Morris D P, Soute B A, Vermeer C, Stafford D W
Department of Biology, University of North Carolina, Chapel Hill 27599-3280.
J Biol Chem. 1993 Apr 25;268(12):8735-42.
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.
从牛肝中纯化得到的维生素K依赖性羧化酶，其性质与粗制、可溶解微粒体中存在的羧化酶活性所报道的性质相似。发现纯化的羧化酶具有维生素K环氧化酶活性，据信这对维生素K依赖性羧化至关重要，但不含有维生素K环氧化物还原酶活性。在特定条件下对羧化酶进行的动力学研究因观察到与两种酶底物（FLEEL和维生素K1对苯二酚）反应呈现非米氏动力学行为而变得复杂。用底物FLEEL进行的初始速率实验表明其行为符合底物抑制，在1 mM FLEEL时活性达到一半最大值。用底物维生素K1对苯二酚进行的实验也显示出非米氏动力学，因为相对于较低浓度下的行为，最大活性过早达到。在35 microM维生素K1对苯二酚时观察到活性达到一半最大值。用不同浓度的NaH14CO3进行的初始速率实验呈现米氏动力学，测得的表观Km为0.29 mM。在选择获得接近最大活性的共底物浓度下，用不同浓度的NaH14CO3进行的初始速率研究表明催化常数kcat接近1.0 s-1。去除第四种底物氧气导致羧化酶活性丧失超过99%。巯基试剂N-乙基马来酰亚胺和抗凝剂华法林一样，不可逆地抑制羧化酶。
