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 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 KH(2). Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K(i) for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V(max)/K(m) toward 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 K(m) for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K(m) for vitamin KH(2). The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH(2) 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.
Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.
V P
VP
Mutucumarana
D W
DW
Stafford
T B
TB
Stanley
D Y
DY
Jin
J
J
Solera
B
B
Brenner
R
R
Azerad
S M
SM
Wu
D019943:Amino Acid Substitution; D001665:Binding Sites; D001778:Blood Coagulation Disorders / Q000201:enzymology / Q000235:genetics; D019735:Carbon-Carbon Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014812:Vitamin K / Q000378:metabolism
D004791:Enzyme Inhibitors; D009842:Oligopeptides; D011994:Recombinant Proteins; D014812:Vitamin K; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase