Department of Biochemistry, University of California, Riverside, California 92521.
Plant Physiol. 1988 Feb;86(2):325-8. doi: 10.1104/pp.86.2.325.
The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V(max) with no apparent effect on K(m). The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation.
由于金属离子存在时草酰乙酸会部分脱羧形成丙酮酸,用常规的与苹果酸脱氢酶偶联的方法测定磷酸烯醇式丙酮酸羧激酶活性会导致结果低估。在常规测定中加入乳酸脱氢酶可以使草酰乙酸生成的丙酮酸还原为乳酸,同时 NADH 被氧化。然后,通过酶促测定底物和产物可以发现,苹果酸脱氢酶和乳酸脱氢酶的联合活性可以解释磷酸烯醇式丙酮酸的全部消耗。改良测定法的净结果是 V(max)升高,而 K(m)没有明显变化。游离二价阳离子浓度似乎是控制草酰乙酸脱羧速率的主要因素。