Since, I was performed the point mutation to overcome the feedback inhibition problems, so now I'd like to try the enzymatic test to my strains in order to compare that enzyme activity. I've got the suitable condition and method from the paper but I'm not sure whether I have to start from optimization of period of incubation, enzyme concentration or whatever to get all desire data for Km calculation. I've never done this before, thanks everyone in advance.
My recommendation is to follow the progress of the reaction with time for each of a range of enzyme concentrations and substrate concentrations. You must measure the initial rate of the reaction, before it starts to slow down because of product inhibition or substrate depletion. The best enzyme concentration at which to make this measurement may differ depending upon the substrate concentration. Plot the initial rate of product formation/enzyme concentration on the y-axis against the substrate concentration on the x-axis (Michaelis plot). Fit the curve to the Michaelis-Menten equation to get the Km and Vmax.
There is an interesting twist to M-M kinetics for this enzyme because its substrate consists of 2 pyruvate molecules. Technically, this could result in 2 different Kms for pyruvate. They could be similar enough as to be indistinguishable, or different enough to result in a weird looking M-M plot that doesn't fit the equation, or vastly different so that only the higher Km is detectable. You also need thiamine diphosphate and Mg2+ as cofactors. It's probably simplest to use these at saturating concentrations.
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue