J. Biochem, 1994, Vol. 116, No. 5 960-966
© 1994 Japanese Biochemical Society
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Interaction of Escherichia coli RecA Protein with ATP and Its Analogues1
Department of Biology, Faculty of Science, Osaka University Toyonaka, Osaka 560
Interactions of Escherichia Coli RecA protein with ATP and its analogues in the absence of DNA were studied by circular dichroic (CD) spectroscopy. The binding of RecA protein to ATP increased the CD band of ATP at around 260 nm. The positive CD band of the RecA protein-ATP complex suggested that the bound ATP was in the anti conformation, in accord with X-ray crystallographic data [Story, R.M. and Steitz, T.A. (1992) Nature 355, 374–376]. At PH 7.5 and at 25°C the dissociation constant (Kd) and thermodynamic parameters for the binding of ATP to RecA protein were 18µM (
G=-6.5 keal·mol-1), H=0 keal·mol-1, and S=22cal.mol-1·K-1. A non-hydrolyzable ATP analogue, adenosine 5'-0-(3-thiotriphosphate) (ATP
S), gave a spectral change similar to that of ATP. The Kd for this analogue, 22µM, was very close to the Km of ATP. These results in the absence of single-stranded DNA were different from those obtained by kinetic analysis [Weinstock, G.M. et al. (1981) J.Biol Chem. 256, 8850–8855], which indicated that the inhibition constant of ATPS was much smaller than the Km of ATP in the presence of DNA. For other ATP analogues (dATP, ADP, and dADP), similar spectral changes were observed, and their Kd values ranged from 19 to 54 µM. UTP, dUTP, and TTP also gave CD spectral changes, but not AMP, GTP, dGTP, CTP, and dCTP. The orders of affinity (1/kd) were: ATP
ATPS ADP»AMP for the number of Phosphate moieties of the nucleotide, ribose > deoxyribose for the sugar moiety, and AUT»G and C for the base moiety.
1This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan.
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