Open AccessStereochemical Studies of the Exchange and Abstraction of Succinate Hydrogen on Succinate Dehydrogenase
Author(s) -
Rétey János,
Seibl Josef,
Arigoni Duilio,
Cornforth John W.,
Ryback George,
Zeylemaker Willem P.,
Veeger Cees
Publication year - 1970
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1970.tb00282.x
Subject(s) - succinic acid , chemistry , incubation , hydrogen , succinate dehydrogenase , enzyme , deuterium , oxide , nuclear chemistry , inorganic chemistry , medicinal chemistry , organic chemistry , biochemistry , physics , quantum mechanics
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 [ 14 C]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.