Glutamyl substrate-induced exposure of a free cysteine residue in the vitamin K-dependent gamma-glutamyl carboxylase is critical for vitamin K epoxidation

The vitamin K-dependent carboxylase catalyzes the posttranslational modific ation of glutamic acid to gamma-carboxyglutamic acid in the vitamin K-depen dent proteins of blood and bone. The vitamin K-dependent carboxylase also c atalyzes the epoxidation of vitamin K hydroquinone, an obligatory step in g amma-carboxylation. Using recombinant vitamin K-dependent carboxylase, puri fied in the absence of propeptide and glutamic acid-containing substrate us ing a FLAG epitope tag, the role of free cysteine residues in these reactio ns was examined. Incubation of the vitamin K-dependent carboxylase with the sulfhydryl-reactive reagent N-ethylmaleimide inhibited both the carboxylas e and epoxidase activities of the enzyme. This inhibition was proportional to the incorporation of radiolabeled N-ethylmaleimide. Stoichiometric analy ses using [H-3]-N-ethylmaleimide indicated that the vitamin K-dependent car boxylase contains two or three free cysteine residues. Incubation with prop eptide, glutamic acid-containing substrate, and vitamin K hydroquinone, alo ne or in combination, indicated that the binding of a glutamic acid-contain ing substrate to the carboxylase makes accessible a free cysteine residue t hat is important for interaction with vitamin K hydroquinone. This is consi stent with our previous observation that binding of a glutamic acid-contain ing substrate activates vitamin K epoxidation and supports the hypothesis t hat binding of the carboxylatable substrate to the enzyme results in a conf ormational change which renders the enzyme catalytically competent.