The Inhibition of the Vitamin K Dependent Carboxylation by Free Radical Scavengers

Prothrombin contains γ-carboxyglutamic acid residues, synthesised at a post-translational step requiring vitamin K quinol, carbon dioxide, molecular oxygen, and a carboxylating system solubilised from liver microsomal fractions of rats (normal and vit.K deficient) and calves by non-ïonic detergents. The reaction is assayed by the incorporation of ¹⁴C into prothrombin precursor or the pentapeptide, Phe-Leu-Glu-Glu-Val. Superoxide dis-mutase reduces the incorporation (50% inhibition at 300μM) and so too do copper chelates with known dismutase activity: 50% inhibition at 60μM copper (II) aspirinate, 127μM copper (II) penicillamine and 152μM copper (II) tyrosine. The superoxide anion is postulated as a primary product of reduced Vitamin K and dioxygen. Catalase also inhibits at high concentrations (> .5mM), possibly by shifting the equilibrium to favour dismutation or b; preventing production of an ‘active carbon” species via hydrogen peroxide. So far it has not been possible to replace Vitamin K with active intermediates, such as t-butyl hydroperoxide, which also inhibits the action of the vitamin, possibly due to the formation of Vitamin K 2,3-epoxide. These results point to the involvement of radical species in the Vitamin K dependent carboxylation of prothrombin.