THE PROPEPTIDE BINDING-SITE OF THE BOVINE GAMMA-GLUTAMYL CARBOXYLASE

gamma-Glutamyl carboxylase is an integral membrane protein required fo r 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 si te resides in the amino terminal third of the gamma-glutamyl carboxyla se molecule (Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., F urie, B., Furie, B. C., and Walsh, C. T. (1995) Biochemistry 34, 481-4 89), In contrast, we found the binding site in the carboxyl half of th e gamma-glutamyl carboxylase, We show that the carboxylase may be clea ved by trypsin into an amino-terminal 30-kDa and a carboxyl-terminal 6 0-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 r esidues 349 and 351. Furthermore, the tryptic fragment that cross-link s to the propeptide also reacts with an antibody specific to the carbo xyl portion of the gamma-glutamyl carboxylase. In addition, cyanogen b romide cleavage of bovine gamma-glutamyl carboxylase cross-linked to t he 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 4 38 and is predicted to be in the lumen of the endoplasmic reticulum.