THE ACTIVE-SITE REGION OF THE VITAMIN-K-DEPENDENT CARBOXYLASE INCLUDES BOTH THE AMINO-TERMINAL HYDROPHOBIC AND CARBOXY-TERMINAL HYDROPHILICDOMAINS OF THE PROTEIN

In order to localize the active site of the vitamin K-dependent carbox ylase, we developed an affinity probe containing the propeptide and th e first two carboxylatable glutamate residues conserved in many native substrates. This probe crosslinked to both the hydrophobic amino-term inal and hydrophilic carboxy-terminal domains of the carboxylase, in c ontrast,vith previous work which localized both the catalytic and the propeptide binding site within the amino-terminal hydrophobic domain, Amino acid analysis revealed that the mass of an amino-terminal fragme nt is seriously underestimated by SDS-PAGE. Reanalysis of the publishe d data in light of this information suggests that a portion of the pro peptide binding site resides within the carboxy-terminal hydrophilic d omain. (C) 1997 Federation of European Biochemical Societies.