Interaction of nitrogenase with nucleotide analogs of ATP and ADP and the effect of metal ions on ADP inhibition.

The interaction of a large number of ATP and ADP analogs with nitrogenase from Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianum has been examined. Only 1,N6-etheno-ATP and 2'-deoxy-ATP served as substrates for acetylene reduction. Other triphosphates including GTP, ITP, 8-Br-ATP, alpha,beta-methylene ATP, beta,gamma-methylene ATP, 6-chloropurine riboside triphosphate, and AMP-PNP were inert, showing less than 50% inhibition at levels up to two- to fivefold greater than ATP. Xanthosine triphosphate behaved simply as a chelator of magnesium, activating the enzyme at low levels but strongly inhibiting at high levels. When nucleotide diphosphates were tested as inhibitors with enzyme from A. vinelandii, GDP, dGDP, and 6-chloropurine riboside diphosphate were ineffective, XDP was three- to fivefold less effective, and dADP and 1,N6-etheno-ADP were about equally as effective as ADP. With enzyme from C. pasteurianum, dADP was twofold less effective than ADP, XDP was fivefold less effective, and IDP and 1,N6-etheno-ADP appeared to be ineffective. Results with enzyme from K. pneumoniae were very similar to those obtained with A. vinelandii. Different metal ions were tested in the presence of both ATP and ADP to determine whether preferential binding to one nucleotide or the other might alter the ADP/ATP ratio needed for 50% inhibition of activity. Magnesium and manganese gave the same ratio, while with Fe and Co, slightly less ADP was required for equivalent inhibition. Nickel appeared to reduce the sensitivity of A. vinelandii nitrogenase to ADP inhibition while increasing that of C. pasteurianum, but both effects were less than twofold. Calcium, strontium, and aluminum ions were inert with enzymes from these organisms. Cd and Zn were also ineffective with K. pneumoniae. Two isomers of ATP beta S were prepared by enzymatic synthesis from ADP beta S. The A form was a more potent inhibitor of A. vinelandii nitrogenase.