Yingjie Peng wrote:
..
After I have solved my strucutre, I have found my target ligand bound at
the potential binding site. Also, I have
found that there are two more ligand molecules bound along the path from
solvent to the binding site. I think this
can enrich the ligand to binding site, enhancing the local concentration
of the ligand, thus reducing the Km of the
ligand.
I've heard this kind of explanation for alternate binding sites before,
but I am skeptical. To the extent that the bound ligands are in equilibrium
with the bulk phase, the local activity of the ligand will be the same as in
the bulk phase- i.e. the bound ligands don't count in figuring the effective
concentration. If anything they lower the activity, competing with the
active site if the concentration of ligand is not >> [enzyme].
There would be a local buffering effect, so if the enzyme is gated by
a nerve impulse or absorption of a quantum of light so that it is
usually inactive and turns on suddenly, the local binding sites could
release their load in response to the local depletion faster than ligand
could diffuse in from the bulk- Then during the next "off" period all the
local binding sites recharge. This would be like the function of a
bypass capacitor in a digital electronic circuit. But during steady-state
turnover I don't see how the bound ligand could help any.
It may be easy to get ligand in physiologically irrelevant low-affinity
sites due to the high concentration of protein in crystallization experiments.
The protein is a good fraction of 1 mM, whereas physiologically important
binding sites are often in the nM to uM range. So if you add a 3-fold excess
of your ligand, and one equivalent binds at the specific site, there will
still be 100 uM or so free ligand, which may bind at low affinity non-specific
sites. Whether this loosely bound ligand will be well-ordered enough to
identify in the density is another question.
Just my thoughts on the matter,
Ed
