Hi there,

You are right : as long as the enzyme concentration used in the assay is far less than the substrate concentration, your kinetics are OK for velocity determination. And you can perfectly compare the calculated Km values.

 Thank you very much Dominique. Your answer gave me a lot of happiness! Now I know that I am doing the things right!

Correct, as long as the substrate is in excess the calculated parameter based on initial velocity data will be independent of protein concentration. 

As mentioned in all the above posts, rather than the enzyme concentration by itself, it is the enzyme to substrate ratio that matters for the kinetics' or initial velocity measurements. Also it may be apt to state that substrate concentration for an enzyme-catalysed reaction should exceed at least 5 times its Km value vis-a-vis the said enzyme.

@Mrinalini Menon : There is absolutely no specific requirement concerning substrate concentration toward Km value. As long as substrate is much more abundant than enzyme it will allow steady state to occur which is the only requirement for initial velocity determination. And there is no Km notion in this proposition whatsoever.

If you measure enzyme activity over a large enough range (0.1 times the lower Km to 5 (better 10) times the higher Km, space data points logarithmically (i.e. 1.0, 1.5, 2.1, 3.2, 4.6, 6.8, 10,...)) you can fit your data to the sum of two hyperbolae: v = Vmax1 * S / (Km1 + S) + Vmax2 * S / (Km2 + S). Most data handling programs (Sigmaplot, gnuplot etc.) can do a non-linear curve fit using the Marquardt-Levenberg algorithm (Simplex would be better, but I don't know ready packages that do that). Always assuming, of course, that you don't get substrate inhibition, that would make things a lot more complicated...

It is true as far as the enzyme is the same native states in the varied concentrations. Some enzyme which may be complicate structure or large ones, in particular, which is allosteric oligomeric proteins,  can alter the conformation of the enzyme in dependence of their concentration at very low or very high. 

I always am recommending the reliable and sensive HPLC-photometric enzyme assay (please see file; J Chrom B BIN LIP Km).   HPLC method requires the substrate in very small amount; i.e., only 0.1 mL of reaction mixture is required to perform the enzymatic reaction.   By the way, Sigma has previously stopped selling our important substrate of BAQ (biotinyl-6-aminoquinoline), but HPLC method can be performed to publish the above article due to minimum use of substrate (measurement of initial velocity at low substrate concentration). 

We have previously developed an HPLC-photometric-carboxylase assay in order to get rid of the use of dangerous radio-isotope (please see file; 4-kinds of carboxylase). 

By the way, Vs of hydrophobic membrane-glycoprotein enzyme such as rat brain biotinidase and rat kidney biotinidase increase by dilution; i.e., series of diluted enzyme solutions are prepared and measured, and graphiclly estimated the V value.   Then precise dilution is required.   Thus, precise HPLC-Surf-SEC protein-determination method is indispensable (please see file; HPLC-Surf-SEC protein-determination method).