Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP
Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP
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DOI：
10.1021/bi049488x
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发表时间：
2004-10-26
期刊：
影响因子：
2.9
通讯作者：
Roche, TE
中科院分区：
文献类型：
--
作者：
Bao, HY;Kasten, SA;Roche, TE
丙酮酸脱氢酶激酶2（PDK 2）活性通过结合PDK 2及其大量丙酮酸脱氢酶（E1）底物的二氢硫辛酰乙酰转移酶核心（E2 60 mer）增强。在E2激活的PDK 2中，90 mM的K+和60 mM的Cl-使PDK 2对ATP的Km和对ADP的竞争性Ki降低了约3倍，并增强了丙酮酸的抑制作用。与PDK 2催化+/- E2相比，E2使PDK 2对ATP的Km增加了近8倍（从5 μ M增加到39 μ M），使k（cat）增加了约4倍，并使对E1的需求减少了至少400倍。通过冷捕集技术测量的ATP结合发生在两个活性位点，K-d为5 μ M，其等于在不存在E2的情况下测量的ATP的PDK 2的K-m和K-d。在E2辅助的催化过程中，PDK 2在其活性位点上结合的ADP比ATP多3倍，丙酮酸类似物二氯乙酸导致结合的ADP比ATP多16倍（没有添加ADP）。丙酮酸作为一个非竞争性抑制剂对ATP，并列入ADP转化丙酮酸抑制非竞争性。在高丙酮酸水平下，丙酮酸是部分抑制剂，但在高ATP水平下也诱导底物抑制。我们的研究结果表明，在生理盐水平下，ADP解离是E2激活的PDK 2催化的限制性步骤，PDK 2。[ADP或ATP].丙酮酸盐复合物形成，并且PDK2.ATP.丙酮酸盐. E1与PDK2.ADP.丙酮酸盐积累反应。
Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K+ at similar to90 mM and Cl- at similar to60 mM decreased the K-m of PDK2 for ATP and competitive K-i for ADP by similar to3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K-m of PDK2 for ATP by nearly 8-fold (from 5 to 39 muM), increased k(cat) by similar to4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K-d of 5 muM, which equals the K-m and K-d of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had similar to3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating.