An on-line method for the collection and analysis of quasi-steady-state kinetic data for enzyme-catalyzed reactions.

A special mixing device for initiating enzyme-catalyzed reactions is used to rapidly achieve an unperturbed quasi-steady state. An on-line computer is employed to sample the initial conditions, the mixing time, and concentrations that change as a function of time during this quasi-steady state phase. A statistical method for estimating initial, quasi-steady state rates from the time course of the enzyme-catalyzed reaction is described. Practical considerations for using this parameter estimation system lead to the conclusion that for the enzyme-catalyzed reaction tested, the extent overall reaction should be above .2% for high initial substrate concentrations, and above 1% for initial substrate concentrations in the range of the Michaelis constant. Application of this method to a typical enzyme-catalyzed reaction suggests that objective estimates of initial rates from a given set of concentrations and corresponding times can be obtained with a standard error in the range of 2–3%, but that reproducibility is not better than about 10%. When this procedure was used to estimate initial rates for the glycerol dehydrogenase-catalyzed oxidation of glycerol by NAD, it was found that this enzyme did not behave according to the classical “Michaelis-Menten” mechanism of enzyme action.