Karpova Tatiana, Danchuk Svitlana, Kolobova Elena, Popov Kirill M
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, 5110 Rockhill Road, Kansas City, MO 64110-2499, USA.
Biochim Biophys Acta. 2003 Dec 1;1652(2):126-35. doi: 10.1016/j.bbapap.2003.08.010.
The activity of mammalian pyruvate dehydrogenase complex (PDC) is regulated by a phosphorylation/dephosphorylation cycle. Dephosphorylation accompanied by activation is carried out by two genetically different isozymes of pyruvate dehydrogenase phosphatase, PDP1c and PDP2c. Here, we report data showing that PDP1c and PDP2c display marked biochemical differences. The activity of PDP1c strongly depends upon the simultaneous presence of calcium ions and the E2 component of PDC. In contrast, the activity of PDP2c displays little, if any, dependence upon either calcium ions or E2. Furthermore, PDP2c does not appreciably bind to PDC under the conditions when PDP1c exists predominantly in the PDC-bound state. The stimulatory effect of E2 on PDP1c can be partially mimicked by a monomeric construct consisting of the inner lipoyl-bearing domain and the E1-binding domain of E2 component. This strongly suggests that the E2-mediated activation of PDP1c largely reflects the effects of co-localization and mutual orientation of PDP1c and E1 component facilitated by their binding to E2. Both PDP1c and PDP2c can efficiently dephosphorylate all three phosphorylation sites located on the alpha chain of the E1 component. For PDC phosphorylated at a single site, the relative rates of dephosphorylation of individual sites are: 2>site 3>site 1. Phosphorylation of sites 2 or 3 in addition to site 1 does not have a significant effect on the rates of dephosphorylation of individual sites by PDP1c, suggesting a random mechanism of dephosphorylation. In contrast, there is a significant decrease in the overall rate of dephosphorylation of pyruvate dehydrogenase by PDP2c under these conditions. This indicates that the mechanism of dephosphorylation of PDC phosphorylated at multiple sites by PDP2c is not purely random. These marked differences in the site-specificity displayed by PDP1c and PDP2c should be particularly important under conditions such as starvation and diabetes, which are associated with a great increase in phosphorylation of sites 2 and 3 of pyruvate dehydrogenase.
哺乳动物丙酮酸脱氢酶复合体（PDC）的活性受磷酸化/去磷酸化循环调控。去磷酸化伴随激活作用由丙酮酸脱氢酶磷酸酶的两种基因不同的同工酶PDP1c和PDP2c完成。在此，我们报告的数据表明PDP1c和PDP2c表现出显著的生化差异。PDP1c的活性强烈依赖于钙离子和PDC的E2组分同时存在。相比之下，PDP2c的活性对钙离子或E2的依赖性很小，即便有依赖也微乎其微。此外，在PDP1c主要以结合于PDC的状态存在的条件下，PDP2c与PDC的结合并不明显。E2对PDP1c的刺激作用可被由E2组分的含硫辛酰胺的内部结构域和E1结合结构域组成的单体构建体部分模拟。这有力地表明，E2介导的PDP1c激活在很大程度上反映了PDP1c和E1组分通过与E2结合而促进的共定位和相互取向的作用。PDP1c和PDP2c都能有效使位于E1组分α链上的所有三个磷酸化位点去磷酸化。对于在单个位点磷酸化的PDC，各个位点的相对去磷酸化速率为：位点2＞位点3＞位点1。除位点1外，位点2或位点3的磷酸化对PDP1c使各个位点去磷酸化的速率没有显著影响，这表明去磷酸化机制是随机的。相比之下，在这些条件下，PDP2c使丙酮酸脱氢酶的总体去磷酸化速率显著降低。这表明PDP2c对多位点磷酸化的PDC进行去磷酸化的机制并非完全随机。在饥饿和糖尿病等与丙酮酸脱氢酶位点2和位点3磷酸化大幅增加相关的条件下，PDP1c和PDP2c所表现出的位点特异性的这些显著差异应该尤为重要。
