The inhibition of vitamin K-dependent carboxylase by cyanide

Abstract

The formation of 7-carboxyglutamic acid residues from glutamic acid residues is a vitamin K-dependent carboxylation reaction. The reaction has been demonstrated in various species and tissues [1,2], but the most extensive studies have been performed in the microsomal fraction of rat liver.It is generally assumed that the carboxylation reaction is coupled to the epoxidation of reduced vitamin K 13,41, but the reaction mechanism is still unclear. The purification of the carboxylating enzyme (carboxylase) from rat liver seems to be difficult and has not been reported until now. The possible invoh'ement of other cofactors, such as haem groups [5-7] is therefore still a matter of dispute. The main argument for the involvement of haem in the carboxylation reaction was the observation that cyanide inhibits rat carboxylase [5,8]. This inhibition has not been further analyzed, however, whereas other common haem ligands such as azide and carbon monoxide do not inhibit the vitamin K-dependent carboxylation I I ]. We have developed a carboxylatingenzyme system from the livers of warfarin-treated cows and obtained a 100-fold purification of the microsomal enzyme by immunospecific adsorption onto antibodies against the endogenous ubstrate [9]. This partly purified enzyme preparation is attached to Sepharose beads and is called solid-phase carboxylase (SP-carboxylase). The enzymatic activity of SP-carboxylase is strictly dependent on the presence of phospholipids and in the presence of an excess of exogenous ubstrate (Phe-ku-Glu-Glu-ku), the carboxylation rate was constant for at least 3 h at 25 C [10]. Here, we describe the results of some more detailed investiga-