Vitamin K-dependent carboxylation. In vitro modification of synthetic peptides containing the gamma-carboxylation recognition site.
Synthetic peptides including the gamma-carboxylation recognition site and acidic amino acids were compared as substrates for vitamin K-dependent gamma-carboxylation by bovine liver carboxylase. The 28-residue proPT28 (proprothrombin -18 to +10) and proFIX28 (pro-Factor IX -18 to +10) were carboxylated with a Km of 3 microM. The Vmax of proPT28 was 2-3 times greater than that of proFIX28. An analog of proFIX28 that contained the prothrombin propeptide had a Vmax 2-3-fold greater than an analog of proPT28 that contained the Factor IX propeptide. proFIX28/RS-1, based upon Factor IX Cambridge, proFIX28/RQ-4, based upon Factor IX Oxford 3, and proFIX28 had equivalent Km and Vmax values. Analogs of proPT28 containing Ala6-Glu7 or Glu6-Ala7 were carboxylated at equivalent rates. A peptide containing Asp6-Asp7 was carboxylated at a rate of about 1% of that of Glu carboxylation. Carboxylation of peptides containing Asp6-Glu7 and Glu6-Asp7 yielded results identical with peptides containing Ala6-Glu7 and Glu6-Ala7. Carboxymethylcysteine was not carboxylated when substituted for Glu6 in a peptide containing Asp7. These results indicate that the prothrombin propeptide is more efficient in the carboxylation process than is the Factor IX propeptide, but that both propeptides direct carboxylation; the gamma-carboxylation recognition site does not include residues -4 and -1; aspartic acid and carboxymethylcysteine are poor substrates for the carboxylase, but aspartic acid does not inhibit the carboxylation of adjacent glutamic acids.
Hubbard
B R
BR
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, Massachusetts.
Jacobs
M
M
Ulrich
M M
MM
Walsh
C
C
Furie
B
B
Furie
B C
BC
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002233:Carbocysteine / Q000378:metabolism; D019735:Carbon-Carbon Ligases; D002417:Cattle; D005164:Factor IX / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D008025:Ligases / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism; D013379:Substrate Specificity; D014812:Vitamin K
D005971:Glutamates; D010446:Peptide Fragments; D011498:Protein Precursors; D014812:Vitamin K; D001224:Aspartic Acid; D018698:Glutamic Acid; D002233:Carbocysteine; D011516:Prothrombin; D005164:Factor IX; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase