Studies of gluconeogenic mitochondrial enzymes. IV. The conversion of oxaloacetate to fumarate by bovine liver mitochondrial malate dehydrogenase and fumarase.
Studies of gluconeogenic mitochondrial enzymes. IV. The conversion of oxaloacetate to fumarate by bovine liver mitochondrial malate dehydrogenase and fumarase.
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糖异生线粒体酶的研究。
DOI：
10.1016/0003-9861(69)90060-5
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发表时间：
1969
影响因子：
3.9
通讯作者：
M. Strmecki
中科院分区：
文献类型：
--
作者：
L. A. Fahien;M. Strmecki
介绍了苹果酸脱氢酶和富马酸酶的纯化方法。从同一牛肝线粒体提取物中可以同时制备谷氨酸-草酰乙酸转氨酶和谷氨酸脱氢酶。对线粒体提取物中这些酶的水平进行了估计。对纯化的苹果酸脱氢酶和富马酸酶的动力学进行了研究，并与前人对纯化的谷氨酸脱氢酶和谷草转氨酶的研究进行了比较。DPNH和草酰乙酸酯的米氏常数明显低于肝脏线粒体苹果酸脱氢酶的米氏常数，而苹果酸的抑制常数高于心脏酶的米氏常数。这表明这两种酶之间存在差异，肝脏酶更适合参与糖异生(还原草酰乙酸酯)。草酰乙酸酯是苹果酸脱氢酶的底物抑制剂，是比苹果酸、α-酮戊二酸或DPN更有效的抑制剂。草酰乙酸酯也是延胡索酸酶反应的抑制剂。这四种酶不能同时在线粒体中发挥最大作用。例如，在糖异生中，如果DPNH、草酸乙酯和谷氨酸水平较高，则会抑制谷氨酸脱氢酶(DPNH)、苹果酸脱氢酶(草酸乙酸酯)和富马酸酶(草酸乙酸酯)。在这些条件下，转氨酶可以发挥最大活性。如果草酰乙酸酯的水平降低(通过转氨酶和苹果酸脱氢酶反应)，那么苹果酸脱氢酶反应可能发挥最大作用。由于苹果酸的米氏常数相对于草酰乙酸酯和富马酸的抑制常数很高，富马酸酶反应(苹果酸转化为富马酸)仍然受到抑制，而谷氨酸脱氢酶反应不会反向进行(DPNH氧化)，因为铵离子的米氏常数很高。该酶将保持抑制状态，直到DPN/DPNH的比率变高。由于草酰乙酸酯在转氨酶反应中的抑制常数较低，转氨酶反应不会向相反方向进行(天冬氨酸的脱氨基)。这些反应的结果将是α-酮戊二酸、天冬氨酸和苹果酸的积累。与DPNH和草酰乙酸酯不同的是，这些化合物可以通过线粒体膜扩散，并在细胞质中用于合成草酰乙酸酯和DPNH，这是糖异生所必需的。
Methods of purifying malate dehydrogenase and fumarase are described. The enzymes glutamate-oxaloacetate transaminase, and glutamate dehydrogenase can be prepared simultaneously from the same bovine liver mitochondria extract. Estimates are made of the levels of these enzymes in mitochondrial extracts. The kinetics of the purified malate dehydrogenase and fumarase are studied and compared with previous studies of the purified glutamate dehydrogenase and glutamate-oxaloacetate transaminase.The values of the Michaelis constants of DPNH and oxaloacetate are considerably lower and the inhibition constant of malate is higher with the liver mitochondrial malate dehydrogenase than these same values with the heart enzyme. This suggests a difference between these two enzymes and that the liver enzyme is better suited to participate in gluconeogenesis (reduction of oxaloacetate).Oxaloacetate is a substrate inhibitor of malate dehydrogenase and is a more potent inhibitor than malate, α-ketoglutarate, or DPN. Oxaloacetate is also an inhibitor of the fumarase reaction.All four of these enzymes could not function maximally in the mitochondria at the same time. For example, in gluconeogenesis if the levels of DPNH, oxaloacetate and glutamate are high there would be inhibition of glutamate dehydrogenase (by DPNH), malate dehydrogenase (by oxaloacetate) and fumarase (by oxaloacetate). Under these conditions the transaminase could be functioning at maximal activity. If the levels of oxaloacetate are reduced (by the transaminase and malate dehydrogenase reactions) then the malate dehydrogenase reaction could function maximally. The fumarase reaction (conversion of malate to fumarate) would remain inhibited since the Michaelis constant of malate is quite high with respect to the inhibition constants of oxaloacetate and fumarate.The glutamate dehydrogenase reaction would not proceed in the reverse (DPNH oxidation) direction because of the high Michaelis constant of ammonium ions. This enzyme would remain inhibited until the ratio of DPN to DPNH became high. The transaminase reaction would not proceed in the reverse direction (deamination of aspartate) because of the low inhibition constant of oxaloacetate in this reaction. The results of these reactions would be accumulations of α-ketoglutarate, aspartate, and malate. These compounds unlike DPNH and oxaloacetate can diffuse through the mitochondrial membrane and be utilized for the synthesis of oxaloacetate and DPNH in the cytoplasm where they are required for gluconeogenesis.