Smith C R, Knowles V L, Plaxton W C
Departments of Biochemistry and Biology, Queen's University, Kingston, Ontario, Canada.
Eur J Biochem. 2000 Jul;267(14):4477-85. doi: 10.1046/j.1432-1327.2000.01494.x.
Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was purified 201-fold to electrophoretic homogeneity and a final specific activity of 51 micromol phosphoenolpyruvate utilized per min per mg protein. SDS/PAGE and gel filtration analyses of the final preparation indicated that this PKc is a 220-kDa homotetramer composed of 56-kDa subunits. The enzyme was relatively heat-stable and displayed a broad pH optimum of pH 6.8. PKc activity was absolutely dependent upon the simultaneous presence of a bivalent and univalent cation, with Mg2+ and K+ fulfilling this requirement. Hyperbolic saturation kinetics were observed for phosphoenolpyruvate, ADP, Mg2+ and K+ (apparent Km values = 0.12, 0.075, 0.21 and 0.48 mM, respectively). Although the enzyme utilized UDP, CDP and IDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate, oxalate, and the flavonoids rutin and quercetin were the most effective inhibitors (I50 values = 4, 0.3, 0.07, and 0.10 mM, respectively). L-Aspartate functioned as an activator (Ka = 0.31 mM) by causing a 40% increase in Vmax while completely reversing the inhibition of PKc by L-glutamate. Reciprocal control by L-aspartate and L-glutamate is specific for these amino acids and provides a rationale for the in vivo activation of PKc that occurs during periods of enhanced NH +4-assimilation. Allosteric features of B. napus PKc are compared with those of B. napus phosphoenolpyruvate carboxylase. A model is presented that highlights the pivotal role of L-aspartate and L-glutamate in the coordinate regulation of these key phosphoenolpyruvate utilizing cytosolic enzymes.
从甘蓝型油菜悬浮细胞中纯化得到的胞质丙酮酸激酶（PKc），纯化倍数为201倍，达到电泳纯，最终比活性为每分钟每毫克蛋白质利用51微摩尔磷酸烯醇丙酮酸。对最终制剂进行的SDS/PAGE和凝胶过滤分析表明，该PKc是一种由56 kDa亚基组成的220 kDa同四聚体。该酶相对耐热，最适pH范围较宽，为pH 6.8。PKc活性绝对依赖于二价阳离子和单价阳离子的同时存在，Mg2+和K+满足这一要求。观察到磷酸烯醇丙酮酸、ADP、Mg2+和K+呈双曲线饱和动力学（表观Km值分别为0.12、0.075、0.21和0.48 mM）。虽然该酶利用UDP、CDP和IDP作为替代核苷酸，但ADP是首选底物。L-谷氨酸、草酸盐以及类黄酮芦丁和槲皮素是最有效的抑制剂（I50值分别为4、0.3、0.07和0.10 mM）。L-天冬氨酸作为激活剂（Ka = 0.31 mM），使Vmax增加40%，同时完全逆转L-谷氨酸对PKc的抑制作用。L-天冬氨酸和L-谷氨酸的相互调控对这些氨基酸具有特异性，为NH +4同化增强期间PKc在体内的激活提供了理论依据。将甘蓝型油菜PKc的别构特征与甘蓝型油菜磷酸烯醇丙酮酸羧化酶的别构特征进行了比较。提出了一个模型，突出了L-天冬氨酸和L-谷氨酸在协调调节这些利用磷酸烯醇丙酮酸的关键胞质酶中的关键作用。
