STUDIES OF GLUCONEOGENIC MITOCHONDRIAL ENZYMES .4. CONVERSION OF OXALOACETATE TO FUMARATE BY BOVINE LIVER MITOCHONDRIAL MALATE DEHYDROGENASE AND FUMARASE

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. © 1969.