THE CATALYTIC ROLE OF THE ACTIVE-SITE ASPARTIC-ACID IN SERINE PROTEASES
THE CATALYTIC ROLE OF THE ACTIVE-SITE ASPARTIC-ACID IN SERINE PROTEASES
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DOI：
10.1126/science.3303334
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发表时间：
1987-08-21
期刊：
影响因子：
56.9
通讯作者：
RUTTER, WJ
中科院分区：
文献类型：
--
作者：
CRAIK, CS;ROCZNIAK, S;RUTTER, WJ
丝氨酸蛋白酶催化三联体Asp、His和Ser中天冬氨酸残基的作用已通过定点诱变用Asn取代胰蛋白酶的Asp 102进行了测试。在异源表达系统中产生天然存在的和突变的酶，纯化至同质，并表征。在中性pH下，突变体酶活性与酯底物和与Ser 195特异性试剂二异丙基氟磷酸是约104倍小于未修饰的酶。与Ser 195反应性的急剧丧失相反，突变体胰蛋白酶与His 57特异性试剂甲苯磺酰基-L-赖氨酸氯甲基酮反应，效率仅为未修饰酶的五倍。因此，His 57与该亲和标记反应的能力并未严重受损。突变酶的催化活性随着pH的增加而增加，因此在pH 10.2时，kcat是胰蛋白酶的6%。动力学分析表明，这种新的活动表明，这是由于部分参与的可滴定的碱或氢氧离子的催化机制。通过证明天冬氨酸残基在催化中的重要性，特别是在生理pH下，这些实验为催化三联体的进化保守提供了合理化。
The role of the aspartic acid residue in the serine protease catalytic triad Asp, His, and Ser has been tested by replacing Asp102 of trypsin with Asn by site-directed mutagenesis. The naturally occurring and mutant enzymes were produced in a heterologous expression system, purified to homogeneity, and characterized. At neutral pH the mutant enzyme activity with an ester substrate and with the Ser195-specific reagent diisopropylfluorophosphate is approximately 104 times less than that of the unmodified enzyme. In contrast to the dramatic loss in reactivity of Ser195, the mutant trypsin reacts with the His57-specific reagent, tosyl-L-lysine chloromethylketone, only five times less efficiently than the unmodified enzyme. Thus, the ability of His57 to react with this affinity label is not severely compromised. The catalytic activity of the mutant enzyme increases with increasing pH so that at pH 10.2 the kcat is 6 percent that of trypsin. Kinetic analysis of this novel activity suggests this is due in part to participation of either a titratable base or of hydroxide ion in the catalytic mechanism. By demonstrating the importance of the aspartate residue in catalysis, especially at physiological pH, these experiments provide a rationalization for the evolutionary conservation of the catalytic triad.