The propeptide binding site of the bovine gamma-glutamyl carboxylase.
gamma-Glutamyl carboxylase is an integral membrane protein required for the posttranslational modification of vitamin K-dependent proteins. The main recognition between the enzyme and its substrates is through an 18-amino acid propeptide. It has been reported that this binding site resides in the amino-terminal third of the gamma-glutamyl carboxylase molecule (Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., Furie, B., Furie, B. C., and Walsh, C. T. (1995) Biochemistry 34, 481-489). In contrast, we found the binding site in the carboxyl half of the gamma-glutamyl carboxylase. We show that the carboxylase may be cleaved by trypsin into an amino-terminal 30-kDa and a carboxyl-terminal 60-kDa fragment joined by a disulfide bond(s), and the propeptide binds to the 60-kDa fragment. The sequence of the amino terminus of the 60-kDa fragment reveals that the primary trypsin-sensitive sites are at residues 349 and 351. Furthermore, the tryptic fragment that cross-links to the propeptide also reacts with an antibody specific to the carboxyl portion of the gamma-glutamyl carboxylase. In addition, cyanogen bromide cleavage of bovine gamma-glutamyl carboxylase cross-linked to the peptide comprising residues TVFLDHENANKILNRPKRY of human factor IX yields a cross-linked fragment of 16 kDa from the carboxyl half of the molecule, the amino-terminal sequence of which begins at residue 438. Thus, the propeptide binding site lies carboxyl-terminal to residue 438 and is predicted to be in the lumen of the endoplasmic reticulum.
Wu
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SM
Biology Department, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.
Mutucumarana
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VP
Geromanos
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S
Stafford
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019735:Carbon-Carbon Ligases; D002417:Cattle; D003432:Cross-Linking Reagents; D006801:Humans; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D013388:Succinimides; D014812:Vitamin K / Q000378:metabolism
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011498:Protein Precursors; D013388:Succinimides; D014812:Vitamin K; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase; C019358:disuccinimidyl suberate