Expression and Characterization of the Naturally Occurring Mutation L394R in Human γ-Glutamyl Carboxylase

Patients with mutation L394R in γ-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IX's propeptide and vitamin KH2. Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher Ki for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower Vmax/Kmtoward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher Km for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher Km for vitamin KH2. The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH2 binding properties are altered in the L394R mutant. Compared with its wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.