Modular mutagenesis of exons 1, 2, and 8 of a glutathione S-transferase from the mu class. Mechanistic and structural consequences for chimeras of isoenzyme 3-3.
Modular mutagenesis of exons 1, 2, and 8 of a glutathione S-transferase from the mu class. Mechanistic and structural consequences for chimeras of isoenzyme 3-3.
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DOI：
10.1021/bi00157a005
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发表时间：
1992-10
期刊：
影响因子：
2.9
通讯作者：
Pinghui Zhang;Suxing Liu;Shu-ou Shan;Xinhua Ji;Gary L. Gilliland;Richard N. Armstrong
中科院分区：
文献类型：
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作者：
Pinghui Zhang;Suxing Liu;Shu-ou Shan;Xinhua Ji;Gary L. Gilliland;Richard N. Armstrong
大鼠MU类GSH转移酶的外显子1、外显子2和外显子8分别编码MU类同工酶的序列可变区1和4。这四个可变区中的两个位于该同工酶的N-末端和C-末端，并影响活性部位。为了评估这些可变区对Mu类同工酶催化多样性的影响，通过将4型亚基序列的可变区移植到3型亚基的相应区域，构建了7种嵌合同工酶。嵌合同工酶表现出独特的催化性能。用类型4亚基的可变区4全部或部分替换类型3亚基的可变区4，可得到在亲核芳香取代反应中具有更高周转数的嵌合催化剂。对同工酶3-3晶体结构的分析[Ji，X.，Zhang，P.，Armstrong，R.N.，&Gillland，G.L.(1992)生物化学(本期前文)]表明，在这类反应中，灵活的C-末端尾部与N-末端结构域的相互作用有助于限制同工酶3-3活性部位的产物释放速度。将亚基3的全部或部分序列可变区1替换为亚基4的序列可变区1，产生嵌合同工酶，这种嵌合同工酶模仿同工酶4-4对α，β-不饱和酮的高立体选择性，但不具有催化效率。
Exons 1 and 2 and exon 8 of the mu class GSH transferases from rat encode sequence-variable regions 1 and 4 of mu class isoenzymes, respectively. These two of four variable regions are located at the N- and C-termini of this isoenzyme class and impinge on the active site. In order to assess the influence of these variable regions on the catalytic diversity of the class mu isoenzymes, seven chimeric isoenzymes were constructed by transplantation of the variable regions of the sequence of the type 4 subunit into the corresponding regions of the type 3 subunit. The chimeric isoenzymes exhibit unique catalytic properties. Replacement of all, or part, of variable region 4 of the type 3 subunit with that of the type 4 subunit results in chimeric catalysts with higher turnover numbers in nucleophilic aromatic substitution reactions. Analysis of the crystal structure of isoenzyme 3-3 [Ji, X., Zhang, P., Armstrong, R. N., & Gilliland, G. L. (1992) Biochemistry (preceding paper in this issue)] suggests that interaction of the flexible C-terminal tail with the N-terminal domain helps limit the rate of product release from the active site of isoenzyme 3-3 in this type of reaction. Substitution of all, or part, of the sequence-variable region 1 of subunit 3 with that of subunit 4 results in chimeric isoenzymes that mimic the high stereoselectivity but not the catalytic efficiency of isoenzyme 4-4 toward alpha,beta-unsaturated ketones.(ABSTRACT TRUNCATED AT 250 WORDS)