ADENOSINE TRIPHOSPHATE INHIBITION OF PYRUVATE KINASE REACTION AND ITS DEPENDENCE ON TOTAL MAGNESIUM ION CONCENTRATION
ADENOSINE TRIPHOSPHATE INHIBITION OF PYRUVATE KINASE REACTION AND ITS DEPENDENCE ON TOTAL MAGNESIUM ION CONCENTRATION
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DOI：
10.1042/bj1120303
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发表时间：
1969-01-01
影响因子：
4.1
通讯作者：
STORM, E
中科院分区：
文献类型：
--
作者：
HOLMSEN, H;STORM, E
1.研究了ATP、PPi和EDTA在各种浓度的镁（其中“镁”是指游离和络合物形式的总Mg 2+）下对肌肉丙酮酸激酶反应的影响。反应速率被确定为在记录的孵育时间内形成的丙酮酸的量。2.在44 mm-镁下，ATP对ADP和磷酸烯醇式丙酮酸的Km值在6.8mm-1的三盐酸缓冲体系或甘氨酰甘氨酸-氢氧化钠缓冲体系中均不受ATP的影响，但在这些体系中Km值不同。一种底物的Km与第二种底物的浓度无关。3.在10 mm-镁的tris-HCl体系中，ATP与ADP和磷酸烯醇式丙酮酸竞争性抑制反应。在甘氨酰甘氨酸-氢氧化钠体系中，抑制作用表现为非竞争性。在10毫米镁的Km值低于在44毫米镁和依赖于所使用的系统。4.在三盐酸体系中，反应速率随镁浓度的增加而增加，当镁浓度为ADP浓度的10-20倍时，反应速率达到最大值。进一步增加抑制反应，在44 mm-镁的速率是其最大值的25-50%。这种抑制作用与增加三甲基氯化铵浓度所产生的抑制作用相反，而不是由于Mg 2+离子的特定作用。5.在6·8 mm-ATP存在下，当镁浓度低于4- 6 mm-Mg时，无反应发生，当反应速率增加时，抑制作用明显消失，并与10- 25 mm-Mg时无ATP时的反应速率相等。镁浓度的进一步增加得到的反应速率，在ATP的存在下比在其不存在下略高。在ATP存在下的最大速率明显低于在其不存在下。当6·8 mm-PPor 6·8 mm-EDTA存在时，反应速率随镁浓度升高的变化与在低于6- 8 mm-镁的ATP存在下获得的变化相似，但镁浓度进一步增加导致速率增加至与对照相当的最大值。因此，纯螯合作用的效果是将反应最大值置换为更高的镁浓度，而不改变最大速率。当对这一效应进行校正后，ATP在44 mm-镁处产生抑制，与ADP竞争（Ki 2·1×10− 2 m）。这种程度的抑制是远远低于先前报道的丙酮酸激酶反应的机制，其重要性进行了讨论。
1. The effects of ATP, PPiand EDTA on the skeletal-muscle pyruvate kinase reaction at various concentrations of magnesium (where ‘magnesium’ refers to total Mg2+, both free and in the form of complexes) were investigated. The reaction rate was determined as the amount of pyruvate formed in a recorded time of incubation. 2. At 44mm-magnesium theKmvalues for ADP and phosphoenolpyruvate were unaltered by the presence of ATP up to 6·8mmin systems buffered with either tris–hydrochloric acid or glycylglycine–sodium hydroxide, but theKmvalues were different in these systems. TheKmfor one substrate was independent of the concentration of the second substrate. 3. At 10mm-magnesium in the tris–hydrochloric acid system ATP inhibited the reaction competitively with respect to ADP and phosphoenolpyruvate. In the glycylglycine–sodium hydroxide system the inhibition appeared to be non-competitive. At 10mm-magnesium theKmvalues were lower than at 44mm-magnesium and dependent on the system used. 4. In the tris–hydrochloric acid system the reaction rate rose with increasing magnesium concentration up to a maximum at a concentration 10–20 times that of ADP. Further increase inhibited the reaction and at 44mm-magnesium the rate was 25–50% of its maximum. This inhibition paralleled that produced by increasing trimethylammonium chloride concentrations and was not due to a specific effect of the Mg2+ion. 5. In the presence of 6·8mm-ATP no reaction occurred below 4–6mm-magnesium, and further increase apparently abolished the inhibition as the reaction rate increased and became equal to those obtained in the absence of ATP at 10–25mm-magnesium. Further increase in magnesium concentration gave reaction rates that were slightly higher in the presence of ATP than in its absence. The maximal rate in the presence of ATP was distinctly lower than in its absence. When 6·8mm-PPior 6·8mm-EDTA was present the variations in reaction rate with rising magnesium concentration were similar to that obtained in the presence of ATP below 6–8mm-magnesium but further increase in the magnesium concentration resulted in an increase in the rate up to a maximum comparable with that of the control. The effect of pure chelation was thus a displacement of the reaction maximum to higher magnesium concentrations without changing the maximal rate. When correction had been made for this effect, ATP gave inhibition at 44mm-magnesium that was competitive with respect to ADP (Ki2·1×10−2m). This degree of inhibition is far less than was reported earlier and its importance for the mechanism of the pyruvate kinase reaction is discussed.