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Change of lactate dehydrogenase function by protein engineering

通过蛋白质工程改变乳酸脱氢酶功能

基本信息

批准号：
08660120
负责人：
TAGUCHI Hayao
金额：
$ 1.34万
依托单位：
Science University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08660120/
关键词：
lactate dehydrogenase catalytic center substrate specificity stereochemistry allosteric enzyme enzyme regulation 3-D protein structure change of enzyme function アロステリック

项目摘要

ln Lactobocillus pentosus D-LDH,replacements of Phe299 induced marked reductions in the affinity to 2-ketoacid substrates and the catalytic velocity, and also changed primary isotope effects on the enzyme reaction, indicating that Phe299 plays essential roles to stimulate the binding of 2-ketoacid substrates and subsequent catalytic reaction. The replacements Particularly markedly damaged the pyruvate reduction among the reactions for 2-ketoacids, indicating that Phe299 plays also important role in the specific recognition of the substrate C3 moiety. Replacement of Serl02 induced a significant positive cooperativity on the substrate binding, indicating that the structure of the substrate binding site is coordinately changed with the whole protein structure. In L.casei allosteric L-LDH,on the other hand, replacement of His205 in the regulatory site induced a great loss of the homotropic regulation by pyruvate, and thereby change the enzyme to a absolutely fructose 1,6-bisphosphate-dependent enzyme. Analysis by the enzyme kinetics and chemical modification strongly suggested that His205 is involved in the pyruvate binding not in an inactive state, but in an active state of the enzyme structure. Therefore, His205 possibly triggered the allosteric transition through the interactions with pyruvate. In L.pentosus non-allosteric L-LDH,which shows high sequence similarity with L.casei L-LDH,the three-dimensional structure could be built through its crystarographic analysis. To also determine three dimensional structure of L.casei L-LDH,in addition, the crystals of the enzyme, the maximal size of which was I.25 mm, were obtained by screening optimal conditions for crystallization.

在戊糖乳杆菌D-LDH中，Phe299的取代导致与2-酮酸底物的亲和力和催化速度显著降低，并改变了酶反应的初级同位素效应，表明Phe299在刺激2-酮酸底物的结合和随后的催化反应中起着关键作用。在2-酮酸的反应中，Phe299对丙酮酸还原反应的破坏尤为明显，表明Phe299在底物C3部分的特异性识别中也起着重要作用。SerL02的取代对底物结合有显著的正协同作用，说明底物结合部位的结构与整个蛋白质结构发生了协调变化。另一方面，在干酪乳杆菌变构的L-乳酸脱氢酶中，调节位点His205的替换导致丙酮酸对同质性调节的极大丧失，从而使该酶转变为绝对果糖1，6-二磷酸依赖的酶。酶动力学和化学修饰的分析有力地表明，His205参与丙酮酸的结合不是处于非活性状态，而是处于酶结构的活性状态。因此，His205可能通过与丙酮酸的相互作用而触发变构转变。与干酪乳杆菌L-乳酸脱氢酶序列高度相似的非变构L-乳酸脱氢酶，通过其冰冻分析可以建立其三维结构。此外，为了确定干酪乳杆菌L-乳酸脱氢酶的三维结构，通过对最佳结晶条件的筛选，得到了最大尺寸为1.25 mm的酶晶体。