Studies on the activating enzyme for iron protein of nitrogenase from Rhodospirillum rubrum.
Removal of ADP-ribose from the iron protein of nitrogenase by activating enzyme resulted in the activation of the inactive iron protein. A radioassay that directly measured the initial velocity of the activation was developed using iron protein radiolabeled with either [8-3H]- or [G-32P]ADP-ribose. The release of radiolabeled ADP-ribose by activating enzyme was linearly correlated with the increase in the specific activity of the iron protein as measured by acetylene reduction. Both ATP and MnCl2 were required for the activation of inactive iron protein. The optimal ratio of [MnCl2]/[ATP] in the radioassay was 2:1, and the optimal concentrations were 4 mM and 2 mM for [MnCl2] and [ATP], respectively. The Km for inactive iron protein was 74 microM and the Vmax was 628 pmol of [32P] ADP-ribose released min-1 microgram of activating enzyme-1. Adenosine, cytidine, guanosine, or uridine mono-, di-, or triphosphates did not substitute for ATP in the activation of native iron protein. Activating enzyme removed ADP-ribose from oxygen-denatured iron protein in the absence of ATP. ADP, ADP-ribose, pyrophosphate, and high concentrations of NaCl inhibited activating enzyme activity.
Saari
L L
LL
Pope
M R
MR
Murrell
S A
SA
Ludden
P W
PW
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002712:Chlorides; D004789:Enzyme Activation; D004798:Enzymes / Q000378:metabolism*; D006026:Glycoside Hydrolases; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D009699:N-Glycosyl Hydrolases; D009591:Nitrogenase / Q000378:metabolism*; D010088:Oxidoreductases; D010100:Oxygen / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D014316:Tritium
D002712:Chlorides; D004798:Enzymes; D017895:Manganese Compounds; D010761:Phosphorus Radioisotopes; D014316:Tritium; D000246:Adenosine Diphosphate Ribose; D008345:Manganese; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C036091:ADP-ribosyl-(dinitrogen reductase) hydrolase; C025340:manganese chloride; D010100:Oxygen