Catalytic cooperativity among subunits of Escherichia coli transcription termination factor Rho. Kinetics and substrate structural requirements.
Escherichia coli transcription termination factor Rho shows a 30-fold faster rate of ATP hydrolysis when all three catalytic sites are filled with ATP than when only a single site is filled (Stitt, B. L. and Xu, Y. (1998) J. Biol. Chem. 273, 26477-26486). To study the structural requirements of the substrate for this catalytic cooperativity, rapid mix/chemical quench experiments using various ATP analogs were performed. The results indicate that it is the configuration of the beta- and gamma-phosphoryl groups of ATP that is of primary importance for the rate enhancement. Our results also show that there are kinetically slow branches of the enzyme mechanism that are not seen when the chemistry step of the catalytic cycle is fast. These branches become prominent, however, when two of the three Rho active sites are empty or bear non-hydrolyzable compounds. A first-order step that is slow compared with V(max) catalysis enables a single ATP molecule bound in any one of the three Rho active sites to be hydrolyzed and defines the kinetically slow branches. This first-order step could be a protein conformation change or a rearrangement of bound RNA. The results reinforce the importance of catalytic cooperativity in normal Rho function and suggest that several protein conformations exist along the catalytic pathway.
Browne
Rebecca J
RJ
Department of Biochemistry and Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Barr
Eric W
EW
Stitt
Barbara L
BL
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008962:Models, Theoretical; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D012234:Rho Factor / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D012234:Rho Factor; D000255:Adenosine Triphosphate