Reversible and irreversible inhibition of sheep liver sorbitol dehydrogenase with Cibacron Blue 3GA and Eriochrome Black T.
Due to the central role of sorbitol dehydrogenase in diabetic cataract, it is important to examine this enzyme's interaction with different inhibitory compounds such as dyes. The aim of the study was to investigate the binding of Cibacron Blue and Eriochrome Black T to the active site in sorbitol dehydrogenase. These dyes' effect on the enzyme was studied by steady state and affinity labelling kinetics. Both dyes were coenzyme competitive inhibitors with KEI values around 0.5 microM. Essentially the same KEI values were obtained using the dyes as protecting ligands against the affinity label D,L-alpha-Bromo-beta-(5-imidazolyl)-propionic acid. Both dyes were also able to inhibit the enzyme irreversibly through an affinity labelling mechanism, with KEI' values for Cibacron Blue and Eriochrome Black T of 2.2 and 3.1 mM, respectively. Dithiothreitol and NADH were competitive protecting ligands against both dyes. The rate of inactivation was fastest for Cibacron Blue at acid pH values, while the opposite was the case with EBT. Both Cibacron Blue and Eriochrome Black T bind to sorbitol dehydrogenase in two different ways. In both cases the complex formed prior to irreversible inhibition is the weakest. The tighter reversible complexes are suggested to share a common epitope in the coenzyme binding region. Both irreversible complexes involve binding close to the zinc ion at the active site and the sugar binding site. Due to different pH dependences it can be concluded that the affinity labelling mechanism is different for the two dyes and in neither case is the inactivation due to removal of the active site zinc ion.
Kvernmo
T
T
Biochemical Institute, University of Oslo, Norway.
Winberg
J O
JO
McKinley-McKee
J S
JS
D000818:Animals; D001391:Azo Compounds / Q000378:metabolism / Q000494:pharmacology*; D004396:Coloring Agents; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007064:L-Iditol 2-Dehydrogenase / Q000037:antagonists & inhibitors*; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D015394:Molecular Structure; D012756:Sheep; D014227:Triazines / Q000378:metabolism / Q000494:pharmacology*
D001391:Azo Compounds; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D014227:Triazines; C012842:Cibacron Blue F 3GA; D007064:L-Iditol 2-Dehydrogenase; C014527:Eriochrome Black T