James,
I think you need to be a little more specific about what you want to
calculate. Keq for a reaction A <=> B will not change with an enzyme
mutation as the thermodynamic relationships between the reactants and
products do not change. As a catalyst, the enzyme impacts only the
free energy of activation delG "double-dagger" and not the reaction
thermodynamics.
However, a mutation can markedly impact the Keq of ligand binding, but
Km here is of no use. As mentioned by others, Km is an amalgam of
many intermediate equilibria in a reaction up to the rate limiting
step. Moreover, Km equals Kd in only the most simplest of enzymes,
which sadly few of us work on. Thus, you need to determine actual Kds
for all your ligands, which in turn requires you to determine the
reaction characteristics of your enzyme (random sequential, ordered,
bi-bi, etc.). It is not easy and real enzymologists are rapidly dying
off (or at least retiring). The old Cantor and Schimmel book
"Biophysical Chemistry Part III" (1971) should help you here. I
actually cannot think of a recent monograph on pure enzymology
published in the last decade or so.
If you want the change in activation energy caused by a mutation (delE
"double-dagger"), the the "k" in RTlnk is the kcat and an Arrhenius
plot is just the thing to use. But as Roger said, you MUST carefully
control for temperature dependent pH changes.
Good luck and fire up the spec for some long days,
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [email protected]
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On Wed, Nov 18, 2009 at 8:24 AM, james09 pruza
<[email protected]>wrote:
Dear All,
Sorry for the non-ccp4 query.
I have solved a crystal structure of an enzyme and woring on its
biochemical aspect. We have a mutant of this enzyme and we are comparing
some thermodynamic parameters of this enzyme with mutant( lke delH and
delS,
delG). we have done the expt at different temp. and know the km value at
these temp. Now the question is how to get the value of delH and delS.
The relation of these parameters is:-
-RTlnK = delH - TdelS
Is the K in this relation is km or kd. If it can be treated as kd, so it
should be 1/km.
All suggestions are welcome.
Thanks
James
