Characterization of the purified vitamin K-dependent gamma-glutamyl carboxylase.
Vitamin K-dependent carboxylase, purified from bovine liver, has properties similar to those reported for the carboxylase activity present in crude, solubilized microsomes. The purified carboxylase was found to possess the vitamin K epoxidase activity, believed to be essential for vitamin K-dependent carboxylation, but did not contain vitamin K epoxide reductase activity. Kinetic studies of the carboxylase done under defined conditions were complicated by the non-Michaelis-Menten kinetic behavior observed for reactions with two of the enzymes substrates, FLEEL and vitamin K1 hydroquinone. Initial rate experiments with the substrate FLEEL demonstrated behavior consistent with substrate inhibition and gave half-maximal activity at 1 mM FLEEL. Experiments with the substrate vitamin K1 hydroquinone also displayed non-Michaelis-Menten kinetics, as maximal activity was reached prematurely in relation to behavior at lower concentrations. Half-maximal activity was observed at 35 microM vitamin K1 hydroquinone. Initial rate experiments with varying NaH14CO3 concentration displayed Michaelis-Menten kinetics and gave a Km(app) of 0.29 mM. At cosubstrate concentrations chosen to obtain near-maximal activity, initial rate studies with varying NaH14CO3 concentration indicated a kcat near 1.0 s-1. Removal of the fourth substrate, oxygen, resulted in the loss of more than 99% of carboxylase activity. The sulfhydryl reagent N-ethylmaleimide inhibited carboxylase irreversibly, as did the anticoagulant warfarin.
Morris
D P
DP
Department of Biology, University of North Carolina, Chapel Hill 27599-3280.
Soute
B A
BA
Vermeer
C
C
Stafford
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D019735:Carbon-Carbon Ligases; D002417:Cattle; D004789:Enzyme Activation; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D014812:Vitamin K / Q000378:metabolism*; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000494:pharmacology
D014812:Vitamin K; D014859:Warfarin; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase