Can you be more specific about the reaction you are interested in? Is it rapidly reversible equilibrium binding of a ligand to a protein, slowly reversible (long residence time) noncovalent binding of a ligand to a protein, reversible covalent binding of a ligand to a protein,  reversible noncovalent complexation between small molecules, or reversible covalent bond formation between small molecules?

I don't know how to calculate such things, but I could suggest ways to measure some of them.

Thank you for your concern, actually ligand interacts with a protein through hydrogen bonding, hydrophobic and electrostatic interactions and the Kd value is about 314nM  and the protein has two binding pockets for the ligand  Kd value of the 2nd binding pocket is very low about 4nM negative co-operativity.    

It seems that you are interested in the dissociation rate of a non-covalently bound ligand from a protein. The system is a bit complicated because there are 2 cooperative binding sites, but the fact that they have very different Kds may be helpful experimentally.

In many cases, the rate of dissociation of a ligand (koff)  is very rapid (milliseconds to seconds) and is defined by the binding equilibrium Kd=koff/kon. In other cases, the dissociation can be very slow (minutes to hours) because there is a slow conformational change in the protein involved.

Two excellent articles that discuss this topic in detail are Copeland et al (2007) Nature Reviews of Drug Discovery 5, 730-739 and Tummino and Copeland (2008) Biochemistry 47, 5481-5492

Thank you  Dr.Adam, I will go through the papers you suggested.