Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, The Netherlands.
FEBS J. 2021 Jan;288(1):293-309. doi: 10.1111/febs.15337. Epub 2020 May 5.
In cells, the breakdown of arginine to ornithine and ammonium ion plus carbon dioxide is coupled to the generation of metabolic energy in the form of ATP. The arginine breakdown pathway is minimally composed of arginine deiminase, ornithine transcarbamoylase, carbamate kinase, and an arginine/ornithine antiporter; ammonia and carbon dioxide most likely diffuse passively across the membrane. The genes for the enzymes and transporter have been cloned and expressed, and the proteins have been purified from Lactococcus lactis IL1403 and incorporated into lipid vesicles for sustained production of ATP. Here, we study the kinetic parameters and biochemical properties of the individual enzymes and the antiporter, and we determine how the physicochemical conditions, effector composition, and effector concentration affect the enzymes. We report the K and V values for catalysis and the native oligomeric state of all proteins, and we measured the effect of pathway intermediates, pH, temperature, freeze-thaw cycles, and salts on the activity of the cytosolic enzymes. We also present data on the protein-to-lipid ratio and lipid composition dependence of the antiporter.
在细胞中，精氨酸分解为鸟氨酸和铵离子以及二氧化碳与以 ATP 形式产生代谢能相偶联。精氨酸分解途径最少由精氨酸脱亚氨酶、鸟氨酸转氨甲酰酶、氨基甲酰磷酸激酶和精氨酸/鸟氨酸反向转运蛋白组成；氨和二氧化碳很可能通过膜被动扩散。酶和转运蛋白的基因已被克隆和表达，并且从乳球菌乳脂亚种 IL1403 中纯化了蛋白质，并将其纳入脂质体中以持续产生 ATP。在这里，我们研究了单个酶和反向转运蛋白的动力学参数和生化特性，并确定了物理化学条件、效应物组成和效应物浓度如何影响酶。我们报告了所有蛋白质的催化 K 和 V 值以及天然寡聚状态，并测量了途径中间产物、pH 值、温度、冻融循环和盐对胞质酶活性的影响。我们还提供了关于蛋白与脂质比例和反向转运蛋白脂质组成依赖性的数据。
