。尽管大的离子强度依赖的动力学参数，测量的反应焓被发现是独立的离子强度。涉及将酶从低盐条件直接转移到高盐条件的额外实验产生可忽略的焓变化，其在整个研究的盐浓度（0-200 mM KCl）内在实验误差内保持恒定。这些结果表明，盐对酶活性的影响是熵的起源，并进一步表明，由于离子强度的变化在酶中的主要构象变化的情况下。在非常低的离子强度下进行的高灵敏度差示扫描量热实验显示了以50.5和60 ℃为中心的两个明确定义的峰为特征的量热转变曲线。在高离子强度下，高温峰向较低温度（57.4 ℃）移动，产生单一但略微不对称的过渡曲线。在所研究的实验条件下，未观察到低温峰的转变温度发生偏移。这些结果表明，离子强度的变化主要影响蛋白质亚基的结构稳定性，产生主要的过渡峰，主要是亚基I和II。
The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to bc -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCI. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCI). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength. High-sensitivity differential scanning calorimetric experiments performed at very low ionic strength show a calorimetric transition profile characterized by two well-defined peaks centered at 50.5 and 60-degrees-C. At high ionic strength, the high-temperature peak shifts to lower temperatures (57.4-degrees-C), giving rise to a single but slightly asymmetric transition profile. No shift in the transition temperature was observed for the low-temperature peak under the experimental conditions studied. These results suggest that variation of ionic strength primarily affects the structural stability of the protein subunits giving rise to the main transition peak, primarily subunits I and II.