Use of site-directed mutagenesis to identify valine-573 in the S'1 binding site of rat neutral endopeptidase 24.11 (enkephalinase).
Use of site-directed mutagenesis to identify valine-573 in the S'1 binding site of rat neutral endopeptidase 24.11 (enkephalinase).
复制标题
使用定点诱变鉴定大鼠中性内肽酶 24.11（脑啡肽酶）S1 结合位点中的缬氨酸 573。
DOI：
10.1021/bi00487a009
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Hersh,LB
中科院分区：
文献类型：
--
作者：
Vijayaraghavan,J;Kim,YA;Jackson,D;Orlowski,M;Hersh,LB
德克萨斯大学西南医学中心生物化学系，德克萨斯州达拉斯，75235，和药理系，纽约西奈山医学院，纽约，10029，收到1990年3月9日；修订稿件收到1990年5月9日摘要：根据细菌溶血素和哺乳动物中性内肽酶24.11活性部位内的一段氨基酸序列的同源性，研究了中性内肽酶24.11的valine-573可能参与底物结合。通过定点突变将-573突变为亮氨酸和丙氨酸。用一系列含有可变P‘l残基的化合物检测了这些突变对抑制剂结合和底物催化的影响。在含有丙氨酸等少量P‘l残基的情况下，两种突变酶表现出与野生型酶基本相同的动力学性质。然而，对于更大的P‘l残基，如苯丙氨酸、酪氨酸和亮氨酸，Val573Leu突变体显示出抑制物亲和力的24-100倍下降。同样，含有大量P‘l残基的底物显示出10-40倍的减少，而Km几乎没有变化。相比之下，Val573Ala突变体在抑制剂结合或底物周转方面只显示出轻微的变化。这些结果支持Valine-573作为中性内肽酶24.11的疏水结合口袋--S结合亚基的一部分。
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235, and Department of Pharmacology, Mount Sinai School of Medicine, New York, New York 10029 Received March 9, 1990; Revised Manuscript Received May 9, 1990 abstract: On the basis of the identity of a segment of the amino acid sequence within the active site of the bacterial enzyme thermolysin and the mammalian enzyme neutral endopeptidase 24.11, the possible involvement of valine-573 of neutral endopeptidase 24.11 in substrate binding was investigated. Valine-573 was changed to leucine and to alanine by site-directed mutagenesis. The effect of these mutations on inhibitor binding and substrate catalysis was examined with a series of compounds containing variable P'l residues. With a small P'l residue such as alanine, both mutant enzymes exhibited kinetic properties essentially the same as the wild-type enzyme. However, with larger P'l residues such as phenylalanine, tyrosine, and leucine, the Val573Leu mutant showed a 24-100-fold decrease in inhibitor affinity. Similarly substrates containing bulky P'l residues showed a 10-40-fold decrease in with little change in Km. In contrast, the Val573Ala mutant showed only modest changes in terms of inhibitor binding or substrate turnover. These results support the proposed role of valine-573 as a part of the hydrophobic binding pocket, S'l binding subsite, of neutral endopeptidase 24.11.