CONFORMATION AND CONFORMATIONAL CHANGES OF REDUCED DIPHOSPHOSPYRIDINE NUCLEOTIDE IN SOLUTION

The binding of reduced diphosphopyridine nucleotide to dehydrogenases can produce changes in its ultraviolet spectrum attributable to both the reduced nicotinamide and adenine moieties. Changes in coenzyme conformation have been proposed to explain the results of nuclear magnetic resonance studies on enzyme-coenzyme binding and fluorometric studies of the denaturation of reduced diphosphopyridine nucleotide in solution. Using solvent perturbation techniques, we described here the exposure of the coenzyme chromophores to solvent in the native conformation and after denaturation. These results, together with information obtained through the resolution of “denaturation” ultraviolet difference spectra into red or blue shifts, hyper- or hypochromicities, and the equivalent of an adenine pH difference spectrum, lead to the conclusion that the adenine ring is “stacked” to the nicotinamide ring, excluding water in the closed conformation of reduced diphosphopyridine nucleotide and reduced triphosphopyridine nucleotide in solution. We suggest that the use of “denaturation” difference spectra resolution components together with knowledge of coenzyme conformation will now facilitate interpretation of difference spectra arising from the binding of coenzyme to dehydrogenases. © 1969, American Chemical Society. All rights reserved.