Chan Maurice, Sim Tiow-Suan
Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore.
Biochem Biophys Res Commun. 2005 Jan 7;326(1):188-96. doi: 10.1016/j.bbrc.2004.11.018.
The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K(m) of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1mM pyridoxal-5'-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K(i) of 0.8mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.
丙酮酸激酶在疟疾感染过程中的重要作用促使人们利用红细胞内期疟原虫的mRNA克隆了编码恶性疟原虫丙酮酸激酶（pfPyrK）的cDNA。全长cDNA编码一种计算分子量为55.6 kDa、等电点为7.5的蛋白质。纯化的重组pfPyrK具有酶活性，以同源四聚体的活性形式存在。该酶对磷酸烯醇丙酮酸和ADP表现出双曲线动力学，K(m)分别为0.19和0.12 mM。pfPyrK不受果糖-1,6-二磷酸（大多数物种丙酮酸激酶的一般激活因子）的影响。6-磷酸葡萄糖（弓形虫酶的激活剂）不影响pfPyrK的活性。与兔丙酮酸激酶类似，pfPyrK易被1 mM磷酸吡哆醛-5'-磷酸灭活，但程度较小。对pfPyrK抑制剂的筛选显示，它受到ATP和柠檬酸盐的显著抑制。详细的动力学分析表明，在柠檬酸盐存在下，PEP的动力学从双曲线转变为S形，并且ATP对PEP具有竞争性抑制行为。柠檬酸盐对ADP表现出非竞争性抑制，K(i)为0.8 mM。总之，恶性疟原虫在其发育周期的红细胞内期表达一种活性丙酮酸激酶，该激酶在感染过程中可能发挥重要的代谢作用。
