# What is the minimum integer n to multiply the Michaelis constant of an enzyme to - (Nov/10/2015 )

I am using a NADP+ stock solution as my cofactor in an enzyme activity assay. The Km of this enzyme for the cofactor is about 2 mM. I just measured the concentration of my NADP+ stock solution to be a little over 4 mM. Would ~ 4 mM be an adequate saturating concentration for my enzyme as I varied the concentration of the enzyme's natural substrate? If not, how might the minimum saturating concentration be determined, if there is a secret rule of thumb for it? I don't want to use more NADP+ powder than necessary as it is too expensive.

-wswr-

have you determined the saturation point for nadp of your enzyme? km is the substrate value at 1/2 maximal velocity, but 2 x km is not necessarily saturating.

-mdfenko-

This is what I wanted to calculate. Is there a formula to figure out the saturation point knowing the Km?

-wswr-

i've always run the complete saturation curve to determine the kinetic constants although you may be able to determine km and vmax by running a few reactions, with different concentrations of substrate, and prepare a lineweaver-burk plot. then, using the determined constants, you may be able to simulate the complete curve and determine the saturating concentration.

personally, i think it would be easier to run the reactions for the complete curve.

-mdfenko-

What if I solve for S with the equation v = 0.9Vmax, where 0.9 = S/(Km + S) and Km is known?

-wswr-

i don't think that will work.

solving for s becomes km/0.11111 or 9 x km (without knowing vmax).

i stand by my first comments.

-mdfenko-