Binding of ligands to horse liver alcohol dehydrogenase lacking zinc ions at the active sites.
The zinc-deficient enzyme binds the fluorescence probes for the enzyme substrate pocket (auramine O, 13-ethylberberine, chlorprothixene and acridine orange) more tightly than the native enzyme, whereas 1-anilinonaphthalene 8-sulphonic acid is bound with comparable affinity. The use of fluorescence probes as reporter ligands revealed that the formation of binary complexes between the zinc-deficient enzyme and aldehydes is possible (as with the native enzyme) and confirmed an increased affinity of coenzymes to the modified enzyme. The absence of catalytic zinc ions brings about a loss of the essential stabilization effect in simultaneous NADH and aldehyde binding to liver alcohol dehydrogenase. 2,2'-Bipyridine, which chelates the active-site zinc ion in the native enzyme, is bound rather loosely to the same site as aldehydes, auramine O and ethylberberine in the case of the zinc-depleted enzyme. The stopped-flow measurements showed that the pH dependence of auramine O and ethylberberine binding to native and zinc-depleted enzyme is essentially similar. These data are compatible with the presence of ionizable groups in the surroundings of the bound probes. This group might be either His-67, bound to the zinc ion, or the zinc-liganding water molecule in the case of the native enzyme (pK close to 9), or the free His-67 residue in the case of the zinc-deficient enzyme (pK about 8).
D015082:2,2'-Dipyridyl / Q000378:metabolism; D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001576:Benzophenoneidum / Q000378:metabolism; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D015032:Zinc / Q000502:physiology*
D000447:Aldehydes; D000817:Anilino Naphthalenesulfonates; D001051:Apoenzymes; D001059:Apoproteins; D003067:Coenzymes; D008024:Ligands; D009243:NAD; D001576:Benzophenoneidum; D015082:2,2'-Dipyridyl; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D015032:Zinc