SITE-DIRECTED MUTAGENESIS AND THE ROLE OF THE OXYANION HOLE IN SUBTILISIN
SITE-DIRECTED MUTAGENESIS AND THE ROLE OF THE OXYANION HOLE IN SUBTILISIN
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DOI：
10.1073/pnas.83.11.3743
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发表时间：
1986-06-01
影响因子：
11.1
通讯作者：
POULOS, T
中科院分区：
文献类型：
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作者：
BRYAN, P;PANTOLIANO, MW;POULOS, T
利用寡核苷酸定向突变研究丝氨酸蛋白酶枯草杆菌蛋白酶BPN“催化机制中过渡态稳定的性质。来自解淀粉芽孢杆菌的这种胞外酶的基因已被克隆并在枯草芽孢杆菌中表达。在过渡态复合物中，被水解的肽键的羰基被认为采用四面体构型而不是基态平面构型。晶体学研究表明，这种活化复合物的稳定化部分是通过从天冬酰胺-155的酰胺侧基到肽底物的羰基氧的氢键的捐赠来完成的。为了具体地测试该假设，在位置155处引入亮氨酸。亮氨酸与天冬酰胺是电子等排的，但不能向四面体中间体提供氢键。发现Leu-155变体具有不变的Km，但当用肽底物测定时，催化速率常数kcat大大降低（小200-300倍）。这些动力学结果与Asn-155介导的活化复合物的稳定性是一致的，并为酶催化的过渡态稳定假说提供了进一步的实验支持。
Oligonucleotide-directed mutagenesis was used to investigate the nature of transition state stabilization in the catalytic mechanism of the serine proteinase, subtilisin BPN''. The gene for this extracellular enzyme from Bacillus amyloliquefaciens has been cloned and expressed in Bacillus subtilis. In the transition state complex, the carbonyl group of the peptide bond to be hydrolyzed is believed to adopt a tetrahedral configuration rather than the ground-state planar configuration. Crystallographic studies suggest that stabilization of this activated complex is accomplished in part through the donation of a hydrogen bond from the amide side group of Asn-155 to the carbonyl oxygen of the peptide substrate. To specifically test this hypothesis, leucine was introduced at position 155. Leucine is isosteric with asparagine but is incapable of donating a hydrogen bond to the tetrahedral intermediate. The Leu-155 variant was found to have an unaltered Km but a greatly reduced catalytic rate constant, kcat, (factor of 200-300 smaller) when assayed with a peptide substrate. These kinetic results are consistent with the Asn-155 mediating stabilization of the activated complex and lend further experimental support for the transition-state stabilization hypothesis of enzyme catalysis.