Vitamin K-dependent oxygenase/carboxylase; Differential inactivation by sulfhydryl reagents

Inhibition of vitamin K-dependent carboxylase and oxygenase by sulfhydryl reagents was compared. Formation of vitamin K epoxide and vitamin K-dependent carboxylation are both strongly (>90%) inhibited by I mM p-hydroxymercuribenzoate, and this inhibition is reversed by dithiothrietol. Both activities are also effectively inhibited by N-ethylmaleimide (NEM). Preincubation with vitamin K hydroquinone prevents NEM inhibition of epoxide formation but not of carboxylation. These data argue that separate active sites are required to support vitamin K-dependent epoxide formation and carboxylation and that the binding site for vitamin K oxygenase contains an active thiol group.