On a general note, it is not unusual at all for a random mutation (i.e.,
one not in the active or regulatory site of an enzyme, and not
significantly connected with the catalytic or regulatory mechanism) to
affect the rate constant of an enzyme-catalyzed reaction (kcat or
kcat/Km) by a factor of 2 or more. Most enzyme kineticists don't get too
excited about assigning an important role to a mutated residue unless
one of the rate constants changes by a factor of 10. One danger in
interpreting mutagenesis experiments is doing only a single kinetics
measurement at a fixed pH and/or substrate concentration. If the
mutation affects the pKa of enzyme catalytic groups or the effective Km
of the substrate, a difference in rate will be noted, even though the
fundamental rate constants kcat or kcat/Km, or their pH-independent
maximal values, may not have significantly changed. Just my $0.02.
--
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Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
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fax: (315)-228-7935
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Mischa Machius wrote:
I assume you are talking about a sugar-binding enzyme ;) I have some
aspects to consider in addition to what Artem raises. Many effects of
a mutation are not recognizable in a static crystal structure or even
in an NMR structure. For example, it is usually difficult to assess
the thermodynamics of substrate binding, not to mention the kinetics.
Multi-valent substrates usually display some sort of cooperativity for
the binding process, which you might have affected by mutating one of
the subsites. You might be able to obtain some hints from a Michaelis-
Menten analysis of the mutant compared to the wild type, but that
would only be a start. Your crystallographic result of a less occupied
substrate-binding site for the mutant serves as a hint as well, but
such results are hardly conclusive. You will have to follow up with
more rigorous methods, such as ITC (thermodynamics of binding) and
time-resolved methods (kinetics of binding).
One example of an effect of a mutation that is usually not
recognizable in a crystal structure has to do with substrate guiding.
In this case, the mutation has changed the surface of the protein,
thus affecting how well the multi-valent substrate can approach and
wiggle itself into the binding site. Once in the binding site, it is
structurally virtually indistinguishable from the wild-type.
Ah, the nightmares of interpreting crystal structures in terms of
biology!
Good luck! Best - MM
On Jun 11, 2008, at 7:21 PM, Narayanan Ramasubbu wrote:
Dear all:
I have a single residue mutant whose enzyme activity is about 50% of
the wild type. Interestingly, the mutation
is in a region that involves a secondary site but not the active
site. The two structures with or without ligands
fit well (0.18 A) and the metal binding and cofactor binding sites
are all preserved in the mutant. The one difference
noticed is that the ligand does not fill the active site (partially
occupied subsites) unlike the wild type where all the
subsites are occupied. Water structure around the actives site
residues are "identical".
I looked at the electrostatics and both surfaces look similar (not
an expert).
There are some residues whose sides chains show some positional
disorder and these residues are at the edges of the
active site.
The resolution of the both data sets are 1.5A.
The mutant enzyme was derived by MR.
One another possibility that I want to look at is to compare the
compactness of the two enzyme structures.
What is the best way to compare that? I am wondering whether the
"breathing" that was mentioned for some enzymes
might be playing a role in the mutant enzyme.
Also, I would appreciate comments on other possible explanations for
this unusual (?) behavior.
Thanks a lot
Subbu
--------------------------------------------------------------------------------
Mischa Machius, PhD
Associate Professor
Department of Biochemistry
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.; ND10.214A
Dallas, TX 75390-8816; U.S.A.
Tel: +1 214 645 6381
Fax: +1 214 645 6353
