Tb. Stanley et al., Amino acids responsible for reduced affinities of vitamin K-dependent propeptides for the carboxylase, BIOCHEM, 38(47), 1999, pp. 15681-15687
The binding of the gamma-glutamyl carboxylase to its protein substrates is
mediated by a conserved 18 amino acid propeptide sequence found in all vita
min K-dependent proteins. We recently found that the apparent affinities of
the naturally occurring propeptides for the carboxylase vary over a 100-fo
ld 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 (K-i = 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, allo
wed 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. T
he reduced binding affinity of these propeptides relative to that of FIX is
due to residues -15 in protein C, -10 and -6 in bone Gla protein, and -9 i
n prothrombin. A role for the -9 position was not previously recognized but
is further shown by our identification of a new, naturally occurring mutat
ion at this position in factor IX which causes a warfarin-sensitive hemophi
lia B phenotype. In addition, we find that propeptides with mutations found
in warfarin-sensitive patients have reduced affinity for the carboxylase,
suggesting a physiological relevance of propeptide binding affinity.