Inhibition of dimeric dihydrodiol dehydrogenases of rabbit and pig lens by ascorbic acid.
The dehydrogenase activity of dimeric dihydrodiol dehydrogenases (DD) purified from pig and rabbit lenses was inhibited by either L-ascorbic acid or its epimer, isoascorbic acid, at pH 7.5. Isoascorbate [IC50 (concn. giving 50% inhibition) = 0.043 mM for the pig enzyme; IC50 = 0.13 mM for the rabbit enzyme] was a more potent inhibitor than ascorbate (IC50 values 0.45 and 0.90 mM respectively), but 1 mM-dehydroascorbate gave less than 30% inhibition. Glucose, glucuronate, gulono-gamma-lactone, glutathione and dithiothreitol did not inhibit the enzyme activity. The inhibition by isoascorbate and ascorbate was instantaneous and reversible, and their inhibitory potency was decreased by addition of ascorbate oxidase. In the reverse reaction, isoascorbate and ascorbate gave low IC50 values of 0.013 and 0.10 mM respectively for the pig enzyme and 0.025 and 0.25 mM for the rabbit enzyme. The inhibition patterns by the two compounds were competitive with respect to dihydrodiols of naphthalene and benzene and uncompetitive with respect to NADP+, but those in the reverse reaction were uncompetitive with respect to both carbonyl substrate and NADPH. The steady-state kinetic measurements in the forward and reverse reactions by the pig enzyme were consistent with an ordered Bi Bi mechanism, in which NADP+ binds to the enzyme first and NADPH leaves last. The results indicate that ascorbate and its epimer directly bind to an enzyme: NADP+ binary complex as dead-end inhibitors. Thus ascorbate may be an important modulator of DD in the lens.
Hara
A
A
Department of Biochemistry, Gifu Pharmaceutical University, Japan.
Shinoda
M
M
Kanazu
T
T
Nakayama
T
T
Deyashiki
Y
Y
Sawada
H
H
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism / Q000494:pharmacology*; D001667:Binding, Competitive; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D046911:Macromolecular Substances; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011817:Rabbits; D013552:Swine
D046911:Macromolecular Substances; C005056:isoascorbic acid; D009249:NADP; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C023125:dihydrodiol dehydrogenases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C116788:cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase; D001205:Ascorbic Acid