Bovine adrenocortical microsomal hydroxylase and thermotropic transition. Substrate-cytochrome P-450 binding reaction versus substrate hydroxylation.

The effect of temperature on steroid C-21 hydroxylation and substrate-cytochrome P-450 binding reaction under turnover conditions (NADPH + O2 are investigated. The Arrhenius activity plot exhibited a single break, while the van 't Hoff plot of the substrate dissociation constant (Ks) exhibited four breaks between 10 and 40 degrees C which corresponded to the characteristic temperatures of the lipids' phase transitions. Unlike the case of the Ks value, the detergent Triton X-114 was without effect on the Arrhenius activity plot. This indicates that the single break in the case of the enzyme activity is distinct from but not necessarily independent of the multiple breaks inthe case of the Ks. At physiologic temperature and concentration of the substrate, the free energy (--9.5 kcal/mol) of the substrate-cytochrome binding reaction is more than sufficient to account for the apparent activation energy (6.6 kcal/mol) of the overall hydroxylation. This suggests that the substrate-cytochrome P-450 binding reaction has the potential of being a source of energy for the overall reaction.