Processive carboxylation of FIX is impaired in the V255M mutant. (A) A complex between the carboxylase and FIX was reacted in the presence of a challenge FIX (FIX_−18/+41), and carboxylation of both FIX forms was monitored. (B-E) Independent gels were used to monitor FIX_−18/+41 and fIX in the wild-type (WT) or V255M complexes because of differences in their size, and [¹⁴C]-standards were included on the gels to allow quantitation of carboxylation products. (F-G) FIX_−18/+41 was carboxylated after FIX in the complex with WT carboxylase but occurred simultaneously with FIX in the complex with V255M carboxylase. FIX carboxylation was much lower in the complex with the V255M mutant than with WT carboxylase. It was also lower than observed in the reaction monitoring catalysis (Figure 2E), which may have been due to the lower KH₂ concentrations used in the processivity assay. (H) S300F carboxylation of both FIX in the complex and FIX_−18/+41 was poor.