Rahmatullah M, Roche T E, Jilka J M, Kazemi M
Eur J Biochem. 1985 Jul 1;150(1):181-7. doi: 10.1111/j.1432-1033.1985.tb09005.x.
The activity of the pyruvate dehydrogenase kinase, which phosphorylates and thereby inactivates the pyruvate dehydrogenase complex, was stimulated by malonyl-CoA. Treatment with [2-14C]malonyl-CoA resulted in acylation of sites in the complex. Both acylation and activation of kinase activity increased in a time-dependent manner with a parallel increase in those activities when the malonyl-CoA:CoA ratio was varied. Protein-bound acyl groups were labilized by performic acid treatment indicating their attachment to protein at thiol residues; however, the product released was volatile, which is not characteristic of malonic acid. While malonyl-CoA was initially free of acetyl-CoA, stimulation of kinase activity and acylation of sites in the complex by malonyl-CoA were shown to be contingent upon enzyme-catalyzed decarboxylation. Decarboxylation appeared to be catalyzed by a trace contaminant present in highly purified preparations of both the pyruvate and 2-oxoglutarate dehydrogenase complexes. Under conditions in which both free CoA was removed (by conversion to succinyl-CoA) and then, after various periods, free acetyl-CoA was removed (by enzymic conversion to acetyl phosphate), both acetylation of sites in the complex and activation of kinase activity increased in a time-dependent manner. Concomitantly there was a decrease in the concentration dependence for activation of the kinase by malonyl-CoA. Our results strongly support the conclusion that activation of kinase activity is associated with acylation of sites in the complex, and that, in the case of malonyl-CoA, those processes depend on enzyme-catalyzed decarboxylation.
丙酮酸脱氢酶激酶可使丙酮酸脱氢酶复合体磷酸化并使其失活，丙二酰辅酶A可刺激该激酶的活性。用[2-¹⁴C]丙二酰辅酶A处理会导致该复合体中位点的酰化。当改变丙二酰辅酶A与辅酶A的比例时，酰化和激酶活性的激活均呈时间依赖性增加，且这些活性也平行增加。经过甲酸处理后蛋白质结合的酰基会不稳定，这表明它们是通过硫醇残基与蛋白质相连；然而，释放的产物是挥发性的，这并非丙二酸的特征。虽然最初丙二酰辅酶A不含乙酰辅酶A，但丙二酰辅酶A对激酶活性的刺激以及该复合体中位点的酰化被证明取决于酶催化的脱羧作用。脱羧作用似乎是由丙酮酸和2-氧代戊二酸脱氢酶复合体的高纯度制剂中存在的微量污染物催化的。在去除游离辅酶A（通过转化为琥珀酰辅酶A），然后在不同时间段后再去除游离乙酰辅酶A（通过酶促转化为乙酰磷酸）的条件下，该复合体中位点的乙酰化和激酶活性的激活均呈时间依赖性增加。同时，丙二酰辅酶A对激酶激活的浓度依赖性降低。我们的结果有力地支持了以下结论：激酶活性的激活与该复合体中位点的酰化有关，并且就丙二酰辅酶A而言，这些过程依赖于酶催化的脱羧作用。
