Roth D A, Whirl M L, Velazquez-Estades L J, Walsh C T, Furie B, Furie B C
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, Massachusetts.
J Biol Chem. 1995 Mar 10;270(10):5305-11. doi: 10.1074/jbc.270.10.5305.
The gamma-glutamyl carboxylase and vitamin K epoxidase activities of a series of mutants of bovine vitamin K-dependent carboxylase with progressively larger COOH-terminal deletions have been analyzed. The recombinant wild-type (residues 1-758) and mutant protein carboxylases, Cbx 711, Cbx 676, and Cbx 572, representing residues 1-711, 1-676, and 1-572, respectively, were expressed in baculovirus-infected Sf9 cells. Wild-type carboxylase had a Km for the substrate Phe-Leu-Glu-Glu-Leu (FLEEL) of 0.87 mM; the carboxylation of FLEEL was stimulated 2.5-fold by proPT18, the propeptide of prothrombin. Its Km for vitamin K hydroquinone was 23 microM and the specific epoxidase activity of the carboxylase was 938 pmol vitamin KO/30 min/pmol of carboxylase. Cbx 711, which was also stimulated by proPT18, had a Km for FLEEL, a Km for vitamin K hydroquinone, and a specific epoxidase activity that was comparable to the wild-type carboxylase. In contrast Cbx 572 lacked both carboxylase and epoxidase activities. Although Cbx 676 had a normal carboxylase active site in terms of the Km for FLEEL and its stimulation by proPT18, the Km for vitamin K hydroquinone was 540 microM, and the specific epoxidase activity was 97 pmol KO/30 min/pmol of Cbx 676. The catalytic efficiencies of Cbx 676 for glutamate carboxylation and vitamin K epoxidation were decreased 15- and 400-fold, respectively, from wild-type enzyme reflecting the requirement for formation of an activated vitamin K species for carboxylation to occur. These data indicate that the truncation of COOH-terminal segments of the carboxylase had no effect on FLEEL or propeptide recognition, but in the case of Cbx 676, selectively affected the interaction with vitamin K hydroquinone and the generation of epoxidase activity. These data suggest that a vitamin K epoxidase activity domain may reside near the COOH terminus while the carboxylase active site domain resides toward the NH2 terminus.
对一系列羧基末端缺失逐渐增大的牛维生素K依赖性羧化酶突变体的γ-谷氨酰羧化酶和维生素K环氧化酶活性进行了分析。重组野生型（第1至758位氨基酸残基）以及分别代表第1至711、1至676和1至572位氨基酸残基的突变体蛋白羧化酶Cbx 711、Cbx 676和Cbx 572在杆状病毒感染的Sf9细胞中表达。野生型羧化酶对底物苯丙氨酸-亮氨酸-谷氨酸-谷氨酸-亮氨酸（FLEEL）的Km值为0.87 mM；凝血酶原前肽proPT18可使FLEEL的羧化反应增强2.5倍。其对维生素K氢醌的Km值为23 μM，羧化酶的特异性环氧化酶活性为938 pmol维生素K0/30分钟/pmol羧化酶。同样受proPT18刺激的Cbx 711对FLEEL的Km值、对维生素K氢醌的Km值以及特异性环氧化酶活性与野生型羧化酶相当。相比之下，Cbx 572既缺乏羧化酶活性也缺乏环氧化酶活性。尽管就FLEEL的Km值及其受proPT18的刺激而言，Cbx 676具有正常的羧化酶活性位点，但其对维生素K氢醌的Km值为540 μM，特异性环氧化酶活性为97 pmol K0/30分钟/pmol Cbx 676。与野生型酶相比，Cbx 676对谷氨酸羧化和维生素K环氧化的催化效率分别降低了15倍和400倍，这反映了羧化反应发生需要形成活化的维生素K物种。这些数据表明，羧化酶羧基末端片段的截短对FLEEL或前肽识别没有影响，但在Cbx 676的情况下，选择性地影响了与维生素K氢醌的相互作用以及环氧化酶活性的产生。这些数据表明，维生素K环氧化酶活性结构域可能位于羧基末端附近，而羧化酶活性位点结构域位于氨基末端附近。
