Metal-directed affinity labeling of zinc(II), cobalt(II), and cadmium(II) horse liver alcohol dehydrogenases.
The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition to bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn greater than Cd greater than Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co greater than Zn greater than Cd.
D000345:Affinity Labels; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000494:pharmacology*; D003035:Cobalt / Q000494:pharmacology*; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011485:Protein Binding; D013053:Spectrophotometry; D015032:Zinc / Q000494:pharmacology*
D000345:Affinity Labels; D002104:Cadmium; D003035:Cobalt; D000429:Alcohol Oxidoreductases; D015032:Zinc