So heres the background, I am currently doing research on one of the luciferase proteins. The luciferase protein creates bioluminescence using the substrates ATP and Luciferan. Its a two step process... but the general sense is that u can use the light to measure the activity of the protein as a unit of the amount of protein in the solution. What this means is that u can measure the km by using the relative values obtained by the light emitted by the protein. This process measures the 2 step reaction as a whole, and allows for an accurate km for the two substrates Luciferan and ATP. My issue is that we created a mutant in which the km for luciferan was much lower than wild type. However, when we tried to do the ATP km, and the km values continually do not make sense, the bioluminescence is greatly reduced, even when using the same concentrations. My boss ssaid possibly its the order of reagents, but why would that reduce the bioluminescence? P.S. I am only a freshman in college, so my knowledge of biochem is limited. WHere should i go to learn more about this type of thing? or could someone help?