Mechanism of Binding of Reduced Nicotinamide Adenine Dinucleotide Phosphate to Vertebrate Fatty Acid Synthetases
SITES AND TYPE OF BONDING, CONFORMATION OF COENZYME, AND BINDING OF ANALOGUES
Richard E. Dugan 1 and John W. Porter 1
From the
1 From the Lipid Metabolism Laboratory, Veterans Administration Hospital, and the Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706
The techniques of fluorescence enhancement and fluorescence polarization were used to study the binding of NADPH and structurally similar pyridine nucleotides to pigeon and rat liver fatty acid synthetases. NADPH, on binding to the enzyme, changes from a closed to an open conformation and the nicotinamide ring becomes associated with hydrophobic groups of the protein. The orientation of NADPH when bound to enzyme is such as to permit the transfer of radiant energy from tryptophan residues of the protein to the nicotinamide moiety of NADPH. The binding affinity of the enzyme for the coenzyme is considerably reduced by removal of the 2'-ribose phosphate (NADH), and by oxidation of the pyridine ring (NADP), but it is only slightly decreased by alteration of substituents on the adenine and pyridine rings (deamino-NADP and acetylpyridine-NADP). Enzyme-bound NADH is not aligned with the enzyme in the same manner as NADPH. The binding of NADPH to enzyme is competitively inhibited by pyridine nucleotide analogues, and at higher concentrations, by the ions of inorganic salts structurally unrelated to NADPH. Hence, electrostatic forces of attraction between charged groups on the coenzyme and the enzyme appear to have an important role in maintaining enzyme-coenzyme association.
Submitted on June 8, 1970
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