MECHANISM OF RAT-LIVER MICROSOMAL STEARYL-COA DESATURASE - STUDIES OF SUBSTRATE-SPECIFICITY, ENZYME-SUBSTRATE INTERACTIONS, AND FUNCTION OF LIPID

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  • 1 January 1976
    • journal article
    • research article
    • Vol. 251  (16) , 5095-5103
Abstract
The 3 purified proteins which are required for microsomal stearyl-CoA desaturation, NADH-cytochrome b5 reductase [EC 1.6.2.2], cytochrome b5 and desaturase, were combined with egg lecithin or dimyristyl lecithin vesicles to reconstruct a functional electron transport system capable of utilizing NADH and O2 in the desaturation of stearyl-CoA. Such preparations appear to consist of phospholipid vesicles which contain the 3 proteins bound to the outer surface of the vesicles. Acyl-CoA derivatives containing 12-19 C fatty acyl chains are required for desaturase activity, while derivatives containing 9-20 C are capable of binding to the enzyme. Shorter chain acyl-CoA derivatives, free CoA and free fatty acids do not appear to bind to the enzyme. Inhibition and analog studies suggest that the methylene chain of stearyl-CoA assumes an eclipsed (gauche) conformation at C atoms 9,10 in the enzyme.cntdot.substrate complex. Isotope rate effects obtained with 2H stearyl-CoA derivatives indicate that H removal is the rate-limiting step of desaturation. Stearyl-CoA binds to pure liposomes and desaturase-containing liposomes, and it is this form of stearyl-CoA which appears to be the substrate for desaturase. The Arrhenius plots of desaturase activity obtained using desaturase bound to egg lecithin liposomes, in which the liquid crystalline to crystalline phase transition temperature is -5.degree., was linear between 15 and 35.degree., while that obtained using desaturase bound to dimyristyl lecithin liposomes showed a break at 24.degree. coinciding with the liquid crystalline to crystalline phase transition temperature for this lipid. The decrease observed in the 2H isotope rate effect below the transition temperature indicates that a step in the reaction sequence other than H abstraction becomes rate-limiting when the lipid is in the crystalline state. In this system translational diffusion does not emerge as the rate-limiting step. The liposomes contained sufficient reductase and cytochrome b5 so that translational diffusion was not rate-limiting.

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