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

Abstract

A soluble preparation of succinate dehydrogenase has been used to study the exchange of hydrogen between succinate and the medium. Anaerobic exchange of hydrogen on this enzyme preparation was faster in the presence of fumarate. 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. 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. 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. Analogous incubation of tetradeuteriosuccinate in water led to RS‐dideuteriosuccinate as the major initial product, along with lesser amounts of S‐trideuteriosuccinate. 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. The implications of these findings are discussed. Copyright © 1970, Wiley Blackwell. All rights reserved