DISSECTING THE CATALYTIC TRIAD OF A SERINE PROTEASE
DISSECTING THE CATALYTIC TRIAD OF A SERINE PROTEASE
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DOI：
10.1038/332564a0
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发表时间：
1988-04-07
期刊：
影响因子：
64.8
通讯作者：
WELLS, JA
中科院分区：
文献类型：
--
作者：
CARTER, P;WELLS, JA
丝氨酸蛋白酶存在于几乎所有的生物体中，在细胞内外都发挥作用1;它们以两个家族存在，“胰蛋白酶样”和“枯草杆菌蛋白酶样”，它们独立地进化出类似的催化装置2，其特征在于Ser、His、Asp三联体、氧阴离子结合位点以及可能稳定过渡态的其他决定因素（图1）2-4。对于解淀粉芽孢杆菌枯草杆菌蛋白酶，这些功能元件赋予酰胺键的非酶促水解至少109至1010倍的总速率增强。我们已经研究了催化的重要性和催化三联体内的残基之间的相互作用，通过单独或多个取代丙氨酸（S），使用定点诱变5，6的克隆B。解淀粉枯草杆菌蛋白酶基因7.选择丙氨酸取代以使不利的空间接触最小化，并避免从取代的侧链施加新的电荷相互作用或氢键。与参与底物结合的残基中的突变的影响8 -10相反，催化三联体中的突变大大降低了周转数，并且仅对米氏常数产生轻微影响。多个突变体的动力学分析表明，三联体内的残基相互作用协同加速酰胺键水解的一个因素为102 × 106。
Serine proteases are present in virtually all organisms and function both inside and outside the cell1; they exist as two families, the 'trypsin-like' and the 'subtilisin-like', that have independently evolved a similar catalytic device2characterized by the Ser, His, Asp triad, an oxyanion binding site, and possibly other determinants that stabilize the transition state (Fig. l)2–4. ForBacillus amyloliquefacienssubtilisin, these functional elements impart a total rate enhancement of at least 109to 1010times the non-enzymatic hydrolysis of amide bonds. We have examined the catalytic importance and interplay between residues within the catalytic triad by individual or multiple replacement with alanine(s), using site-directed mutagenesis5,6of the clonedB. amyloliquefacienssubtilisin gene7. Alanine substitutions were chosen to minimize unfavourable steric contacts and to avoid imposing new charge interactions or hydrogen bonds from the substituted side chains. In contrast to the effect of mutations in residues involved in substrate binding8–10, the mutations in the catalytic triad greatly reduce the turnover number and cause only minor effects on the Michaelis constant. Kinetic analyses of the multiple mutants demonstrate that the residues within the triad interact synergistically to accelerate amide bond hydrolysis by a factor of ∼2×l06.