ENGINEERING ENZYME SPECIFICITY BY SUBSTRATE-ASSISTED CATALYSIS
ENGINEERING ENZYME SPECIFICITY BY SUBSTRATE-ASSISTED CATALYSIS
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DOI：
10.1126/science.3299704
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发表时间：
1987-07-24
期刊：
影响因子：
56.9
通讯作者：
WELLS, JA
中科院分区：
文献类型：
--
作者：
CARTER, P;WELLS, JA
提出了一种新的工程酶特异性的方法，其中酶的催化基团首先通过定点诱变引起失活而被去除。活性然后通过含有缺失的催化官能团的底物部分恢复。用N-琥珀酰-L-Phe-L-Ala-L-Ala-L-Phe-p-nitroanilide（sFAAF-pNA）测定时，通过定点诱变将解淀粉芽孢杆菌枯草杆菌蛋白酶基因中的催化His 64替换为Ala（突变体命名为His 64 Ala），使催化效率（kcat/Km）降低一百万倍。模型建立的研究表明，在P2位置的底物结合在枯草杆菌蛋白酶的活性位点的His侧链几乎可以叠加在催化的丝氨酸蛋白酶的His侧链。因此，在pH 8.0下，His 64 Ala突变体水解His P2底物（sFAHF-pNA）比同源Ala P2或Gln P2底物（sFAAF-pNA或sFAQF-pNA）快高达400倍。相比之下，野生型酶水解这三种底物具有相似的催化效率。从substrate-dependentpH配置文件和水解的大多肽的其他数据表明，His 64 Ala突变体酶可以恢复部分失去的催化组氨酸的功能，从HisP 2侧链上的基板。这种“底物辅助催化”为工程酶提供了一个新的基础，具有非常狭窄的和潜在有用的底物特异性。这些研究还表明，在丝氨酸蛋白酶的催化三联体的进化可能的功能中间体。
A novel approach to engineering enzyme specificity is presented in which a catalytic group from an enzyme is first removed by site-directed mutagenesis causing inactivation. Activity is then partially restored by substrates containing the missing catalytic functional group. Replacement of the catalytic His64with Ala in theBacillus amyloliquefacienssubtilisin gene (the mutant is designated His64Ala) by site-directed mutagenesis reduces the catalytic efficiency (kcat/Km) by a factor of a million when assayed withN-succinyl-L-Phe-L-Ala-L-Ala-L-Phe-p-nitroanilide (sFAAF-pNA). Model building studies showed that a His side chain at the P2 position of a substrate bound at the active site of subtilisin could be virtually superimposed on the catalytic His side chain of this serine protease. Accordingly, the His64Ala mutant hydrolyzes a His P2 substrate (sFAHF-pNA) up to 400 times faster than a homologous Ala P2 or Gln P2 substrate (sFAAF-pNA or sFAQF-pNA) atpH 8.0. In contrast, the wild-type enzyme hydrolyzes these three substrates with similar catalytic efficiencies. Additional data from substrate-dependentpH profiles and hydrolysis of large polypeptides indicate that the His64Ala mutant enzyme can recover partially the function of the lost catalytic histidine from a His P2 side chain on the substrate. Such "substrate-assisted catalysis" provides a new basis for engineering enzymes with very narrow and potentially useful substrate specificities. These studies also suggest a possible functional intermediate in the evolution of the catalytic triad of serine proteases.