Molecular polymorphism and mechanisms of activation and deactivation of the hydrolytic function of the coupling factor of oxidative phosphorylation

Abstract

The 13S coupling factor of oxidative phosphorylation from Alcaligenes faecalis has a latent ATPase function that is activated by heating at 55.degree. C for 10 min at pH 8.5 in 50% glycerol. The specific activity increases from 0.1 to 20-30 .mu.mol min-1 mg-1. ATP is not required for stabilization at 55.degree. C when glycerol is present. Activation involves displacement of the endogenous ATPase inhibitor subunit (.epsilon. subunit), and readdition of this subunit results in deactivation. In the deactivation process the ATPase inhibitor subunit is replaced by other cationic proteins such as protamine, histones or poly(lysine). Mg2+ and H+ also are effective deactivators. Since every positively charged substance tested deactivated the enzyme, the inhibitor subunit is probably complexed with the enzyme at a site containing a surplus of negative charges. The activated enzyme is not cold labile, but it is salt labile, having a half-life of 2-3 min in 0.1 M KI at 25 or 0.degree. C. The activated ATPase is also inhibited by aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) and by the cross-linking agent dimethyl suberimidate. Evidence for polymorphism comes from finding that the properties of the unactivated enzyme (intrinsic ATPase) are different in many ways from the properties of activated ATPase. The coupling factor''s ability to hydrolyze ATP suggests that there are at least 4 distinct functional allomorphs of this enzyme: the latent enzyme which has no kinetically measurable ATPase activity, intrinsic ATPase, which is catalyzed by a small percentage of the molecular population that was activated by some natural mechanism, activated ATPase which has properties different from those of intrinsic ATPase, and aged activated ATPase in which some of the properties (Km for substrate, sensitivity to deactivation by Mg2+ and H+) spontaneously change within 30 min.