Vitamin K-dependent carboxylation of peptide-bound glutamate. The active species of "CO2" utilized by the membrane-bound preprothrombin carboxylase.
Vitamin K participates in the post-translational carboxylation of peptide-bound glutamate to form the gamma-carboxy-glutamate residues of prothrombin. The reaction requires reduced vitamin K, bicarbonate, oxygen, and a membrane-bound carboxylase. The active species of "CO2," i.e. CO2 or HCO3-, utilized in this carboxylation was determined by the low temperature method of Filmer and Cooper ((1970) J. Theor. Biol. 29, 131-145), taking advantage of the fact that menaquinone-2, in contrast to phylloquinone, is very active at 10 degrees. Microsomes from livers of vitamin K-deficient rats, were incubated in the presence of cycloheximide, avidin, NADH, menaquinone-2, 1 mM acetazolamide (to inhibit carbonic anhydrase), and either 14CO2 or H14CO3-. At 1-min intervals aliquots were removed from the reaction mixture. gamma-Carboxyglutamate was isolated from these samples by ion exchange chromatography after alkaline hydrolysis. After 1 min the incorporation of 14CO2 into gamma-carboxyglutamate was 8 to 10 times as great as that found with H14CO3-. When the carbonic anhydrase inhibitor was omited (with or without addition of exogenous carbonic anhydrase) the two incorporation curves approximated each other at a rate near that exhibited by bicarbonate alone. Similar results were obtained in a microsomal carboxylase system solubilized with Triton X-100. It is concluded that CO2 is the active species of "CO2" initially participating in the vitamin K-dependent carboxylation of preprothrombin and that neither ATP nor biotin is required for the reaction.
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000378:metabolism*; D005971:Glutamates; D007700:Kinetics; D008297:Male; D008566:Membranes / Q000201:enzymology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D011498:Protein Precursors; D011516:Prothrombin; D051381:Rats; D014812:Vitamin K / Q000494:pharmacology*; D014813:Vitamin K Deficiency / Q000201:enzymology*
D001639:Bicarbonates; D005971:Glutamates; D011498:Protein Precursors; D014812:Vitamin K; D002245:Carbon Dioxide; D011516:Prothrombin; D002262:Carboxy-Lyases; C022436:preprothrombin carboxylase