Jones J P, Gardner E J, Cooper T G, Olson R E
J Biol Chem. 1977 Nov 10;252(21):7738-42.
Vitamin K participates in the post-translational carboxylation of peptide-bound glutamate to form the gamma-carboxy-glutamate residues of prothrombin. The reaction requires reduced vitamin K, bicarbonate, oxygen, and a membrane-bound carboxylase. The active species of "CO2," i.e. CO2 or HCO3-, utilized in this carboxylation was determined by the low temperature method of Filmer and Cooper ((1970) J. Theor. Biol. 29, 131-145), taking advantage of the fact that menaquinone-2, in contrast to phylloquinone, is very active at 10 degrees. Microsomes from livers of vitamin K-deficient rats, were incubated in the presence of cycloheximide, avidin, NADH, menaquinone-2, 1 mM acetazolamide (to inhibit carbonic anhydrase), and either 14CO2 or H14CO3-. At 1-min intervals aliquots were removed from the reaction mixture. gamma-Carboxyglutamate was isolated from these samples by ion exchange chromatography after alkaline hydrolysis. After 1 min the incorporation of 14CO2 into gamma-carboxyglutamate was 8 to 10 times as great as that found with H14CO3-. When the carbonic anhydrase inhibitor was omited (with or without addition of exogenous carbonic anhydrase) the two incorporation curves approximated each other at a rate near that exhibited by bicarbonate alone. Similar results were obtained in a microsomal carboxylase system solubilized with Triton X-100. It is concluded that CO2 is the active species of "CO2" initially participating in the vitamin K-dependent carboxylation of preprothrombin and that neither ATP nor biotin is required for the reaction.
维生素K参与肽结合型谷氨酸的翻译后羧化反应，以形成凝血酶原的γ-羧基谷氨酸残基。该反应需要还原型维生素K、碳酸氢盐、氧气和一种膜结合羧化酶。利用菲尔默和库珀的低温方法（(1970) J. Theor. Biol. 29, 131 - 145），基于与叶绿醌不同，甲萘醌-2在10℃时非常活跃这一事实，确定了该羧化反应中所利用的“CO₂”的活性形式，即CO₂或HCO₃⁻。将维生素K缺乏大鼠肝脏的微粒体在环己酰亚胺、抗生物素蛋白、NADH、甲萘醌-2、1 mM乙酰唑胺（抑制碳酸酐酶）以及¹⁴CO₂或H¹⁴CO₃⁻存在的情况下进行孵育。每隔1分钟从反应混合物中取出等分试样。碱性水解后，通过离子交换色谱法从这些样品中分离出γ-羧基谷氨酸。1分钟后，¹⁴CO₂掺入γ-羧基谷氨酸的量是H¹⁴CO₃⁻的8至10倍。当省略碳酸酐酶抑制剂时（无论是否添加外源碳酸酐酶），两条掺入曲线以接近单独碳酸氢盐所表现出的速率相互接近。在用 Triton X - 100增溶的微粒体羧化酶系统中也获得了类似结果。结论是，CO₂是最初参与凝血酶原前体维生素K依赖性羧化反应的“CO₂”的活性形式，并且该反应既不需要ATP也不需要生物素。
