Protein-lipid interactions in cytochrome oxidase from Saccharomyces cerevisiae. Effects of detergents and reconstitution of enzyme activity by phospholipids by using cholate-mediated exchange.
Cytochrome oxidase, purified from the yeast Saccharomyces cerevisiae, was shown to have associated phospholipid, cholate or detergent, which could be varied by dialysis or (NH4)2SO4 precipitation of the protein. Cholate and the detergents Triton X-100 and Tween 80 were shown to differ in their ability to support enzyme activity. Changes in the Vmax, but not the Km, for ferrocytochrome c as the cholate concentration was varied indicate that cholate increases the number of exposed active sites of the enzyme. Cholate was used to introduce chosen phospholipids into the lipid environment of yeast cytochrome oxidase. Kinetic studies clearly showed that cholate can mediate exchange of exogenous for endogenous phospholipid. All phospholipids screened supported activity up to the basal value for the unsubstituted enzyme, whereas mitochondrial phosphatidylethanolamine and various phosphatidlycholines (except 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) produced enhanced activity. A detailed kinetic examination revealed that the major effect of phosphatidylethanolamine is to increase k+1, whereas the major effect of phosphatidylcholine is to increase K+2 in the minimal kinetic scheme E + S k+1 in equilibrium k-1 ES k+2 leads to E + P Cardiolipin, although supporting activity, does not give any enhancement of k+1 or k+2 over the values for the cholate control. The relevance of these observations to protein-lipid interactions in cytochrome oxidase is discussed.
D002793:Cholic Acids / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism* / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D002793:Cholic Acids; D003574:Cytochrome c Group; D003902:Detergents; D010743:Phospholipids; D003576:Electron Transport Complex IV