Studies on the mechanism of hydrogen exchange between succinate and water catalyzed by the soluble succinic dehydrogenase of Singer from bovine heart.

The equilibrium exchange of hydrogen between the 2,3methylene positions of succinate and water was studied under aerobic conditions with specifically tritiated and deuterated succinates and soluble succinic dehydrogenase prepared by the method of Singer from bovine heart. The exchange of tritium from racemic 2-tritiosuccinate (racemic-T1), racemic 2,3-ditritiosuccinate (racemic-T?), and meso-2,3-ditritiosuccinate (meso-Ts) proceeds to completion and exhibits simple, first order kinetics. The rates of exchange of tritium from racemic-Ti and racemic-Tz are identical and 1.35 times faster than that from meso-T,. The enantiomorphs of racemic-T, and racemic-Tz are not distinguished kinetically. Hydrogen exchange products of deuterated succinates were identified by their infrared spectra. In the exchange of deuterium from 2,2,3,3-tetradeuterosuccinate (D4) to water, meso-2,3-dideuterosuccinate (meso-DB) is the only major intermediate detected between Dq and the fmal product, succinate. 2-Deuterosuccinate is detected as an intermediate during the exchange of deuterium from racemic 2,3-dideuterosuccinate, but not from meso-D?. In the exchange of deuterium from D20 to succinate, meso-D:! is again the major intermediate detected between succinate and the final exchange product, Dq. The exchange of deuterium from succinate to water goes to completion regardless of the extent and stereochemistry of deuteration. Hydrogen exchange proceeds mainly through the exchange of trans (RS) pairs of methylene hydrogens, and is analogous to the trans elimination of a proton pair during the oxidation of succinate to fumarate. Kinetic isotope effects appear to relate only to the nature of trans hydrogen pairs and to be unrelated to the stereochemical differences between labeled