Hunt M, Köhler P
Institute of Parasitology, University of Zürich, Switzerland.
Biochim Biophys Acta. 1995 May 18;1249(1):15-22. doi: 10.1016/0167-4838(95)00061-x.
ATP-dependent phospho enol pyruvate carboxykinase (EC 4.1.1.49; PEPCK, ATP) was purified from glycosomes of cultured procyclic Trypanosoma brucei to electrophoretic homogeneity. The purified enzyme exhibited a mean specific activity of 83 units mg-1, as measured in the carboxylation direction at 30 degrees C. A similar activity was obtained for the decarboxylation reaction. The enzyme was shown to be a homodimer in solution with a subunit molecular mass of 59 kDa. Amino acid sequence analysis suggested that the PEPCK (ATP) is identical to the trypanosomal protein p60, the sequence of which was previously predicted from the corresponding nucleotide sequence by other investigators. The basic nature of the enzyme was indicated by a high isoelectric point (pH 8.9). The enzyme was found to be strictly dependent on adenosine nucleotides for activity, as well as on the presence of Mn2+. Mg2+ was found to be ineffective as activator of the trypanosomal enzyme, but a combination of subsaturating (< or = 300 microM) concentrations of Mn2+ and high concentrations of Mg2+ caused a synergistic effect on the carboxylation activity, indicating a dual cation requirement. Mn2+ is necessary to activate the enzyme and Mn2+ or Mg2+ most likely forms the cation-nucleotide complex as the active form of the substrate. Relatively high (5 mM) levels of ATP were required to produce a significant inhibition of the carboxylation reaction. Quinolinic acid, a structural analogue of oxaloacetate, completely inhibited the decarboxylation reaction at a 1 mM concentration. The apparent Michaelis constants of the enzyme were 490 microM for PEP, 37 microM for oxaloacetate, 40 microM for ADP, 10.3 microM for ATP, 970 microM for Mn2+ and 26 mM for HCO3-. Endogenous substrate concentrations were found to be 327 nmol PEP, 1486 nmol ADP, 4200 nmol ATP and 11.5 nmol Mn2+ (ml cell volume)-1. Our kinetic data suggest that under physiological conditions PEPCK (ATP) in T. brucei is bidirectional and that its activity is regulated primarily by mass action. The physiological relevance of the enzyme in procyclic T. brucei is discussed.
从培养的布氏锥虫前循环期糖体中纯化出依赖ATP的磷酸烯醇丙酮酸羧激酶（EC 4.1.1.49；PEPCK，ATP），达到电泳纯。在30℃下以羧化方向测定，纯化后的酶平均比活性为83单位mg-1。脱羧反应也获得了类似的活性。该酶在溶液中显示为同型二聚体，亚基分子量为59 kDa。氨基酸序列分析表明，PEPCK（ATP）与锥虫蛋白p60相同，其序列先前已由其他研究人员根据相应的核苷酸序列预测得出。该酶的高碱性由高的等电点（pH 8.9）表明。发现该酶的活性严格依赖于腺苷核苷酸以及Mn2+的存在。发现Mg2+对锥虫酶无激活作用，但亚饱和（≤300 microM）浓度的Mn2+与高浓度的Mg2+组合对羧化活性产生协同作用，表明需要两种阳离子。Mn2+是激活该酶所必需的，并且Mn2+或Mg2+很可能形成阳离子 - 核苷酸复合物作为底物的活性形式。需要相对较高（5 mM）水平的ATP才能对羧化反应产生显著抑制。喹啉酸是草酰乙酸的结构类似物，在1 mM浓度下完全抑制脱羧反应。该酶对PEP的表观米氏常数为490 microM，对草酰乙酸为37 microM，对ADP为40 microM，对ATP为10.3 microM，对Mn2+为970 microM，对HCO3-为26 mM。发现内源性底物浓度为327 nmol PEP、1486 nmol ADP、4200 nmol ATP和11.5 nmol Mn2+（每毫升细胞体积）-1。我们的动力学数据表明，在生理条件下，布氏锥虫中的PEPCK（ATP）是双向的，其活性主要受质量作用调节。讨论了该酶在布氏锥虫前循环期的生理相关性。
