Physiological consequences of loss of allosteric activation of yeast NAD+-specific isocitrate dehydrogenase
Physiological consequences of loss of allosteric activation of yeast NAD+-specific isocitrate dehydrogenase
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DOI：
10.1074/jbc.m512281200
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发表时间：
2006-06-23
影响因子：
4.8
通讯作者：
McAlister-Henn, Lee
中科院分区：
文献类型：
--
作者：
Hu, Gang;Lin, An-Ping;McAlister-Henn, Lee
基于变构调节特性，NAD（+）特异性异柠檬酸脱氢酶（IDH）被认为控制体内三羧酸循环的通量。为了在酿酒酵母中区分与该酶调控功能障碍相关的生长表型，我们分析了表达明确的IDH突变形式或非变构细菌NAD（+）特异性异柠檬酸脱氢酶（IDHa）的菌株。如前所述，具有严重催化缺陷但调控特性完整的IDH突变形式的表达导致无法以乙酸为碳源生长，并且产生小菌落的频率急剧增加，缺乏IDH的菌株（idh1 Delta idh2 Delta）也表现出表型。在乙酸培养基上，具有严重调节缺陷的酶或细菌IDHa酶的表达也降低了生长速度，这表明变抗调节对这种碳源上的最佳生长也很重要。然而，IDHa的表达对微小频率没有影响，这表明在表达IDH调节突变形式的菌株中观察到的中等微小频率可能与这些酶的催化效率略有降低有关。最后，在从葡萄糖培养基到乙醇作为碳源培养基的培养过程中，测量了氧气消耗的增长率。表达野生型或催化缺陷型IDH突变型的菌株表现出快速的呼吸过渡，而表达IDH调节突变型或细菌IDHa酶的菌株表现出慢得多的呼吸过渡。这表明在环境条件的变化中，IDH的变构激活具有重要的生理作用。
Based on allosteric regulatory properties, NAD(+)-specific isocitrate dehydrogenase (IDH) is believed to control flux through the tricarboxylic acid cycle in vivo. To distinguish growth phenotypes associated with regulatory dysfunction of this enzyme in Saccharomyces cerevisiae, we analyzed strains expressing well defined mutant forms of IDH or a non-allosteric bacterial NAD(+)-specific isocitrate dehydrogenase (IDHa). As previously reported, expression of mutant forms of IDH with severe catalytic defects but intact regulatory properties produced an inability to grow with acetate as the carbon source and a dramatic increase in the frequency of generation of petite colonies, phenotypes also exhibited by a strain (idh1 Delta idh2 Delta) lacking IDH. Reduced growth rates on acetate medium were also observed with expression of enzymes with severe regulatory defects or of the bacterial IDHa enzyme, suggesting that allosteric regulation is also important for optimal growth on this carbon source. However, expression of IDHa produced no effect on petite frequency, suggesting that the intermediate petite frequencies observed for strains expressing regulatory mutant forms of IDH are likely to correlate with the slight reductions in catalytic efficiency observed for these enzymes. Finally, rates of increase in oxygen consumption were measured during culture shifts from medium with glucose to medium with ethanol as the carbon source. Strains expressing wild-type or catalytically deficient mutant forms of IDH exhibited rapid respiratory transitions, whereas strains expressing regulatory mutant forms of IDH or the bacterial IDHa enzyme exhibited much slower respiratory transitions. This suggests an important physiological role for allosteric activation of IDH during changes in environmental conditions.