Vulbfson P L, Skolysheva L K, Severin S E
Biokhimiia. 1980 Nov;45(11):1923-33.
The interaction of phosphorylase B with the SH-reagents, i.e. 2-chloromercuri-4-nitrophenol and ethylmercurichloride was studied. It was shown that phosphorylase B inhibition obeys the pseudo-first-order kinetics, the inactivation rate constants being equal to 11 M-1 s-1 and 17,5 M-1 s-1, respectively. Data from the SH-group titration with 2-chloromercuri-4-nitrophenol and p-chloromercuri benzoate suggest that the number of modified cysteine residues and the amount of bound 2-chloromercuri-4-nitrophenol in the phosphorylase B dimer is equal to 2. In the modified phosphorylase B the absorption maximum of pyridoxal phosphate is decreased at 330 nm and is increased at 410 nm. The binding of 2-chloromercuri-4-nitrophenol is accompanied by quenching of the protein and coenzyme fluorescence. Upon interaction with ethylmercurichloride only the pyridoxalphosphate fluorescence is quenched. The increase of the spin label mobility in the modified enzyme calculated from the EPR spectra of the spin-labelled preparations is indicative of the changes in the protein conformation coupled with the blocking of one SH-group in the enzyme monomer. The rate of enzyme inactivation under effects of the SH-reagents is a function of pH and is considerably increased within the pH range of 5.7-6.7. The pH-optimum of activity of partly modified enzyme remains practically unchanged; however, at the pH shift towards the acidic values the activity is drastically decreased as compared to that of the native enzyme. The data obtained suggest that the enzyme inactivation is due to modification of one SH-group in the phosphorylase B monomer vicinal to the pyridoxal phosphate binding site and probably involved in the enzymatic reaction.
研究了磷酸化酶B与巯基试剂，即2-氯汞基-4-硝基苯酚和乙基氯化汞的相互作用。结果表明，磷酸化酶B的抑制作用符合准一级动力学，失活速率常数分别为11 M-1 s-1和17.5 M-1 s-1。用2-氯汞基-4-硝基苯酚和对氯汞基苯甲酸进行巯基滴定的数据表明，磷酸化酶B二聚体中被修饰的半胱氨酸残基数量和结合的2-氯汞基-4-硝基苯酚量均为2。在修饰后的磷酸化酶B中，磷酸吡哆醛的最大吸收峰在330 nm处降低，在410 nm处升高。2-氯汞基-4-硝基苯酚的结合伴随着蛋白质和辅酶荧光的猝灭。与乙基氯化汞相互作用时，只有磷酸吡哆醛的荧光被猝灭。根据自旋标记制剂的电子顺磁共振光谱计算得出的修饰酶中自旋标记流动性的增加表明，蛋白质构象发生了变化，同时酶单体中的一个巯基被封闭。巯基试剂作用下酶的失活速率是pH的函数，在pH 5.7-6.7范围内显著增加。部分修饰酶的活性pH最佳值实际上保持不变；然而，与天然酶相比，当pH向酸性值移动时，活性会急剧下降。所获得的数据表明，酶的失活是由于磷酸化酶B单体中与磷酸吡哆醛结合位点相邻且可能参与酶促反应的一个巯基被修饰所致。
