Active site occupancy required for catalytic cooperativity by Escherichia coli transcription termination factor Rho

Escherichia coli transcription termination factor Rho exhibits the phenomenon of catalytic cooperativity. The catalytic rate per site is 30-fold faster when all three sites are filled with substrate ATP than when only a single site is occupied (Stitt, B. L., and Xu, Y. (1998) J. Biol. Chem. 273, 26477-26486). Experiments presented here investigate whether all three active sites must be filled or whether only two occupied sites are required for catalytic cooperativity. The results indicate that all three Rho catalytic sites must be filled with substrate to achieve the enhanced catalytic rate, both in pre-steady-state and in steady-state hydrolysis. They further suggest that, once the enzyme is saturated with ATP, a V(max) enzyme conformation is achieved that is stable for at least three catalytic cycles.