Amino acids responsible for reduced affinities of vitamin K-dependent propeptides for the carboxylase.

The binding of the γ-glutamyl carboxylase to its protein substrates is mediated by a conserved 18 amino acid propeptide sequence found in all vitamin K-dependent proteins. We recently found that the apparent affinities of the naturally occurring propeptides for the carboxylase vary over a 100-fold range and that the propeptide of bone Gla protein has severely impaired affinity for the carboxylase [Stanley, T. B., et al. (1999) J. Biol. Chem. 274, 16940−16944 (1)]. Here we report a consensus propeptide sequence that binds tighter (Ki = 0.43 nM) to the carboxylase than any known propeptide sequence. Comparing the factor IX propeptide to the propeptides of protein C, bone Gla protein, and prothrombin, the weakest binding propeptides, allowed us to predict which residues might be responsible for these substrates' relatively weak binding to the carboxylase. We then made propeptides with the predicted amino acid changes and determined their binding affinities. The reduced binding affinity of these propeptides r...