Abstract
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 M and V MAX 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.
Highlights
The arginine deiminase pathway is one of the simplest routes for the generation of ATP and alkalinization of the internal pH
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
We had to work with ArcD2 as ArcD1 from L. lactis IL1403 misses the 14th transmembrane helix when aligned to ArcD1 from L. lactis MG1363 [39], rendering the transporter inactive
Summary
The arginine deiminase pathway is one of the simplest routes for the generation of ATP and alkalinization of the internal pH. The ADI pathway is widely used in bacteria to generate metabolic energy [1,2] and to protect cells in acidic environments [3,4]; per molecule of arginine metabolized three protons are used (see reaction equation). The anabolic OTCs have a strongly reduced cooperativity and lower apparent KM for carbamoyl-Pi. In mammalian cells arginine metabolism is rather complex, as arginine is involved in synthesis of proteins, urea, creatine, polyamines, nitric oxide, proline, glutamate and agmatine [12]. Mammalian cells use arginases that catalyze the reaction of arginine plus water into ornithine plus urea, and they have anabolic but not catabolic OTCs [13]. Mammalian cells do not use CKs but have AOAs in their mitochondria and make use of arginine uniporters [14,15]
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