Role of α-Asp181, β-Asp192, and γ-Asp190 in the distinctive subunits of human NAD-specific isocitrate dehydrogenase
Role of α-Asp181, β-Asp192, and γ-Asp190 in the distinctive subunits of human NAD-specific isocitrate dehydrogenase
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DOI：
10.1021/bi700061t
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发表时间：
2007-05-08
期刊：
影响因子：
2.9
通讯作者：
Colman, Roberta F.
中科院分区：
文献类型：
--
作者：
Bzymek, Krzysztof P.;Colman, Roberta F.
人NAD依赖性异柠檬酸脱氢酶（IDH）通过降低异柠檬酸的Km被ADP变构激活。该酶具有三种亚基类型，其可区分的序列以大约2 α：1 β：1 γ的比例存在，并且每个四聚体结合2 mol的每个配体。为了评估这些亚基是否也具有不同的功能，我们用天冬酰胺取代了等价的天冬氨酸，一次一个亚基;每个表达的纯化酶由一个突变体和两个野生型亚基组成。之所以选择天冬氨酸盐，是因为β-Asp（192）和γ-Asp（190）先前已被反应性ADP类似物亲和标记，而α-Asp（181）基于序列比对是等效的。α-D181 N IDH突变体显示V-max降低2000倍，Mn（II）和NAD的K（m）s增加15倍，而异柠檬酸的K-m变化小得多。相比之下，β-D192 N和γ-D190 N IDH的Vmax值与野生型酶相比仅降低4-5倍。β-D192 N酶对NAD的Km是正常酶的9倍，对Mn（II）或异柠檬酸的亲和力几乎没有影响或没有影响，而γ-D190 N酶对辅酶和Mn（II）的Km分别是正常酶的19倍和72倍，对异柠檬酸的Km的影响小得多。最后，所有三种突变酶都不能通过降低异柠檬酸的Km来响应ADP，尽管它们确实结合ADP。因此，这些柠檬酸盐靠近但不在ADP位点中，并且是ADP和异柠檬酸盐位点之间的通信所需的。这些结果表明，α-天冬氨酸（181）是唯一的一个必要的催化这些谷氨酸盐。β-Asp（192）是酶对NAD的亲和力的决定因素，γ-Asp（190）也是如此，而γ-Asp（190）也影响酶对金属离子的亲和力。我们的结论是，α亚基和β亚基以及α亚基和γ亚基之间共享NAD和ADP位点，α亚基和γ亚基之间共享Mn（II）位点，而α亚基对于催化至关重要。虽然α-天冬氨酸（181）、β-天冬氨酸（192）和γ-天冬氨酸（190）可能来自一个共同的祖先，但这三个亚基的同源物具有不同的功能。
Human NAD-dependent isocitrate dehydrogenase (IDH) is allosterically activated by ADP by lowering the K-m for isocitrate. The enzyme has three subunit types with distinguishable sequences present in the approximate ratio 2 alpha:1 beta:1 gamma and, per tetramer, binds 2 mol of each ligand. To evaluate whether the subunits also have distinct functions, we replaced equivalent aspartates, one subunit at a time, by asparagines; each expressed, purified enzyme was composed of one mutant and two wild-type subunits. The aspartates were chosen because beta-Asp(192) and gamma-Asp(190) had previously been affinity labeled by a reactive ADP analogue and alpha-Asp(181) is equivalent based on sequence alignments. The alpha-D181N IDH mutant exhibits a 2000-fold decrease in V-max, with increases of 15-fold in the K(m)s for Mn(II) and NAD and a much smaller change in the K-m for isocitrate. In contrast, the V-max values of the beta-D192N and gamma-D190N IDHs are only reduced 4-5-fold as compared to wild-type enzyme. The K-m for NAD of the beta-D192N enzyme is 9 times that of the normal enzyme with little or no effect on the affinity for Mn(II) or isocitrate, while the K(m)s for coenzyme and for Mn(II) of the gamma-D190N enzyme are 19 and 72 times, respectively, that of the normal enzyme with a much smaller effect on the K-m for isocitrate. Finally, all three mutant enzymes fail to respond to ADP by lowering the K-m for isocitrate, although they do bind ADP. Thus, these aspartates are close to but not in the ADP site and are required for communication between the ADP and isocitrate sites. These results demonstrate that alpha-Asp(181) is the only one of these aspartates essential for catalysis. beta-Asp(192) is a determinant of the enzyme's affinity for NAD, as is gamma-Asp(190), while gamma-Asp(190) also influences the enzyme's affinity for metal ion. We conclude that the NAD and ADP sites are shared between alpha- and beta- and alpha- and gamma-subunits, and the Mn(II) site is shared between alpha- and gamma-subunits, while the alpha-subunit is essential for catalysis. Although alpha-Asp(181), beta-Asp(192), and gamma-Asp(190) may have derived from a common progenitor, these aspartates of the three subunits have evolved distinct functions.