Fluorinated ligands as nuclear magnetic resonance probes of active-site nonequivalence in abortive ternary complexes of horse liver alcohol dehydrogenase.
The interactions of the dimeric horse liver alcohol dehydrogenase (LADH) with ligands in two nonreactive ternary complexes have been examined by using nuclear magnetic resonance and fluorescence techniques. One complex contains the enzyme, NADH, and the abortive alcohol substrate p-(trifluoromethyl) benzyl alcohol. The trifluoromethyl group allows the bound environment and bound to free exchange kinetics of this alcohol to be examined by using 19F NMR. Binding isotherms of the coenzyme and the abortive substrate were examined by using fluorescence. Similar measurements were made with the enzyme, NADH, and the aldehyde analogue p-(trifluoromethyl) benzamide. For both complexes there was no evidence of cooperative equilibrium binding of any ligand. Careful measurements of the exchange kinetics of the fluorinated alcohol or amide when binding to the enzyme NADH complex using NMR techniques showed that a single lifetime describes the exchange of ligands from both subunits of the protein. These results appear to rule out any site-site interactions in this system and support the notion that the observed biphasic kinetics observed in transient reactions of LADH with NAD and alcohols is a property of a single site in this system.[1]References
- Fluorinated ligands as nuclear magnetic resonance probes of active-site nonequivalence in abortive ternary complexes of horse liver alcohol dehydrogenase. Anderson, D.C., Wilson, M.L., Dahlquist, F.W. Biochemistry (1982) [Pubmed]
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