calculating Ki from IC50 - (Feb/12/2010 )

I am trying to calculate Ki from IC50 using the following formula: Ki= IC50/(S/Km+1), but in the web which I use http://botdb.abcc.ncifcrf.gov/toxin/kiCalE...5B4C8CE1C1D5F85 they ask me to put the concentration of enzyme, and if IC50 is lower than C/2 of enzyme, the Ki can't be done because it is not stechiometric.
My compounds are very active and my IC50 is 0,4nM , while concentration of enzyme 4nM. I can't use less, because I won't have enough activity and reaction will not go (colorimetric assay). What can I do? Is it important or I can calculate Ki from the equation by myself?
Please, help!

kamilaf on Feb 13 2010, 09:44 AM said:

It’s to early on Monday morning to get my brain round this properly . I think the short answer is yes it is important, because you appear to have knocked out 50% of the enzyme activity with only 1/10 the relative concentration of inhibitor?? This seems strange. What is the Km for your substrate?

Here’s the original reference for converting IC50 to KI. Note that the equation you have is only valid if you have a competitive inhibiting compound.
Cheng Y, Prusoff WH. Biochem Pharmacol. 1973 Dec 1;22(23):3099-108. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.

My Km is 100uM and this concentration of substrate I am using in assay.
I am wondering if my IC50 can't be higher than C\2 os enzyme, how can I check compounds with very good activity??

kamilaf on Feb 15 2010, 06:49 PM said:

Even the most active compounds will be limited by the ‘at least one inhibitor molecule to one enzyme molecule’ rule of classic kinetics.

The only exceptions I can think of:
The enzyme concentration is wrong; does the activity possibly come from a minor component? Or is activity associated with multimeric states?
The inhibitor is working as a general denaturant. Often these are characterised by steep hill slopes.
The inhibitor is large enough to bind multiple enzymes.

If it is about the concentration of protein is was bought from the company, and they give the concentration, maybe it is lower than they say. The other thing that the protein can easily lose activity, it is not very stable, maybe this is the problem? It is decomposed during reaction?

The inhibitor can´t be general denaturant because I have checked it on few other very similar enzymes and it wasn´t so active.

Any other suggestion?

It’s got me stumped. Looking on the bright side, you may be onto something novel .

I guess in answer to your original post. You still should not use IC50 to calculate Ki, instead go through the usual steps of determining the type of inhibition using different inhibitor/substrate ratios.

Good luck.

I will try to answer your question. It is because the test compound is very potent. If you google "tight binding inhibitor", you may find some equations to convert the Ic50 to Ki.
You may try Munson-Rodbard equation.