Enzyme kinetics

Phil Potter phil.potter at stjude.org
Thu Jan 3 17:58:44 EST 2002


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?



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