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The gamma-glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout the multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M, S300F) whose combined heterozygosity causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation: complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX (fIX)) or calcification (Matrix Gla Protein (MGP)) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of VKD protein in the complex. The VKD protein in the complex with wild type carboxylase was carboxylated before challenge protein carboxylation occurred, and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time, and did not completely carboxylate fIX in the complex. S300F carboxylation was poor with both fIX and MGP. Additional studies analyzed fIX and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The V255M mutant generated more carboxylated peptide than wild type GGCX, however the peptides were partially carboxylated. Analysis of the V255M mutant in fIX-HEK293 cells lacking endogenous GGCX revealed poor fIX clotting activity. The studies show that disrupted processivity causes disease, and explain the defect in the patient. The kinetic analyses also suggest that disrupted processivity may occur in wild type carboxylase under some conditions, e.g. warfarin therapy or vitamin K deficiency.