The cooperativity of the single-stranded DNA dependent nucleoside triphosphatase activity of the recA protein was investigated by examining the influence of a good substrate (ATP) on the hydrolysis of a poor substrate (GTP). At pH 7.5 and 37 °C, both ATP and GTP are hydrolyzed with a turnover number of 17.5 min-1. The S0.5for GTP (750 μM), however, is nearly 20-fold higher than the S0.5for ATP (45 μM). Low concentrations of ATP activate the GTPase activity of the recA protein by lowering the S0.5for GTP; in the presence of 50 μM ATP, the S0.5for GTP is reduced from 750 μM to 200 μM. Concentrations of ATP greater than 50 μM result in competitive inhibition of the ATP-activated GTPase activity. Although GTP is a substrate for hydrolysis, it will not substitute for ATP as a high-energy cofactor in the standard recA protein promoted three-strand exchange reaction. To account for these results, a minimal kinetic model is presented in which ATP binding induces specific conformational changes in the recA protein that do not occur with GTP binding.
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