In vivo and in vitro kinetics of nitrogenase.
We measured some of the kinetic parameters of nitrogenase to intact systems of Clostridium pasteurianum and Klebsiella pneumoniae to compare them with the kinetics of the enzyme in vitro. We found that the enzyme showed multiple apparent Km values for acetylene reduction in vivo, as it does in vitro. Carbon monoxide was a noncompetitive inhibitor of acetylene reduction; azide was a noncompetitive inhibitor of acetylene reduction, and nitrogen was a partial inhibitor of acetylene reduction. Cyanide was a noncompetitive inhibitor of acetylene reduction in C. pasteurianum but it was a metabolic poison in K. pneumoniae, in addition to being an inhibitor of nitrogenase. The partial nature of nitrogen inhibition was apparent in assays where both nitrogen and CO were present. Nitrogen did not alter the apparent Ki for CO, nor did the presence of CO enhance the competitive effectiveness of nitrogen. By using recombined nitrogenase fractions, we found that the ability of nitrogen to inhibit hydrogen evolution or acetylene reduction varied with the ratio of protein components. The in vivo inhibition of acetylene reduction by dinitrogen was comparable to that obtained with an excess of the Fe protein in vitro. We conclude that there is an effective excess of the Fe protein available under active growth conditions in vivo.
D000114:Acetylene / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D001395:Azotobacter / Q000201:enzymology*; D002248:Carbon Monoxide / Q000494:pharmacology; D003013:Clostridium / Q000201:enzymology*; D003486:Cyanides / Q000494:pharmacology; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D009584:Nitrogen / Q000494:pharmacology; D009591:Nitrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001386:Azides; D003486:Cyanides; D002248:Carbon Monoxide; D009591:Nitrogenase; D009584:Nitrogen; D000114:Acetylene