[Rohini]

So experimentally I changed the substrate concentration and measured the absorbance for the reaction (after adding enzyme and stopping the reaction after 10 minutes) but from this I am unsure as to how to work out the velocity of the reaction, so that I can determine Km and Vmax. Any help would be appreciated thank you.

[Babcock_Hall]

You appear to have done an end-point or stop-time assay.  You need more information than you have provided so far.  At the very least, you would need to have done this experiment multiple times at different concentrations of substrate.  If you want to calculate velocities in micro moles per minute (my preference, but not always necessary), then you will need additional information about the experiment.  Finally, it is a forum rule that you show an attempt first, then we help you.

[Rohini]

Sorry, yes, I measured the absorbance at different concentrations of PNP (the substrate) and I'm trying to plot graph for the Substrate Concentration vs. Velocity. So I can use it to plot the Lineweaver-Burk plot to calculate the Km, but what additional information would I need to calculate the micromole/minute/ml?

[Babcock_Hall]

How well do you understand Beer's law?  Can you write out the key mathematal equation for us?