Hydrolysis of nucleoside triphosphates other than ATP by nitrogenase.
The hydrolysis of ATP to ADP and P(i) is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron (Fe) protein and the molybdenum-iron (MoFe) protein. The oxidation state of nitrogenase was found to greatly influence the nucleotide hydrolysis rates. MgATP hydrolysis rates were 20 times higher under dithionite reducing conditions (approximately 4,000 nmol of MgADP formed per min/mg of Fe protein) as compared with indigo disulfonate oxidizing conditions (200 nmol of MgADP formed per min/mg of Fe protein). In contrast, MgGTP, MgITP, and MgUTP hydrolysis rates were significantly higher under oxidizing conditions (1,400-2,000 nmol of MgNDP formed per min/mg of Fe protein) as compared with reducing conditions (80-230 nmol of MgNDP formed per min/mg of Fe protein). The K(m) values for MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330-540 microM) for both the reduced and oxidized states of nitrogenase. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF(4)(-) resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF(4)(-) x MgNDP. The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed.
Ryle
M J
MJ
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Seefeldt
L C
LC
D000255:Adenosine Triphosphate / Q000378:metabolism; D017607:Aluminum Compounds / Q000494:pharmacology; D016948:Azotobacter vinelandii / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology; D004227:Dithionite / Q000378:metabolism; D005459:Fluorides / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008983:Molybdoferredoxin; D009591:Nitrogenase / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D014544:Uridine Triphosphate / Q000378:metabolism
D017607:Aluminum Compounds; D001426:Bacterial Proteins; D008983:Molybdoferredoxin; D009711:Nucleotides; D007293:Inosine Triphosphate; D004227:Dithionite; C050992:tetrafluoroaluminate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D005459:Fluorides; D014544:Uridine Triphosphate