Mechanism of carbamyl phosphate inhibition of nitrogenase of Clostridium pasteurianum.
Carbamyl phosphate caused a maximal inhibition of 50% of the in vitro nitrogenase activity measured by acetylene reduction and dinitrogen reduction. The addition of 1 mM carbamyl phosphate to a N(2)-fixing culture caused a rapid decrease of 30% of the acetylene reduction activity and also repression of nitrogenase biosynthesis. However, carbamyl phosphate had no effect on the reductant-dependent adenosine triphosphate hydrolysis and H(2) evolution reactions catalyzed by nitrogenase. Studies on the binding of carbamyl phosphate to nitrogenase and each of its two components (azoferredoxin and molybdoferredoxin) indicated that optimal binding was obtained only in the presence of an operating nitrogenase system. Moreover, the binding seemed to be on the molybdoferredoxin component rather than azoferredoxin. From a Scatchard plot and a reciprocal plot of the data, the values of n = 2 and dissociation constant (K) of approximately 5 x 10(-5) M were obtained. The value for the dissociation constant was of the same order of magnitude as the endogenous level of carbamyl phosphate in a N(2)-fixing cell. The carbamyl phosphate pool in NH(3)-grown cells was twice that of N(2)-fixing cells.
D000114:Acetylene / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000641:Ammonia / Q000096:biosynthesis; D002219:Carbamates / Q000378:metabolism / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D002474:Cell-Free System; D003013:Clostridium / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D004794:Enzyme Repression; D005288:Ferredoxins / Q000378:metabolism; D006859:Hydrogen / Q000096:biosynthesis; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D009586:Nitrogen Fixation / Q000187:drug effects; D009591:Nitrogenase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D010084:Oxidation-Reduction; D010710:Phosphates / Q000378:metabolism / Q000494:pharmacology*; D010725:Phosphocreatine / Q000378:metabolism; D011485:Protein Binding
D002219:Carbamates; D002250:Carbon Radioisotopes; D005288:Ferredoxins; D010710:Phosphates; D010725:Phosphocreatine; D000641:Ammonia; D006859:Hydrogen; D000255:Adenosine Triphosphate; D009591:Nitrogenase; D008274:Magnesium; D000114:Acetylene