Enzymology of the pathway for ATP production by arginine breakdown
2021
Pols, Tjeerd | Singh, Shubham | Deelman‐Driessen, Cecile | Gaastra, Bauke F. | Poolman, Bert
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 KM and VMAX 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.
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