The active site region of the vitamin K-dependent carboxylase includes both the amino-terminal hydrophobic and carboxy-terminal hydrophilic domains of the protein

In order to localize the active site of the vitamin K-dependent carboxylase, we developed an affinity probe containing the propeptide and the first two carboxylatable glutamate residues conserved in many native substrates. This probe crosslinked to both the hydrophobic amino-terminal and hydrophilic carboxy-terminal domains of the carboxylase, in contrast,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 fragment is seriously underestimated by SDS-PAGE. Reanalysis of the published data in light of this information suggests that a portion of the propeptide binding site resides within the carboxy-terminal hydrophilic domain. (C) 1997 Federation of European Biochemical Societies.