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.