Rahmatullah M, Roche T E
J Biol Chem. 1985 Aug 25;260(18):10146-52.
The activation of pyruvate dehydrogenasea kinase activity by CoA esters has been further characterized. Half-maximal activation of kinase activity was achieved with about 1.0 microM acetyl-CoA after a 20-s preincubation in the presence of NADH. More than 80% of the acetyl-CoA was consumed during this period in acetylating sites in the pyruvate dehydrogenase complex as a result of the transacetylation reaction proceeding to equilibrium. At 1.0 microM acetyl-CoA, this resulted in more than a 4-fold higher level of CoA than residual acetyl-CoA. Activation of kinase activity could result either from acetylation of specific sites in the complex or tight binding of acetyl-CoA. Removal of CoA enhanced both acetylation and activation, suggesting acetylation mediates activation. For allosteric binding of acetyl-CoA to elicit activation, an activation constant, Ka, less than 50 nM would be required. To further distinguish between those mechanisms, the effects of other CoA esters as well as the reactivity of most of the effective CoA esters were characterized. Several short-chain CoA esters enhanced kinase activity including (in decreasing order of effectiveness) malonyl-CoA, acetoacetyl-CoA, propionyl-CoA, and methylmalonyl-CoA. Butyryl-CoA inhibited kinase activity as did high concentrations of long-chain acyl-CoAs. Inhibition by long-chain acyl-CoAs may result, in part, from detergent-like properties of those esters. Malonyl-CoA, propionyl-CoA, butyryl-CoA, and methylmalonyl-CoA, obtained with radiolabeled acyl groups, were shown to acylate sites in the complex. Propionyl-CoA and butyryl-CoA were tested, in competition with acetyl-CoA or pyruvate, as alternative substrates for acylation of sites in the complex and as competitive effectors of kinase activity. Propionyl-CoA alone rapidly acylated sites in the complex at low concentrations, and low concentrations of propionyl-CoA were effective in activating kinase activity although only a relatively small activation was observed. When an equivalent level (20 microM) of acetyl-CoA and propionyl-CoA was used, marked activation of kinase activity due to a dominant effect of acetyl-CoA was associated with acetylation of a major portion of sites in the complex and with a small portion undergoing acylation with propionyl-CoA. Those results were rapidly achieved in a manner independent of the order of addition of the two CoA esters. That indicates that tight slowly reversible binding of acetyl-CoA is not involved in kinase activation. High levels of propionyl-CoA greatly reduced acetylation by acetyl-CoA and nearly prevented activation of kinase activity by acetyl-CoA.(ABSTRACT TRUNCATED AT 400 WORDS)
辅酶A酯对丙酮酸脱氢酶激酶活性的激活作用已得到进一步研究。在NADH存在的情况下预孵育20秒后，约1.0微摩尔/升的乙酰辅酶A可使激酶活性达到半数最大激活。在此期间，由于转乙酰化反应达到平衡，超过80%的乙酰辅酶A在丙酮酸脱氢酶复合物的乙酰化位点被消耗。在1.0微摩尔/升的乙酰辅酶A浓度下，这导致辅酶A的水平比残留的乙酰辅酶A高4倍以上。激酶活性的激活可能是由于复合物中特定位点的乙酰化或乙酰辅酶A的紧密结合。去除辅酶A可增强乙酰化和激活作用，表明乙酰化介导了激活过程。对于乙酰辅酶A的变构结合以引发激活，所需的激活常数Ka应小于50纳摩尔/升。为了进一步区分这些机制，研究了其他辅酶A酯的作用以及大多数有效辅酶A酯的反应性。几种短链辅酶A酯可增强激酶活性，包括（按有效性递减顺序）丙二酰辅酶A、乙酰乙酰辅酶A、丙酰辅酶A和甲基丙二酰辅酶A。丁酰辅酶A抑制激酶活性，高浓度的长链酰基辅酶A也有同样作用。长链酰基辅酶A的抑制作用可能部分源于这些酯类的类似去污剂的性质。用放射性标记的酰基获得的丙二酰辅酶A、丙酰辅酶A、丁酰辅酶A和甲基丙二酰辅酶A被证明可使复合物中的位点酰化。测试了丙酰辅酶A和丁酰辅酶A与乙酰辅酶A或丙酮酸竞争，作为复合物中位点酰化的替代底物以及激酶活性的竞争性效应物。单独使用丙酰辅酶A在低浓度下能迅速使复合物中的位点酰化，低浓度的丙酰辅酶A能有效激活激酶活性，尽管观察到的激活作用相对较小。当使用等量（20微摩尔/升）的乙酰辅酶A和丙酰辅酶A时，由于乙酰辅酶A的主导作用，激酶活性显著激活，这与复合物中大部分位点的乙酰化以及一小部分位点被丙酰辅酶A酰化有关。这些结果能以与两种辅酶A酯添加顺序无关的方式迅速实现。这表明乙酰辅酶A的紧密缓慢可逆结合不参与激酶激活。高浓度的丙酰辅酶A大大降低了乙酰辅酶A的乙酰化作用，并几乎阻止了乙酰辅酶A对激酶活性的激活。（摘要截断于400字）
