Stereochemical Studies of the Exchange and Abstraction of Succinate Hydrogen on Succinate Dehydrogenase

1 A soluble preparation of succinate dehydrogenase has been used to study the exchange of hydrogen between succinate and the medium. 2 Anaerobic exchange of hydrogen on this enzyme preparation was faster in the presence of fumarate. 3 When succinate was incubated anaerobically with the enzyme in deuterium oxide in the presence of [14C]fumarate, incorporation of deuterium into succinate was much faster than incorporation of radioactivity. 4 Anaerobic incubation of succinate with the enzyme and fumarate in deuterium oxide led initially to the appearance of mono- and di-deuteriated succinic acids in a ratio around 2 to 1. Infrared and polarimetric analysis indicated that the mono-deuteriated succinic acid was largely R and the di-deuteriated acid was largely RS. After prolonged incubation there was evidence for the presence of RR-dideuteriosuccinic acid and of more highly deuteriated species. 5 Analogous incubation of succinate in aqueous deuterium oxide gave qualitatively similar results. A higher proportion of mono- to di-deuteriosuccinic acid was formed initially and polarimetric measurements indicated that not all the monodeuterio-succinic acid was R. 6 Analogous incubation of tetradeuteriosuccinate in water led to RS-dideuteriosuccinate as the major initial product, along with lesser amounts of S-trideuteriosuccinate. 7 Oxidation of stereospecifically deuteriated succinic acids on the enzyme, with potassium hexacyanoferrate(III) as electron acceptor, showed a large kinetic isotope effect for removal of a pro-R hydrogen and a smaller effect for removal of the complementary pro-S-hydrogen. 8 The implications of these findings are discussed.