Effect of nucleotides on the activity of dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum.
The mechanism by which MgADP stimulates the activity of dinitrogenase reductase ADP-ribosyltransferase (DRAT) has been examined by using dinitrogenase reductases from Rhodospirillum rubrum, Klebsiella pneumoniae, and Azotobacter vinelandii as acceptor substrates. In the presence of 0.2 mM NAD, maximal rates of ADP-ribosylation of all three acceptors were observed at an ADP concentration of 150 microM; in the absence of added ADP, DRAT activity with the dinitrogenase reductases from R. rubrum and K. pneumoniae was less than 5% of the maximal rate, but the A. vinelandii protein was ADP-ribosylated at 40% of the maximal rate. Of eight dinucleotides tested, only ADP, 2'-deoxy-ADP, and ADP-beta S served as activators of the DRAT reaction; ADP, 2'-deoxy-ADP, and ADP-beta S were also the only dinucleotides found which inhibited acetylene reduction activity by dinitrogenase reductase. The dinucleotide specificities for both DRAT activation and acetylene reduction inhibition were the same for all three dinitrogenase reductases. In the DRAT reaction with the dinitrogenase reductases from K. pneumoniae and A. vinelandii, the Km for NAD was 30-fold higher in the absence of ADP than in its presence; the Km for NAD with the R. rubrum acceptor was not measurable. In the presence of saturating ADP, ADP-ribosylation of dinitrogenase reductase from R. rubrum was inhibited 63% by 1.5 mM ATP. It is concluded that MgADP stimulates DRAT activity by lowering the Km for NAD and that MgADP exerts its effect by binding to dinitrogenase reductase. MgATP inhibits DRAT activity by competing with MgADP for binding to dinitrogenase reductase.
Lowery
R G
RG
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Ludden
P W
PW
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D017037:Dinitrogenase Reductase; D004789:Enzyme Activation / Q000187:drug effects; D005288:Ferredoxins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D012247:Rhodospirillum rubrum / Q000201:enzymology*
D017037:Dinitrogenase Reductase; D005288:Ferredoxins; D009711:Nucleotides; D009243:NAD; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases; D008274:Magnesium