Abstract
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.
Highlights
In some enzymes that have multiple active sites, the rate constants for events at individual sites increase as substrate occupies more of the catalytic sites
Oxygen exchange experiments [10] revealed only a single water-derived oxygen in the product phosphate. These findings eliminate the possibility of a phosphorylated enzyme or RNA intermediate during catalysis and demand that the hydrolysis energy be used to drive a cycle of enzyme conformation changes
Size of the Initial ATP Hydrolysis Burst—We have previously shown that the rate of ATP hydrolysis by Rho is enhanced when all three active sites are occupied [7]
Summary
(x/3) erativity in F1 are the ongoing subject of intense study. The enzyme is stable and of simpler structure than F1, and all of its nucleotide binding sites are catalytic sites. The rate constants for ATP hydrolysis by Rho make it highly amenable to available chemical quench approaches. In the accompanying paper [11], we examined what features of the ATP substrate are important to achieve the catalytic rate enhancement at saturating substrate concentrations and found that the configuration of the - and ␥-phosphoryl groups is key. This work addresses the question of how many of the three Rho active sites must be filled to enhance the hydrolysis rate, whether the activated enzyme state is persistent and kinetically relevant, and whether an ordered sequential catalytic sequence is operative during the steady state
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