Malliopoulou T B, Rakitzis E T
Department of Biological Chemistry, University of Athens Medical School, Greece.
J Enzyme Inhib. 1988;2(2):99-115. doi: 10.3109/14756368809040716.
Bovine liver rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) is modified by 2,4,6-trinitrobenzenesulphonic acid, by the use of modifying agent concentrations in large excess over enzyme protein concentration. The end-point of the reaction, viz., the number, n, per enzyme protein molecule, of modifiable amino groups was determined graphically by the Kézdy-Swinbourne procedure. It was found that the value for n depends on the pH of the reaction medium, and ranges from 2, at pH 7.00, to 10.66, at pH 9.00. Again, the value for n increases with an increase in the concentration of 2,4,6-trinitrobenzenesulphonic acid used, with values ranging from 3.52, at 0.10 mM modifying agent, to 8.96, at 2 mM modifying agent. Rhodanese primary amino groups modification by 2,4,6-trinitrobenzenesulphonic acid is described by a summation of exponential functions of reaction time at pH values of 8.00 or higher, while at lower pH values it is described by a single exponential function of reaction time. However, the log of the first derivative, at initial reaction conditions, of the equation describing protein modification, is found to be linearly dependent on the pH of the reaction. An identical linear dependence is also found when the log of the first derivative, at the start of the reaction, of the equation describing modification-induced enzyme inactivation is plotted against the pH values of the medium used. In consequence, the fractional concentration of rhodanese modifiable amino groups essential for enzyme catalytic function is equal to unity at all reaction pH values tested. It is accordingly concluded that, when concentrations of 2,4,6-trinitrobenzenesulphonic acid in excess of protein concentration are used, all rhodanese modifiable amino groups are essential for enzyme activity. A number of approaches were used in order to establish a mechanism for the modification-induced enzyme inactivation observed. These approaches, all of which proved to be negative, include the possible modification of enzyme sulfhydryl groups, disulphide bond formation, enzyme inactivation due to sulphite released during modification, modification-induced enzyme protein polymerization, syncatalytic enzyme modification and hydrogen peroxide-mediated enzyme inactivation.
通过使用大大过量于酶蛋白浓度的修饰剂浓度，牛肝硫氰酸酶（硫代硫酸盐硫转移酶，EC 2.8.1.1）可被2,4,6 - 三硝基苯磺酸修饰。反应终点，即每个酶蛋白分子上可修饰氨基的数量n，通过凯兹迪 - 斯温伯恩方法以图形方式确定。发现n值取决于反应介质的pH值，在pH 7.00时为2，在pH 9.00时为10.66。同样，n值随着所用2,4,6 - 三硝基苯磺酸浓度的增加而增加，在0.10 mM修饰剂时为3.52，在2 mM修饰剂时为8.96。在pH值为8.00或更高时，2,4,6 - 三硝基苯磺酸对硫氰酸酶伯氨基的修饰由反应时间的指数函数之和描述，而在较低pH值时由反应时间的单一指数函数描述。然而，发现在初始反应条件下，描述蛋白质修饰的方程的一阶导数的对数与反应的pH值呈线性相关。当将描述修饰诱导的酶失活的方程在反应开始时的一阶导数的对数与所用介质的pH值作图时，也发现了相同的线性相关性。因此，在所有测试的反应pH值下，对于酶催化功能至关重要的硫氰酸酶可修饰氨基的分数浓度等于1。据此得出结论，当使用过量于蛋白质浓度的2,4,6 - 三硝基苯磺酸浓度时，所有硫氰酸酶可修饰氨基对于酶活性都是必不可少的。为了建立观察到的修饰诱导的酶失活的机制，使用了多种方法。所有这些方法都被证明是阴性的，包括酶巯基的可能修饰、二硫键形成、修饰过程中释放的亚硫酸盐导致的酶失活、修饰诱导的酶蛋白聚合、同步催化酶修饰以及过氧化氢介导的酶失活。
