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 modification of glutamic acid to gamma-carboxyglutamic acid in the vitamin K-dependent proteins of blood and bone. The vitamin K-dependent carboxylase also catalyzes the epoxidation of vitamin K hydroquinone, an obligatory step in gamma-carboxylation. Using recombinant vitamin K-dependent carboxylase, purified in the absence of propeptide and glutamic acid-containing substrate using a FLAG epitope tag, the role of free cysteine residues in these reactions was examined. Incubation of the vitamin K-dependent carboxylase with the sulfhydryl-reactive reagent N-ethylmaleimide inhibited both the carboxylase and epoxidase activities of the enzyme. This inhibition was proportional to the incorporation of radiolabeled N-ethylmaleimide. Stoichiometric analyses using [(3)H]-N-ethylmaleimide indicated that the vitamin K-dependent carboxylase contains two or three free cysteine residues. Incubation with propeptide, glutamic acid-containing substrate, and vitamin K hydroquinone, alone or in combination, indicated that the binding of a glutamic acid-containing substrate to the carboxylase makes accessible a free cysteine residue that is important for interaction with vitamin K hydroquinone. This is consistent with our previous observation that binding of a glutamic acid-containing substrate activates vitamin K epoxidation and supports the hypothesis that binding of the carboxylatable substrate to the enzyme results in a conformational change which renders the enzyme catalytically competent.
Bouchard
B A
BA
Center for Hemostasis & Thrombosis Research, Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
Furie
B
B
Furie
B C
BC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019735:Carbon-Carbon Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004789:Enzyme Activation; D004852:Epoxy Compounds / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005033:Ethylmaleimide / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014812:Vitamin K / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004852:Epoxy Compounds; D009842:Oligopeptides; D010455:Peptides; D011498:Protein Precursors; D013439:Sulfhydryl Reagents; D014812:Vitamin K; D018698:Glutamic Acid; C085706:FLAG peptide; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase; D003545:Cysteine; D005033:Ethylmaleimide