Koeppe R E, Stroud R M
Biochemistry. 1976 Aug 10;15(16):3450-8. doi: 10.1021/bi00661a009.
The pKa of aspartyl-102 in trypsin is shown to be 6.8+/-0.2 by difference infrared titration. All but 2.5 of the carboxyls in bovine trypsin were first modified with semicarbazide. The modified enzyme still retains full activity toward nonspecific substrates. The remaining free carboxyls include one equivalent each of Asp-102 and Asp-194. The absorbances associated with the C=O and C=O stretching modes at 1570 and 1710 cm-1 were used to monitor the proportion of ionized or protonated carboxyl present in the enzyme as a function of pD. The pKa of 6.8 was assigned to Asp-102 using copper ions that bind to trypsin between Asp-102 and histidine-57, so lowering the pKapp of Asp-102. The implication of this result for the ionization of the active site, and for the mechanism of serine proteases, is discussed. Asp-194 and the C terminus are shown to titrate with an average pKa of 2.9.
通过差示红外滴定法测得，胰蛋白酶中天冬氨酸-102的pKa为6.8±0.2。先用氨基脲对牛胰蛋白酶中除2.5个羧基外的所有羧基进行修饰。修饰后的酶对非特异性底物仍保留全部活性。剩余的游离羧基包括一个天冬氨酸-102和一个天冬氨酸-194。利用在1570和1710 cm-1处与C=O和C=O伸缩模式相关的吸光度，监测酶中离子化或质子化羧基的比例随pD的变化。利用结合在天冬氨酸-102和组氨酸-57之间的铜离子，将6.8的pKa归属于天冬氨酸-102，从而降低了天冬氨酸-102的表观pKa。讨论了该结果对活性位点电离以及丝氨酸蛋白酶作用机制的影响。天冬氨酸-194和C末端的滴定平均pKa为2.9。
