Localization of the factor IX propeptide binding site on recombinant vitamin K dependent carboxylase using benzoylphenylalanine photoaffinity peptide inactivators.

The propeptide binding/activation site on the vitamin K dependent carboxylase has been localized to a region of carboxylase between residues Arg +50 and Glu +225 by photoinactivation studies using [125I]tyrosyl-labeled benzoylphenylalanine (Bpa)-containing analogs of proFIX19, a peptide containing residues -18 to +1 of factor IX. Four proFIX19 analogs with Bpa substituents at -16, -13, -7, and -6 were synthesized. These peptides were specific photoinactivators of carboxylase and were used to label a His6-carboxylase construct produced in baculovirus-infected insect cells. Fragments of the labeled carboxylase produced by V8 protease digestion were analyzed by peptide-specific antibodies and by autoradiography. The propeptide recognition site was localized to the N-terminal one-third of the 94 kDa carboxylase. This is consistent with previous studies using a carboxylase substrate affinity label, N-(bromoacetyl)-FLEELY [Kuliopulos, A., Nelson, N.P., Yamada, M., Walsh, C.T., Furie, B., Furie, B.C., & Roth, D.A. (1994) J. Biol. Chem. 269, 21364-21370], indicating that the propeptide binding site and the FLEEL binding site are both located within the N-terminal one-third of the vitamin K dependent carboxylase.