I've been looking into the kinetics of polyphenoloxidase catalysis of the oxidation of catechol. I've run the reaction at seven different catechol concentrations () both uninhibited and in the presence of trypsin (measuring using spectrophotometry). The data came out nicely and I've been able to calculate Vmax and Km values that I'm pretty comfortable are accurate. However, I am hitting a wall on kcat.

 

Since I obtained my PPO directly from potatoes, I don't have a value for [Etotal]. My question is, is there any way to work around this? Since kcat=k2 in this case, I've tried setting up simultaneous equations using kcat = Vmax/[Etotal] and k2 = v0/[ES], however, [ES] is also unknown (I think) so that doesn't really work. Any other ideas? Is there a way to determing [Etotal] or [ES], or to relate them to one another? Any other ways to approach this?

 

Thanks!

I think that if you want to find k_cat, you would need both an accurate protein concentration and knowledge that your enzyme was pure.