In an attempt to plot the michaelis- menten curve, it requires the initial rate as the y-axis and the substrate contration as the x-axis.
Now how do you get the substrate concentration of the x-axis given the volume of each assay and the buffer volume.
It is not possible to figure out the substrate concentration by knowing how much enzyme is added in a known volume. If you want to characterize a simple one substrate enzyme-catalyzed reaction, where Km and Vmax need to be determined, the enzyme concentration is fixed and the subtrate is increased. Depending on the enzyme activity you need to use substrate concentrations in the range of nM or uM or mM or M. The more concentration points you take within the scale the finer the accuracy of your Km and Vmax values become.
Generally, you know the substrate concentrations because you prepared the stock solution and diluted it into the enzyme reactions. The enzyme concentration is not relevant to the simple substrate dilution calculation. Did you mean the substrate concentration is 6 mM?
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