Identification of the five hydrophilic residues (Lys-217, Lys-218, Arg-359, His-360, and Arg-513) essential for the structure and activity of vitamin K-dependent carboxylase.
Vitamin K-dependent carboxylase catalyzes the posttranslational conversion of glutamic acid to gamma-carboxyglutamic acid in vitamin K-dependent proteins. The clustered charged-to-alanine scanning mutagenesis of bovine carboxylase has identified five distinct candidate regions (I. Sugiura et al., J. Biol. Chem. 271, 17837-17844, 1996) with significant loss-of-function phenotype. To further specify the residues essential for the structure and function of the enzyme, Lys-217, Lys-218, Arg-359, His-360, Lys-361, Arg-513, and Lys-515 were analyzed by substituting to alanine individually. All the mutants except for K217A were expressed in Chinese hamster ovary cells. The carboxylase activities of R359A, H360A, and R513A decreased in parallel with the vitamin K epoxidase activities. Both carboxylations by R359A and H360A were stimulated saturatively at 1 microM factor IX propeptide (proFIX18) concentration, but that by R513A was not at a concentration up to 128 microM. K218A completely lost the enzyme activities but it cross-linked to the propeptide, suggesting that Lys-218 is critical for enzyme activity without affecting propeptide binding. We conclude that Lys-218, Arg-359, and His-360 are involved in the catalytic event, and Arg-513 participates in propeptide binding.
Shimizu
A
A
First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan.
Sugiura
I
I
Matsushita
T
T
Kojima
T
T
Hirai
M
M
Saito
H
H
D000409:Alanine / Q000235:genetics; D000601:Amino Acids, Essential / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000302:isolation & purification; D016466:CHO Cells; D019735:Carbon-Carbon Ligases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D004852:Epoxy Compounds / Q000378:metabolism; D005164:Factor IX / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000302:isolation & purification; D008239:Lysine / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013329:Structure-Activity Relationship; D014812:Vitamin K / Q000502:physiology*
D000601:Amino Acids, Essential; D003432:Cross-Linking Reagents; D004852:Epoxy Compounds; D011498:Protein Precursors; D011994:Recombinant Proteins; C049838:factor IX Cambridge; D014812:Vitamin K; D006639:Histidine; D005164:Factor IX; D001120:Arginine; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase; D008239:Lysine; D000409:Alanine