Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state.
The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational conversion of glutamic acid to gamma-carboxyglutamic acid in precursor proteins containing the gamma-carboxylation recognition site (gamma-CRS). During this reaction, glutamic acid is converted to gamma-carboxyglutamic acid while vitamin KH2 is converted to vitamin K 2,3-epoxide. Recombinant bovine carboxylase was purified free of gamma-CRS-containing propeptide and endogenous substrate in a single-step immunoaffinity procedure. We show that in the absence of gamma-CRS-containing propeptide and/or glutamate-containing substrate, carboxylase has little or no epoxidase activity. Epoxidase activity is induced by Phe-Leu-Glu-Glu-Leu (FLEEL) (9.2 pmol per min per pmol of enzyme), propeptide, residues -18 to -1 of proFactor IX (3.4 pmol per min per pmol of enzyme), FLEEL and propeptide (100 pmol per min per pmol of enzyme), and proPT28 (HVFLAPQQARSLLQRVRRANTFLEEVRK, residues -18 to +10 of human acarboxy-proprothrombin), (5.3 pmol per min per pmol of enzyme). These results indicate that in the absence of propeptide or glutamate-containing substrate, oxygenation of vitamin K by the carboxylase does not occur. Upon addition of propeptide or glutamate-containing substrate, the enzyme is converted to an active epoxidase. This regulatory mechanism prevents the generation of a highly reactive vitamin K intermediate in the absence of a substrate for carboxylation.
Sugiura
I
I
Center for Hemostasis and Thrombosis Research, Tufts University School of Medicine, Boston, MA 02111, USA.
Furie
B
B
Walsh
C T
CT
Furie
B C
BC
D015055:1-Carboxyglutamic Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019735:Carbon-Carbon Ligases; D002417:Cattle; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D013379:Substrate Specificity
D010455:Peptides; D018698:Glutamic Acid; D015055:1-Carboxyglutamic Acid; D010088:Oxidoreductases; C023111:epoxidase; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase