One doesn't usually measure the Kd (equilibrium dissociation constant,

(k-1/k+1) of an enzyme-substrate pair, except in special cases or with special techniques. The usual measurement is the Michaelis constant Km [(k-1 + k+2)/k-1]. The main reason for this is that the reaction is occurring during the measurement, so the substrate gets used up, hence it's concentration is decreasing all the time.

The special case is if the value of k+2 (the rate constant for conversion of the enzyme-substrate complex to enzyme+product) is much lower than k-1 (the rate constant for dissociation of the enzyme-substrate complex to enzyme+substrate), in which case the Km is essentially the same as the Kd. You don't usually have sufficient information to make this assumption, unless it's a very slow enzyme reaction.

The special technique is pre-steady-state kinetics. You need special stop-flow or quench-flow apparatus for this because the measurement is made on a millisecond time scale. You also need some optical (absorbance or fluorescence) readout of substrate binding.

To measure Km kinetically in the usual manner, you must measure the initial rate of the reaction at several substrate concentrations. Plot the product measurements at multiple evenly-spaced time points at each substrate concentration. Draw a straight line starting from the origin and tangent to the earliest time point measurements, including all the data until the reaction starts to slow down. The slope of the tangent line is the initial rate.

Next plot the initial rate on the y-axis versus the substrate concentration on the x-axis. Use a suitable computer program to perform a nonlinear regression to the Michaelis-Menten equation to get the value of Km and Vmax. The substrate concentrations used for the analysis should cover a range below and above the Km to show the curvature of the plot.