> 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).
That's nice.
> 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.
Unfortunately, there is more than one possibility here, including what D.K.
has suggested. It's not always easy to figure out why the enzyme kinetic
profile does not fit expectations. Since enzymatic mechanisms in general are
insufficiently understood (especially role of dynamics on specificity etc.)
the conclusions can be quite non-trivial.
> 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?
Perhaps you need to provide a bit more details as to what kind of mutations
you made and what were you expecting to achieve. I bet that depending on how
one interprets the roles of individual residues in this particular
catalysis, the expected results would vary. You could have also mutated a
structural residue which would mean that your model is not additive even in
the zeroeth-approximation.
A.G.E.
