Localization of the affinity peptide-substrate inactivator site on recombinant vitamin K-dependent carboxylase.
A recombinant His6-tagged vitamin K-dependent gamma-glutamyl carboxylase has been produced in baculovirus-infected insect cells. The His6-carboxylase shares nearly identical kinetic properties with the wild-type enzyme from bovine liver microsomes. The His6-carboxylase was irreversibly inactivated by the N-bromoacetyl-FLEEL-125I-Y peptide substrate/affinity label under pseudo-first order conditions. This inactivation could be abolished by coincubation with a high affinity peptide substrate consistent with an active site-directed inactivation. The inactivated His6-carboxylase-Ac-FLEEL-125I-Y, purified under denaturing conditions by Ni-chelation chromatography followed by preparative polyacrylamide gel electrophoresis, was subjected to proteolytic digestions with either Glu-C or Lys-C endoproteinases. The resulting polypeptide fragments were probed with three regiospecific antibodies which recognized epitopes present at the extreme N terminus (residues -23 to -13), at the hydrophobic N-terminal region (residues 86-99), and at the hydrophilic C-terminal region (residues 661-673). The site of attachment to the 125I-affinity label is located within the first 218 amino acid residues of the 758-residue carboxylase. This is the first evidence for the involvement of either the putative membrane-anchoring hydrophobic region (residues 50-314) or possibly the N-terminal hydrophilic region (residues 1-50) in gamma-carboxylation of glutamate-peptide substrates.
Kuliopulos
A
A
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Nelson
N P
NP
Yamada
M
M
Walsh
C T
CT
Furie
B
B
Furie
B C
BC
Roth
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D019735:Carbon-Carbon Ligases; D002417:Cattle; D002478:Cells, Cultured; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase