Increased conformational stability of mitochondrial soluble ATPase (F1) by substitution of H2O for D2O.

Between 20 and 40 °C D₂O inhibits the hydrolytic activity of soluble mitochondrial ATPase F₁. The effect of D₂O is proportional to its concentration in the incubation mixture and at nearly 100% D₂O in the incubation mixture the ATPase activity is inhibited by 50–60%. The effect of D₂O is mainly on the V of the reaction. At temperatures above 45 °C, D₂O does not inhibit the activity. D₂O protects against the denaturation of the enzyme that is observed at relatively high temperatures and against the cold-induced inactivation of F₁. The intensity of fluorescence of 8-anilino-1-naphthalene sulfonate incubated with F₁ increases as the enzyme becomes inactivated by low temperatures; in D₂O the changes of fluorescence are almost nil. These observations indicate that H (or D) bonding between the solvent and the protein as well as the strength of the hydrophobic interactions within the enzyme as determined by the solvent are of central importance in determining the overall activity of F₁ and the stability of the enzyme to denaturing conditions. Moreover, the data indicate that the enzyme may exist in two different conformations, each with a characteristic activation energy. It is also proposed that D₂O may be employed with success in the isolation and purification of labile enzymes.