Portela Paula, Howell Steven, Moreno Silvia, Rossi Silvia
Laboratory of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
J Biol Chem. 2002 Aug 23;277(34):30477-87. doi: 10.1074/jbc.M201094200. Epub 2002 Jun 12.
Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HA-Tpk1.Bcy1) and to be phosphorylated in a cAMP-dependent process. Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GST-Pyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK gene with an attenuated mutation (tpk1(w1)). The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mm. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6-bisphosphate it shows an n(H) value of 1.4, as compared with an n(H) of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.
酿酒酵母丙酮酸激酶1（Pyk1）被证明与酵母蛋白激酶A全酶（HA-Tpk1.Bcy1）的免疫沉淀物相关，并在cAMP依赖性过程中被磷酸化。谷胱甘肽S-转移酶（GST）-Pyk1和GST-Pyk2在体外均被牛心蛋白激酶A（PKA）催化亚基和固定化酵母HA-Tpk1磷酸化。牛催化亚基对GST-Pyk1和GST-Pyk2磷酸化的特异性常数在亮氨酸-精氨酸-精氨酸-丙氨酸-丝氨酸-亮氨酸-甘氨酸（肯普肽）的值范围内。当使用野生型菌株时，两种融合蛋白在体内、在过表达该蛋白的完整细胞中或在体外使用粗提物作为蛋白激酶A的来源时均被磷酸化，但当使用仅具有一个TPK基因且有减弱突变（tpk1(w1)）的菌株时则未被磷酸化。在来自三种菌株的部分纯化制剂中测定了磷酸化对Pyk活性的影响，这些制剂含有不同水平的内源性蛋白激酶A活性。在不存在或存在1.5 mM激活剂果糖1,6-二磷酸的情况下，在不同磷酸烯醇丙酮酸浓度下测量Pyk1活性。从具有最高与最低蛋白激酶A活性的提取物中获得的Pyk1的比较得出的初步动力学结果表明，该酶在磷酸化条件下更具活性；在不存在激活剂的情况下，它显示磷酸烯醇丙酮酸滴定曲线向左移动且希尔系数增加，而在存在果糖1,6-二磷酸的情况下，与从几乎无蛋白激酶A活性的提取物中获得的Pyk1的n(H)值为2相比，它显示n(H)值为1.4。
