Wikstrom P, Kirschke H, Stone S, Shaw E
Friedrich Miescher-Institut, Basel, Switzerland.
Arch Biochem Biophys. 1989 Apr;270(1):286-93. doi: 10.1016/0003-9861(89)90030-1.
Earlier work has demonstrated the irreversible inactivation of serine and cysteine proteinases by peptides with a C-terminal chloromethyl ketone group. With a C-terminal diazomethyl ketone, on the other hand, peptides become reagents specific for cysteine proteinases. We have now synthesized and examined the properties of reagents with an additional methyl side chain near the reactive grouping with the goal of diminishing side reactions in a cellular environment. Derivatives of neutral amino acids as well as of lysine and arginine have been prepared. The chloroethyl ketones are about 60% less reactive to chemical nucleophiles than the chloromethyl ketones. However, the susceptibilities of the proteases examined varied remarkably. Cathepsins B and L of the papain family of cysteine proteinases were much less susceptible (about 2 orders of magnitude less) to both peptidyl diazoethyl and chloroethyl ketones. In marked contrast, clostripain, a cysteine proteinase of a separate family was decisively more susceptible to chloroethyl ketones. The serine proteinases showed a drop in susceptibility to the chloroethyl ketones generally, and this was similar to the drop in chemical reactivity in proceeding from the chloromethyl to the chloroethyl ketone.
早期的研究工作已证明，带有C端氯甲基酮基团的肽可使丝氨酸和半胱氨酸蛋白酶发生不可逆失活。另一方面，带有C端重氮甲基酮的肽则成为半胱氨酸蛋白酶的特异性试剂。我们现已合成并研究了在反应基团附近带有额外甲基侧链的试剂的性质，目的是减少细胞环境中的副反应。已制备了中性氨基酸以及赖氨酸和精氨酸的衍生物。氯乙基酮对化学亲核试剂的反应性比氯甲基酮低约60%。然而，所检测的蛋白酶的敏感性差异很大。木瓜蛋白酶家族的半胱氨酸蛋白酶组织蛋白酶B和L对肽基重氮乙基酮和氯乙基酮的敏感性都要低得多（约低2个数量级）。与之形成鲜明对比的是，另一个家族的半胱氨酸蛋白酶梭菌蛋白酶对氯乙基酮的敏感性明显更高。丝氨酸蛋白酶对氯乙基酮的敏感性总体上有所下降，这与从氯甲基酮到氯乙基酮时化学反应性的下降情况相似。
