General Properties of the Hydrogen Exchange Reaction Catalyzed by the Soluble Succinic Dehydrogenase of Singer from Bovine Heart

Kinetic studies are reported on the catalysis of tritium exchange from meso-2,3-ditritiosuccinate to water by soluble succinic dehydrogenase from bovine heart. Tritium exchange does not proceed for the most part via the equilibrium dismutation of succinate and fumarate; rather, exchange appears to proceed directly from an enzyme-succinate complex. Time-dependent changes in the exchange activity were observed in the presence of fumarate. Fumarate and fumarate analogues can either enhance or inhibit the exchange reaction depending on concentration and other conditions. Oxygen enhances the exchange rate several-fold in an easily reversible manner which cannot be attributed to the autoxidation of succinate. Attempts to detect a transfer of tritium from succinate to succinic dehydrogenase directly and indirectly by the transfer of tritium from succinate to 2-methylfumarate to yield tritiated 2-methylsuccinate via the succinate-fumarate dismutation reaction were unsuccessful. Exchange rates are dependent on pH, and the maximum in the pH rate profile occurs at pH 6.9 with an apparent turnover number for meso-2,3-ditritiosuccinate of about 5 moles of succinate x min-1 x moles of flavin-1 at 24°. Succinate Km is relatively insensitive to pH and values of 2 ± 0.5 x 10-4 m were obtained from pH 6.8 to 7.6. Ki values for oxalacetate and malonate are 0.5 to 1 µm and 60 to 70 µm, respectively, at pH 6.9. Solvent isotope effects arise to a large extent from an apparent shift in the pH rate profile toward lower pH values in deuterated water. Succinate Km remains essentially unaffected in deuterated water.