The Michaelis plot is a graph of initial rate of an enzyme reaction on the y-axis versus substrate concentration on the x-axis. So the first thing you must do is measure the initial rates from your fluorescence versus time progress curves, each of which should have been measured at a different substrate concentration. The initial rate is the slope of the RFU versus time curve that is tangent to the curve at time zero.

Once you have made the Michaelis plot, you can measure the Km and Vmax kinetic constants by using nonlinear regression to fit the data to the Michaelis-Menten equation. This is preferable to using the Lineweaver-Burk double reciprocal plot.

Thank you very much ....Adam B Shapiro