Urbina J A
Centro de Biología Celular, Facultad de Ciencias, Universidad Central de Venezuela, Caracas.
Arch Biochem Biophys. 1987 Oct;258(1):186-95. doi: 10.1016/0003-9861(87)90335-3.
The phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) of the epimastigote form of Trypanosoma (Schizotrypanum) cruzi has been purified to homogeneity. The enzyme is composed of two apparently identical 42,000 +/- 500 subunits, is highly specific for adenine nucleotides, and has a strict requirement of Mn2+ ions for activity; the activation of the enzyme by ionic Mn2+ reveals that one Mn2+ ion required for each 42,000 subunit. Hyperbolic kinetics are observed for all substrates in the carboxylation reaction with Km (phosphoenolpyruvate) of 0.36 +/- 0.08 mM, Km (HCO-3) of 3.7 +/- 0.2 mM, and Km (Mg-ADP) of 39 +/- 1 microM. In the decarboxylation reaction the kinetics with respect to oxalacetic acid are also hyperbolic with a Km of 27 +/- 3 microM, but towards Mg-ATP there is a biphasic response: hyperbolic at low (less than 250 microM) concentrations with a Km of 39 +/- 1 microM, but at higher concentrations the nucleotide produces a strong inhibition of the enzyme activity. This inhibition is also observed with Mg-GTP and Mg-ITP which are not substrates of the reaction. The results are consistent with an important regulatory function of the enzyme in the amino-acid catabolism of T. cruzi.
克氏锥虫（克鲁斯锥虫属）前鞭毛体形式的磷酸烯醇丙酮酸羧激酶（ATP：草酰乙酸羧基裂解酶（转磷酸化），EC 4.1.1.49）已被纯化至同质。该酶由两个明显相同的42,000±500亚基组成，对腺嘌呤核苷酸具有高度特异性，并且对活性严格要求Mn2+离子；离子Mn2+对酶的激活表明每个42,000亚基需要一个Mn2+离子。在羧化反应中，所有底物均呈现双曲线动力学，磷酸烯醇丙酮酸的Km为0.36±0.08 mM，HCO-3的Km为3.7±0.2 mM，Mg-ADP的Km为39±1 microM。在脱羧反应中，相对于草酰乙酸的动力学也是双曲线的，Km为27±3 microM，但对于Mg-ATP存在双相反应：在低（小于250 microM）浓度下为双曲线，Km为39±1 microM，但在较高浓度下核苷酸会强烈抑制酶活性。用不是该反应底物的Mg-GTP和Mg-ITP也观察到这种抑制作用。这些结果与该酶在克氏锥虫氨基酸分解代谢中的重要调节功能一致。
