<p>It is commonly believed that enzymatic catalysis is such a complex process, that even a small change in any physicochemical property of the enzyme results in a complete loss of the catalytic and ligand-binding capacity of the molecule. Therefore, when the enzyme sample is not fully active, but is electrophoretically pure, the inactive fraction is often considered equal to the non-binding fraction, and constants characterizing ligand binding by such an enzyme and ligand-induced inhibition are determined under this assumption. Here, we present an enzyme, hexameric purine nucleoside phosphorylase, whose gradual loss of a catalytic activity towards natural substrates does not correlate with the loss of the ability to bind ligands, substrates, and inhibitors. The values of dissociation constants characterizing ligand binding depend on the specific activity of the enzyme used in the experiment. Furthermore, there is a stable state of the enzyme, in which it is no longer able to catalyse reaction with natural substrates, but can still catalyse the same reaction if a substrate resembling the transition state is used. The active site conformations of individual subunits in the X-ray structure of this hexameric molecule reflect the presence of intermediate states observed in the enzyme activity decline profiles.</p>