Acid-catalyzed hydration of reduced nicotinamide adenine dinucleotide and its analogs

Abstract

The rate of the primary acid modification reaction of NADH and 1,4-dihydro-3-acetylpyridine adenine dinucleotide (APADH) and their analgoues was studied over a wide pH range (pH 1-7) with a variety of general acid catalysts. The rate depends on [H+] [H+ concentration] at moderate pH and becomes independent of [H+] at low pH. This behavior is attributed to substrate protonation at the carbonyl group (pK of NADH = 0.6). The reaction is general acid catalyzed; large solvent 2H isotope effects are observed for the general acid and lyonium ion terms. Most buffers cause a linear rate increase with increasing buffer concentration, but certain buffers cause a hyperbolic rate increase. The nonlinear buffer effects are due to complexation of the buffer with the substrate, rather than to a change in rate-limiting step. The rate-limiting step is a H+ transfer from the general acid species to the C5 position of the substrate. Anomerization is not a necessary 1st step in the case of the primary acid modification reaction of .beta.-NADH, in which .beta. to .alpha. anomerization takes place.