how to get good kinetic data?

Bob Steinberg rsteinbe at etowah.uokhsc.edu
Thu Jul 3 18:04:41 EST 1997


john gately wrote:
> 
> Folks--I've got an enzyme that transfers an activated group (PO4) from a
> cofactor (cosubstrate to exact) to a substrate.  The assay measures the
> production of isotopically-labelled product resulting from the transfer
> of the isotope from the cofactor to the substrate.  So if I want kinetic
> data for the substrate, i.e., Vmax, Km, the [cofactor] cannot be
> limiting.  Is that right?  But I can't put that much hot cofactor into
> my reactions.  I'd need my own vault under the mountains in Nevada.  But
> if add to the reactions enough cold cofactor to get uM concentrations,
> it seems I'd have trouble getting back enough counts to measure the
> activity.  I'm a bit mixed up on how to do this experiment.  Can anyone
> help me.  Thanks much.--John
This is essentially the same situation as found with protein kinase
assays using transfer of 32PO4 from ATP to a peptide or protein. To get
good kinetic data for the substrate you want cofactor concentrations of
at least about 5X Km (e.g., for protein kinase reactions where the Km
for ATP is about 10-20 micromolar, we use 100 micromolar 32P-ATP). The
dilution of radioactive cofactor is not a major problem unless the Km
for the substrate is much lower than that for the cofactor, since you
can run the reaction to consume up to 5% or so of the cofactor without
significant depletion. This means you should think about the total cpm
in your sample rather than the specific activity in deciding how much
label to use. (If the Km for the substrate is much lower than that for
the cofactor, I think you are screwed, but maybe someone else has
another solution.) I hope this helps.



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