Mechanism of action of t-butyl hydroperoxide in the inhibition of vitamin K-dependent carboxylation

Abstract

t-Butyl hydroperoxide has been studied as a possible competitive inhibitor of the vitamin K-dependent carboxylation of the pentapeptide PheLeuGluGluIle. Under standard carboxylating conditions the concentrations of reduced phylloquinone and phylloquinone were followed by high-pressure liquid chromatography during 30-min incubations of Triton-solubilized microsomes from rat liver. Under these conditions supporting linear rates of carbon dioxide fixation for 20–30 min, the vitamin KH 2 concentration decreased exponentially to less than 5% of its initial value in 30 min principally due to autooxidation. In the presence of 10 m m t-butyl-OOH, however, the oxidation of vitamin KH 2 was greatly accelerated with none being detected after 7 min. In general, the rate of carboxylation of peptide paralleled the KH 2 concentration. After cessation of carboxylation in the presence of t-butyl-OOH the readdition of KH 2 stimulated additional 14CO 2 fixation. A known competitive inhibitor of vitamin K, 2-chlorophylloquinone, did not accelerate the oxidation of KH 2 but nonetheless inhibited the vitamin K-dependent carboxylation in a competitive manner. These data have led us to conclude that t-butyl-OOH is not a competitive inhibitor of the vitamin K-dependent carboxylase at the active site of the enzyme but merely acts to promote the oxidation of KH 2.