Work

Localization of the affinity peptide-substrate inactivator site on recombinant vitamin K-dependent carboxylase.

Year: 1994

Type: article

Abstract: A recombinant His,-tagged vitamin K-dependent y-glutamyl carboxylase has been produced in baculovirus-infected insect cells.The His,-carboxylase shares nearly identical kinetic properties with the wild-type enzyme from bovine liver microsomes.The His,-carboxylase was irreversibly inactivated by the N-bro-moa~etyl-FLEEL-'~~1-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 His,-carboxylase-Ac-FLEEL-"'I-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 '2SI-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 y-carboxylation of glutamate-peptide substrates.The vitamin K-dependent y-glutamyl carboxylase is a membrane-associated microsomal enzyme responsible for the essential post-translational modification of glutamate residues to y-carboxyglutamate (Gla)' residues of the vitamin K-dependent HL08882-01 (to A. K.) and National Institutes of Health Physician Sci-*

Source: Journal of Biological Chemistry

DOI: 10.1016/s0021-9258(17)31971-3