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.
Yamada
M
M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Kuliopulos
A
A
Nelson
N P
NP
Roth
D A
DA
Furie
B
B
Furie
B C
BC
Walsh
C T
CT
D000345:Affinity Labels; D000595:Amino Acid Sequence; D019735:Carbon-Carbon Ligases; D005164:Factor IX / Q000378:metabolism*; D008025:Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives; D010777:Photochemistry; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D014812:Vitamin K / Q000378:metabolism
D000345:Affinity Labels; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; D014812:Vitamin K; D010649:Phenylalanine; D005164:Factor IX; C053764:benzoylphenylalanine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase