Comparing mutant and wild type enzyme kinetics - (Nov/02/2015 )

I am attempting to characterize a change in Km in a particular enzyme after mutating one of the active site amino acids (I learned kinetics in school - over 10 years ago now - but have never done kinetics research!). I have wild-type enzyme, and I know how to calculate Units and initial velocity and approximately how many Units of enzyme will give me reliable readings (first order kinetics) to determine Km. I have now made and purified my mutant protein, but I'm a confused as to how to directly compare the Km. Should I start with the same number of Units enzyme/mL of the mutant that I did with the wild type? Wouldn't the apparent Units be much lower since I'm expecting Km to increase? Should I do a protein assay to determine mg/mL of my wild type and mutant and assume there are no significant differences in the preps that would affect their activity, other than the one amino acid mutation? Basically, I'm trying to figure out how much mutant enzyme to add when I characterize the mutant reaction at different substrate concentrations. Thoughts/advice appreciated!

you should determine the specific activity of both the wild type and mutated enzyme.

i would perform substrate saturation curve assays with both enzymes in parallel, using the same amount of protein in each assay, to make direct comparisons of the kinetic values.

So to make sure I understand you, you recommend to use the same Units of mutant protein when making the substrate curve? Or did you mean the same mg amount of protein? Thank you very much!

yes, mg of protein (more likely ug) so that each assay has the same final concentration of protein (mg/ml or ug/ml).