Interaction of drugs with a model membrane protein: Effect of dibucaine on cytochrome oxidase proteoliposomes

Abstract

Cytochrome oxidase is a mitochondrial trans-membrane protein which catalyzes the vectorial transfer of electrons from cytochrome c to molecular oxygen. When the oxidase was incorporated into liposomes composed of saturated phospholipids, enzymatic activity was reduced as compared to the activity of either the isolated enzyme or the enzyme incorporated into soy bean phospholipid (asolectin) liposomes. This reduced activity probably resulted from partial replacement of retained oxidase boundary lipid with exogenously added lipid and an unfavourable orientation of a portion of the oxidase molecules for reaction with externally added substrate. On the other hand, substrate binding at the low affinity site was enhanced by incorporation of the oxidase into vesicles composed of either saturated phospho-lipids or asolectin. At pH 7.4 the local anesthetic dibucaine behaved as an uncompetitive inhibitor of the enzyme, while at pH 6.0 the inhibition pattern became mixed in type. Dibucaine had similar effects on both the isolated and incorporated enzyme except that, in general, the anesthetic caused less inhibition of the incorporated oxidase. It is postulated that positively charged anesthetic molecules act predominantly by competing with substrate for binding while non-charged anesthetic molecules interact with the oxidase boundary lipid to form non-productive complexes.