dk at no.email.thankstospam.net
Thu Jan 3 21:28:22 EST 2002
In article <20020103225844.22290.qmail at ww02.jatek.com>, phil.potter at stjude.org wrote:
>We have expressed a carboxylesterase in coli and are monitoring kinetics of
>nitrophenyl acetate metabolism. The wildtype protein give nice values that
>fit Michaelis Menton parameters (r2 for hyperbolic curve fit =0.98).
>We have made two point mutants that we predicted from the 3D structure would
>affect catalysis. However these proteins apparently do not follow Michaelis
>Menton kinetics (r2 ~0.68). We considered this might be an allosteric
>problem since the data seemed to fit a sigmoidal pattern.
>Is there a good way of ensuring that the data we have will fit the latter
>and is it possible to determine conventional paramaters (Km and Vmax) from
>these curve fits? Basically, how do I know if a sigmoidal fit is approrpiate
>for this data?
First you fit to MM with cooperativity (substrate and Km to the power of "n")
and if regression coefficient is good, you say "aha, that's it". Then you do
experiments aimed at undestanding what real things lead the system
to behave so that it can be described by allosteric formalism.
Another possibility is that you have not sigmoid but two hyperbolas.
In that case, your expressed mutants are not homogenous.
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