Inhibition of recA protein promoted ATP hydrolysis. 1. ATP gamma S and ADP are antagonistic inhibitors.
ADP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) inhibit recA protein promoted ATP hydrolysis by fundamentally different mechanisms. In both cases, at least two modes of inhibition are observed. For ADP, the first mode is competitive inhibition. The second mode is manifested by dissociation of recA protein from DNA. These are readily distinguished in a comparison of ATP hydrolyses that are activated by (a) DNA and (b) high (approximately 2 M) salt concentrations. Competitive inhibition with a significant degree of cooperativity is observed under both sets of conditions, although the DNA-dependent activity is more sensitive to ADP than the high-salt reaction. The reaction in the presence of poly(deoxythymidylic acid) or duplex DNA ceases when about 60% of the available ATP is hydrolyzed, reflecting an ADP-mediated dissociation of recA protein from the DNA that is governed by the ADP/ATP ratio. In contrast, ATP hydrolysis proceeds nearly to completion at high salt concentrations. At high concentrations of ATP and ATP gamma S, ATP gamma S also acts as a competitive inhibitor. At low concentrations of ATP gamma S and ATP, however, ATP gamma S activates ATP hydrolysis. These patterns are observed for recA-mediated ATP hydrolysis with either high salt concentrations or a poly(deoxythymidylic acid) [poly(dT)] cofactor, although the activation is observed at much lower ATP and ATP gamma S concentrations when poly(dT) is used. ATP gamma S can also relieve the inhibitory effect of ADP under some conditions. ATP gamma S and ADP are antagonistic inhibitors, reinforcing the idea that they stabilize different conformations of the protein and suggesting that these conformations are mutually exclusive. The ATP gamma S (ATP) conformation is active in ATP hydrolysis. The ADP conformation is inactive.
Lee
J W
JW
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Cox
M M
MM
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D010582:Bacteriophage lambda / Q000235:genetics; D001667:Binding, Competitive; D004279:DNA, Viral / Q000378:metabolism; D004926:Escherichia coli / Q000032:analysis; D006868:Hydrolysis; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000378:metabolism*
D004279:DNA, Viral; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases