[Oxaloacetate keto-enol tautomerase from bovine heart mitochondrial matrix]. / Oksaloatsetat keto-enol tautomerazy matriksa mitokhondrii serdtsa byka.

Bovine heart mitochondrial matrix contains two proteins possessing the oxaloacetate keto-enol tautomerase (EC 5.3.2.2) activity. A procedure for the isolation and purification of the enzymes to an electrophoretically homogeneous state has been developed. The purified proteins have molecular masses of 37 kD and 80 kD and catalyze the keto-enol oxaloacetate tautomerization reaction with the turnover numbers of approximately 3000 and approximately 2000 min-1. The both enzymes were found to differ significantly in all their physicochemical and kinetic properties. Fractionation of rat liver mitochondria revealed that the oxaloacetate keto-enol tautomerase activity is predominantly localized in the mitochondrial matrix. The essential role of oxaloacetate keto-enol tautomerase in the operation of the Krebs cycle is discussed.

Regulation of succinate dehydrogenase and tautomerization of oxaloacetate.

Highly purified succinate-ubiquinone reductase catalyzes the oxidation of L- or D-malate with a Km and initial Vmax equal to approximately 10(-3) M and approximately 100 nmol/min/mg of protein, respectively. The malate dehydrogenase activity of succinate dehydrogenase rapidly decreases regardless of the presence of glutamate plus glutamate-oxaloacetate transaminase. The inhibitor trapping system, however, prevents the inactivation of succinate dehydrogenase under the conditions when the rate of tautomeric oxaloacetate enol in equilibrium oxaloacetate ketone interconversion is high. These results suggest that enol oxaloacetate is an immediate product of malate oxidation at the succinate dehydrogenase active site. Two proteins (Mr 37 and 80 kD) which catalyze the oxaloacetate tautomerase reaction were isolated from the mitochondrial matrix. Some physico-chemical and kinetic properties of these enzymes were characterized. The larger protein was identified as inactive aconitase. The system containing succinate dehydrogenase, L-malate, glutamate plus transaminase and oxaloacetate tautomerase was reconstituted. Such a system is capable of oxidizing malate to aspartate without rapid inactivation of succinate dehydrogenase. Taken together, the data obtained emphasize a significant role of enzymatic oxaloacetate tautomerization in the control of the succinate dehydrogenase activity in the mitochondrial matrix.

Isolation and properties of oxaloacetate keto-enol-tautomerases from bovine heart mitochondria.

Two highly purified proteins with quite different properties capable of oxaloacetate keto-enol-tautomerase activity (oxaloacetate keto-enol-isomerase, EC 5.3.2.2) were isolated from the bovine heart mitochondrial matrix. The first protein has an apparent molecular mass of 37 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-200 gel filtration. It is quite stable upon storage at 40 degrees C and reaches the maximal catalytic activity at pH 8.5 with a half-maximal activity at pH 7.0. The enzyme is specifically inhibited by oxalate and diethyloxaloacetate. When assayed in the enol----ketone direction at 25 degrees C (pH 9.0), the enzyme obeys a simple substrate saturation kinetics with Km and Vmax values of 45 microM and 74 units per mg of protein, respectively; the latter value corresponds to the turnover number of 2700 min-1. The second protein has an apparent molecular mass of 80 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-300 gel filtration. The enzyme is rapidly inactivated at 40 degrees C and shows a sharp pH optimum of activity at pH 9.0. The enzyme can be completely protected from thermal inactivation by oxaloacetate and dithiothreitol. The kinetic parameters of the enzyme as assayed in the enol----ketone direction at 25 degrees C (pH 9.0) are: Km = 220 microM and Vmax = 20 units per mg of protein; the latter corresponds to the turnover number of 1600 min-1. The enzyme activity is specifically inhibited by maleate and pyrophosphate. About 30% of the total oxaloacetate tautomerase activity in crude mitochondrial matrix is represented by the 37 kDa enzyme and about 70% by the 80 kDa protein.