Johnson F A, Lewis S D, Shafer J A
Biochemistry. 1981 Jan 6;20(1):44-8. doi: 10.1021/bi00504a008.
Proton NMR spectroscopy was used to study the ionization behavior of His-159 in a derivative of papain (papain-S-SCH3). In this catalytically inactive derivative of papain, the active-site thiol group of Cys-25 is S-methyl-thiolated so that it cannot form a thiolate anion. The pH dependence of the chemical shift of the C epsilon 1 H resonance of His-159 indicated a pK of 3.45 +/- 0.07 at 45 degrees C in 2H2O with no added ions other than those required for titration. In acetate buffers at an ionic strength of 0.05, the pK increased to 3.87 +/- 0.12. Conversion of papain-S-SCH3 to active papain at pH* 4.17 (at 45 degrees C and an ionic strength of 0.05) caused the position of the C epsilon 1 H resonance to change from a position indicative of partial protonation of His-159 to a position indicative of full protonation, consistent with the existence of an imidazolium-thiolate ion-pair interaction between His-159 and Cys-25 in the active enzyme.
质子核磁共振光谱法被用于研究木瓜蛋白酶衍生物（木瓜蛋白酶-S-SCH3）中His-159的电离行为。在这种木瓜蛋白酶的催化无活性衍生物中，Cys-25的活性位点硫醇基团被S-甲基硫醇化，因此它不能形成硫醇阴离子。His-159的Cε1 H共振化学位移对pH的依赖性表明，在45℃的2H2O中，除滴定所需的离子外不添加其他离子时，其pK为3.45±0.07。在离子强度为0.05的醋酸盐缓冲液中，pK增加到3.87±0.12。在pH* 4.17（45℃，离子强度为0.05）下，木瓜蛋白酶-S-SCH3转化为活性木瓜蛋白酶，导致Cε1 H共振位置从指示His-159部分质子化的位置变为指示完全质子化的位置，这与活性酶中His-159和Cys-25之间存在咪唑鎓-硫醇阴离子离子对相互作用一致。
