Martin-Requero A, Corkey B E, Cerdan S, Walajtys-Rode E, Parrilla R L, Williamson J R
J Biol Chem. 1983 Mar 25;258(6):3673-81.
The mechanism of inhibition of pyruvate carboxylase, pyruvate dehydrogenase, and carbamyl phosphate synthetase induced by alpha-ketoisovalerate metabolism has been investigated in isolated rat hepatocytes incubated with lactate, pyruvate, ammonia, and ornithine as substrates. Half-maximum inhibitions of flux through each of these enzyme steps were obtained with 0.3 mM alpha-ketoisovalerate. The inhibition of pyruvate carboxylase flux by alpha-ketoisovalerate was largely reversed by oleate addition, but pyruvate dehydrogenase flux was inhibited further. Inhibition of flux through pyruvate carboxylase could be attributed mainly to the fall of its allosteric activator, acetyl-CoA, with some additional effect due to inhibition by methylmalonyl-CoA. Tissue acetyl-CoA levels decrease as a result of an inhibition of the active form of pyruvate dehydrogenase. Kinetic studies with the purified pig heart pyruvate dehydrogenase complex showed that methyl-malonyl-CoA, propionyl-CoA, and isobutyryl-CoA were inhibitory, the latter noncompetitive with CoASH with an apparent Ki of 90 microM. The observed inhibition of pyruvate dehydrogenase flux correlated with increases of the acetyl-CoA/CoASH and propionyl-CoA/CoASH ratios and isobutyryl-CoA levels, while increases of the mitochondrial NADH/NAD+ ratio explained differences between the effects of alpha-ketoisovalerate and propionate. Carbamyl phosphate synthetase I purified from rat liver was shown to be inhibited directly by methylmalonyl-CoA (apparent Ki of 5 mM). Inhibition of flux through carbamyl phosphate synthetase during alpha-ketoisovalerate metabolism could be attributed both to a direct inhibitory effect of methyl-malonyl-CoA and to a diminished activation by N-acetylglutamate. Direct effects of various acyl-CoA metabolites on these key enzymes may explain symptoms of hypoglycemia and hyperammonemia observed in patients with inherited disorders of organic acid metabolism.
在以乳酸、丙酮酸、氨和鸟氨酸为底物孵育的离体大鼠肝细胞中，研究了α-酮异戊酸代谢诱导丙酮酸羧化酶、丙酮酸脱氢酶和氨甲酰磷酸合成酶抑制的机制。通过这些酶步骤的通量达到半数最大抑制时，α-酮异戊酸的浓度为0.3 mM。添加油酸可在很大程度上逆转α-酮异戊酸对丙酮酸羧化酶通量的抑制，但丙酮酸脱氢酶通量被进一步抑制。通过丙酮酸羧化酶的通量抑制主要可归因于其变构激活剂乙酰辅酶A的减少，同时甲基丙二酰辅酶A的抑制也有一定额外作用。由于丙酮酸脱氢酶活性形式受到抑制，组织中乙酰辅酶A水平降低。对纯化的猪心丙酮酸脱氢酶复合物进行的动力学研究表明，甲基丙二酰辅酶A、丙酰辅酶A和异丁酰辅酶A具有抑制作用，后者与辅酶A非竞争性结合，表观抑制常数（Ki）为90 μM。观察到的丙酮酸脱氢酶通量抑制与乙酰辅酶A/辅酶A和丙酰辅酶A/辅酶A比值以及异丁酰辅酶A水平的升高相关，而线粒体烟酰胺腺嘌呤二核苷酸（NADH）/烟酰胺腺嘌呤二核苷酸（NAD⁺）比值的升高解释了α-酮异戊酸和丙酸作用的差异。从大鼠肝脏纯化的氨甲酰磷酸合成酶I被证明可被甲基丙二酰辅酶A直接抑制（表观Ki为5 mM）。α-酮异戊酸代谢过程中通过氨甲酰磷酸合成酶的通量抑制既可以归因于甲基丙二酰辅酶A的直接抑制作用，也可以归因于N-乙酰谷氨酸激活作用的减弱。各种酰基辅酶A代谢产物对这些关键酶的直接作用可能解释了有机酸代谢遗传性疾病患者中观察到的低血糖和高氨血症症状。
