Time-dependent inhibition of recA protein-catalyzed ATP hydrolysis by ATPgammaS: evidence for a rate-determining isomerization of the recA-ssDNA complex.
The ATP analog ATPgammaS is a competitive inhibitor of the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction. The degree of inhibition by ATPgammaS, however, changes in a time-dependent manner and is consistent with a two step binding mechanism. In the first step, ATPgammaS binds to the recA-ssDNA complex in a rapid equilibrium step (KD = 50 microM). This initial binding step is followed by an isomerization of the recA-ssDNA-ATPgammaS complex to a new conformational state in which ATPgammaS is bound with a significantly higher affinity (overall K(D) = 0.3 microM). This isomerization is followed by the slow hydrolysis of ATPgammaS to ADP and thiophosphate (0.01 min(-1)). The first-order rate constant for the ATPgammaS-mediated isomerization step (20 min(-1)), although significantly greater than the rate of ATPgammaS hydrolysis, is identical to the steady-state rate constant for the recA protein-catalyzed ATP hydrolysis reaction. These results are consistent with a kinetic model in which an ATP-mediated isomerization of the recA-ssDNA complex represents the rate-determining step on the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction pathway.
Paulus
B F
BF
Department of Biochemistry, The Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205, USA.
Bryant
F R
FR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D002384:Catalysis; D004277:DNA, Single-Stranded / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D011938:Rec A Recombinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004277:DNA, Single-Stranded; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011938:Rec A Recombinases