Inhibition and inactivation of horse liver alcohol dehydrogenase with the imidazobenzodiazepine Ro 15-4513.
The imidazobenzodiazepine ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine -3-carboxylate (Ro 15-4513) is important as a potential "drink and drive" drug due to effects on receptors in brain neurones, resulting in alcohol intoxication-antagonistic properties. Because of the molecule's importance its effect on alcohol metabolism in liver has been investigated. Ro 15-4513 was found to be, like its parent compound the 8-fluoro analogue flumazenil, a reversible alcohol competitive inhibitor of horse liver alcohol dehydrogenase (EC 1.1.1.1) with a dissociation constant of 345 microM at pH 7.0. Due to its azido group Ro 15-4513 was developed as a potential photoaffinity-labeling reagent for benzodiazepine receptors. Used with horse liver alcohol dehydrogenase, the enzyme is chemically modified and inactivated in a Michaelis-Menten type reaction via a reversible enzyme-Ro 15-4513 complex with a dissociation constant of 8.6 mM at pH 7.0. The inactivation reaction has been studied over the pH 6.0-10.0 range. The dissociation constants for the binding of Ro 15-4513 to the enzyme and the first-order rate constants for inactivation have been determined as a function of pH. These give pKa values of 7.2 and 8.8 for the free enzyme, the latter being assigned to the zinc-water ionization. The enzyme is protected from inactivation in a competitive manner by flumazenil and by many heterocyclic and thiol compounds which combine with the active-site zinc. Flumazenil has a similar binding affinity as Ro 15-4513 with an enzyme-flumazenil dissociation constant of 6.0 mM at pH 7.0. Ro 15-4513 may also have potential as a photoaffinity-labeling reagent for other metallo enzymes. Whether the effects of Ro 15-4513 on alcohol-metabolizing enzymes are also of clinical significance remains to be determined.
Langeland
B T
BT
Biokjemisk Institutt, Universitetet i Oslo, Norway.
McKinley-McKee
J S
JS
D000345:Affinity Labels / Q000494:pharmacology; D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors*; D000818:Animals; D001386:Azides / Q000737:chemistry / Q000494:pharmacology*; D001569:Benzodiazepines / Q000737:chemistry / Q000494:pharmacology*; D005442:Flumazenil / Q000494:pharmacology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D009243:NAD; D001569:Benzodiazepines; D005442:Flumazenil; C042957:Ro 15-4513; D000426:Alcohol Dehydrogenase