POTENTIOMETRIC DETERMINATION OF IONIZATIONS AT ACTIVE-SITE OF PAPAIN
POTENTIOMETRIC DETERMINATION OF IONIZATIONS AT ACTIVE-SITE OF PAPAIN
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DOI：
10.1021/bi00668a010
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发表时间：
1976-01-01
期刊：
影响因子：
2.9
通讯作者：
SHAFER, JA
中科院分区：
文献类型：
--
作者：
LEWIS, SD;JOHNSON, FA;SHAFER, JA
[来自木瓜乳胶] 木瓜蛋白酶活性位点基团的电离行为是根据木瓜蛋白酶质子含量差异和木瓜蛋白酶活性位点硫醇基团甲硫衍生物 (木瓜蛋白酶-S-SCH3) 的 pH 依赖性来确定的。质子含量的这种差异是通过两种独立的方法直接测定的。一种方法涉及用二硫苏糖醇对木瓜蛋白酶-S-SCH3 进行脱甲基硫基化时释放的质子进行电位测量，作为 pH 的函数。另一种方法涉及对木瓜蛋白酶与甲硫基磺酸甲酯甲基硫基化时释放的质子进行类似测量。这些测量产生的甲硫基 pH 差滴定表明木瓜蛋白酶活性位点硫醇基团的电离与 His-159 的电离有关。 His-159 在 29°C 去质子化时，硫醇基团的 pK 从 3.3-7.6 变化。 C、T/2 0.05。同样，当活性位点硫醇基团去质子化时，His-159 的 pK 从 4.3 变为 8.5。本工作测定的Cys-25和His-159的微观电离常数表明，质子从Cys-25转移到His-159的平衡常数为8-12，并且在生理pH范围内，活性位点硫醇基团主要以硫醇阴离子形式存在。
The ionization behavior of groups at the active site of papain [from papaya latex] was determined from the pH dependence of the difference in proton content of papain and the methylthio derivative of the thiol group at the active site of papain (papain-S-SCH3). This difference in proton content was determined directly by 2 independent methods. One method involved potentiometric measurements of the protons released on demthylthiolation of papain-S-SCH3 with dithiothreitol, as a function of pH. The other method involved analogous measurements of the protons released on methylthiolation of papain with methyl methanethiosulfonate. The methylthio pH-difference titrations generated by these measurements indicate that ionization of the thiol group at the active site of papain is linked to the ionization of His-159. The pK of the thiol group changes from 3.3-7.6 on deprotonation of His-159 at 29.degree. C, T/2 0.05. Similarly, the pK of His-159 shifts from 4.3-8.5 when the active site thiol group is deprotonated. The microscopic ionization constants determined in this work for Cys-25 and His-159 indicate the equilibrium constant for transfer of a proton from Cys-25 to His-159 is 8-12, and that in the physiological pH range the active site thiol group exists mainly as a thiol anion.