Novel allosteric properties produced by residue substitutions in the subunit interface of yeast NAD+-specific isocitrate dehydrogenase
Novel allosteric properties produced by residue substitutions in the subunit interface of yeast NAD+-specific isocitrate dehydrogenase
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DOI：
10.1016/j.abb.2006.06.022
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发表时间：
2006-09-15
影响因子：
3.9
通讯作者：
McAlister-Henn, Lee
中科院分区：
文献类型：
--
作者：
Hu, Gang;McAlister-Henn, Lee
酵母NAD（+）特异性异柠檬酸脱氢酶（IDH）是由4个IDH1和4个IDH2亚基组成的八聚体，酶的基本结构单元是IDH1/IDH2异二聚体。为了研究IDH1和IDH2之间相互作用的一个方面，异源二聚体界面疏水区域的残基（IDH1中的Val-216、Ser-220和Val-224； IDH2中的Ile-221、Val-225和Val-229）被每个亚基和两个亚基中的丙氨酸残基取代。凝胶过滤和沉降速度分析表明，残基取代不会破坏IDH的八聚体结构。然而，这些取代产生了新的动力学性质，包括在辅助因子方面，AMP的正变构调节和在没有AMP的情况下的协同性。这些变构性质在NAD（+）结合实验中也很明显。尽管在这两个亚基中含有残基取代的突变酶具有大量可测量的活性，但该酶的表达产生的生长表型表明体内IDH功能障碍。(c) 2006爱思唯尔公司版权所有。
Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octamer of four IDH1 and four IDH2 subunits, and the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer. To investigate one aspect of the interaction between IDH1 and IDH2, residues in a hydrophobic region at the heterodimer interface (Val-216, Ser-220, and Val-224 in IDH1; Ile-221, Val-225, and Val-229 in IDH2) were replaced by alanine residues in each and in both subunits. Gel filtration and sedimentation velocity analyses demonstrated that the residue substitutions do not disrupt the octameric structure of IDH. However, these substitutions produce novel kinetic properties including, with respect to cofactor, positive allosteric regulation by AMP and cooperativity in the absence of AMP. These allosteric properties are also apparent in NAD(+)-binding experiments. Despite substantial measurable activity for the mutant enzyme containing residue substitutions in both subunits, expression of this enzyme produces growth phenotypes indicative of IDH dysfunction in vivo. (c) 2006 Elsevier Inc. All rights reserved.