Dear colleagues:
I am trying to interpret the results of the substitution of a Methionine
with Alanine. Following is the kinetic data on the mutation -
1. Km increased by 0.5 fold
2. Vmax decreased by 3.5 fold
3. Kcat decreased by 4 fold
4. Kcat/Km decreased by 10 fold.
5. Activity at saturating concentration of substrate - only 15 % of the wild
type.
Is it possible to conclude from the data that the methionine is involved in
stabilization of the transition state (the methionine is located inside the
putative active site; we do not have a structure of the enzyme-substrate
complex)? Is this even possible atall?
Although impossible, on second thought, given the ability of the
micro-environment to alter/bring down pKa s, is there any instance or
possibility that a methionine could or has acted as a nucleophile?
In addition to all other routine precautions to avoid experimental errors, a
CD analysis of the mutant and wild type has been performed and there are no
obvious differences in the spectrum. The wild type and mutant enzyme show
almost identical size exclusion profiles.
I would appreciate any suggestions or comments.
Sincerely,
Deepak
