Analysis of one enzyme recyclization system with low enzyme concentration

One enzyme recyclization reactions in the presence of two substrates and a small amount of a coenzyme are characterized by a long prestationary phase, in the course of which the distribution of coenzyme forms varies. The kinetics of the coenzyme prestationary phase was analyzed theoretically and some equations characterizing mainly the start and the end of this phase were derived. To verify experimentally the derived equations two recyclization systems with horse liver alcohol dehydrogenase, NAD or NADH, and corresponding substrate pairs (p-nitrosodimethylaniline and 1-butanol, or furyl alcohol and acetaldehyde) were investigated. Consequences for the analytical practice and utilization of these reactions for the determination of small coenzyme and enzyme concentrations are discussed.