Bovine adrenocortical microsomal hydroxylase and thermotropic transitions substrate-cytochrome P-450 binding reaction versus substrate hydroxylation

The effect of temperture 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 (K_s) exhibited four breaks between 10 and 40°C which corresponded to the characteristic temperatures of the lipids' phase transitions. Unlike the case of the K_s 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 in the case of the K_s. 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.