| Abstract: | The substrate-cytochrome P-450C-21 binding reaction has been investigated in detail by using the purified cytochrome. The apparent substrate dissociation constant (KDapp) depended on the enzyme concentration, indicating that the binding reaction does not follow simple two-component mass action equilibrium. However, the binding data fit reasonably well to a model in which the P-450C-21 exists in a monomer-dimer equilibrium and the substrate does not bind to the dimer. The intrinsic dissociation constant (K1) and the dissociation constant for the dimerization reaction (K2) were calculated from the titration data by a pattern search procedure. K1 and K2 were found to be essentially independent of the enzyme concentration, indicating the appropriateness of the assumed model. In the present study, all factors that increased the dissociation of the dimer, as indicated by an increase in K2, decreased KDapp so that it approached the intrinsic constant K1. These results suggest that there is mutual interaction of the substrate binding and self-association reactions of cytochrome P-450C-21 in the purified preparation. |
