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 carb oxylase has been localized to a region of carboxylase between residues Arg +50 and Glu +225 by photoinactivation studies using [I-125]tyrosy l-labeled benzoylphenylalanine (Bpa)-containing analogs of proFIX19, a peptide containing residues -18 to +1 of factor IX. Four proFIX19 ana logs with Bpa substituents at -16, -13, -7, and -6 were synthesized. T hese peptides were specific photoinactivators of carboxylase and were used to label a His(6)-carboxylase construct produced in baculovirus-i nfected insect cells. Fragments of the labeled carboxylase produced by V8 protease digestion were analyzed by peptide-specific antibodies an d by autoradiography. The propeptide recognition site was localized to the N-terminal one-third of the 94 kDa carboxylase. This is consisten t with previous studies using a carboxylase substrate affinity label, N-(bromoacetyl)-FLEELY [Kuliopulos, A., Nelson, N. P., Yamada, M., Wal sh, C. T., Furie, B., Furie, B. C., and Roth, D. A. (1994) J. Biol. Ch em. 269, 21364-21370], indicating that the propeptide binding site and the FLEEL binding site are both located within the N-terminal one-thi rd of the vitamin K dependent carboxylase.