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 enzymecatalyzed 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 dehydrogenasecatalyzed oxidation of glycerol by NAD, it was found that this enzyme did not behave according to the classical "Michaelis-Menten" mechanism of enzyme action.
Quasi-steady-state (1) enzyme kinetic data usually consists of a series of substrate or product concentrations, c,, obtained at discrete times, I*, or of a continuous record of c(t) plotted on a stripchart recorder. The usual procedure in enzyme studies is to use the kinetic data to obtain a single parameter called the "initial, quasi-steady-state rate" (v(O)). v(0) obtained in this way is not an experimental observable. It is a parameter that was estimated from observables. As in the case of other estimated ' This work was supported in part by grants from the NSF (GB-7 110 and GB-20612) the Robert A. Welch Foundation (G517), and the NIH (CA-l 1430).