Expression and characterization of the naturally occurring mutation L394R in human gamma-glutamyl carboxylase

Patients with mutation L394R in gamma -glutamyl carboxylase have a severe b leeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially correc ts this deficiency. To characterize L394R, we purified recombinant mutant L 394R and wild-type carboxylase expressed in baculovirus-infected insect cel ls. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor M's propeptide and vitamin KM2. Mutant L394R diff ers from its wild-type counterpart as follows: 1) 110-fold higher K-i for B oc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fol d lower V-max/K-m toward the substrate FLEEL in the presence of the propept ide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-f old higher K-m for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) B-fold higher K-m, for vitamin KH2. The primary defect in mutant L394R appears to be in its glutamate-binding s ite. To a lesser degree, the propeptide and KH2 binding properties are alte red in the L394R mutant. Compared with its wildtype counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propepti de.