The kinetic mechanism of sheep liver sorbitol dehydrogenase.
The relations between the kinetic parameters for both sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase show that a Theorell-Chance compulsory order mechanism operates from pH 7.4 to 9.9. This is supported by many parallels with the kinetics of horse liver alcohol dehydrogenase, which operates by this classical mechanism. An isotope-exchange study using D-(2H8)sorbitol confirmed the existence of ternary complexes and that, under maximum velocity conditions, their interconversion is not rate-determining. Substrate inhibition at high concentrations of D-sorbitol or D-fructose confirmed rate-determining enzyme--coenzyme product dissociation, slowed by the existence of more stable abortive ternary enzyme-coenzyme product complexes with substrate. The effect of the inhibitor/activator 2,2,2-tribromoethanol showed the existence of enzyme-NAD-CBr3CH2OH complexes inhibiting the first phase of reaction and enzyme-NADH-CBr3CH2OH complexes dissociating more rapidly than the usual rate-determining enzyme-NADH coenzyme product dissociation in the final phase. Inhibition studies with dithiothreitol also confirmed an ordered binding of coenzymes and second substrates to sorbitol dehydrogenase. Neither D-sorbitol nor D-fructose had any effect on enzyme inactivation by the affinity labelling reagent DL-2-bromo-3-(5-imidazolyl)propionic acid, thus giving no evidence for their existence as binary enzyme-substrate complexes. Several alternative polyol substrates for sorbitol dehydrogenase gave the same maximum velocity as sorbitol. This indicated a common rate-limiting binary enzyme-NADH product dissociation and a similarity of mechanism. An enzyme assay for pH 7.0 and 9.9 is given which enables the concentration of sorbitol dehydrogenase to be determined from initial rate measurements of enzyme activity.
Lindstad
R I
RI
Institute of Biochemistry, University of Oslo, Blindern, Norway.
Hermansen
L F
LF
McKinley-McKee
J S
JS
D000818:Animals; D003903:Deuterium; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000031:analogs & derivatives / Q000494:pharmacology; D005632:Fructose / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007064:L-Iditol 2-Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D011108:Polymers / Q000378:metabolism; D012756:Sheep; D013012:Sorbitol / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity
D011108:Polymers; C024617:polyol; C062527:tribromoethanol; D005632:Fructose; D000431:Ethanol; D013012:Sorbitol; D003903:Deuterium; D007064:L-Iditol 2-Dehydrogenase; D004229:Dithiothreitol