PMID:10551822
| Citation |
Kim, DE and Patel, SS (1999) The mechanism of ATP hydrolysis at the noncatalytic sites of the transcription termination factor Rho. J. Biol. Chem. 274:32667-71 |
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| Abstract |
Escherichia coli transcription termination factor rho is a hexamer with three catalytic subunits that turnover ATP at a fast rate and three noncatalytic subunits that turnover ATP at a relatively slow rate. The mechanism of the ATPase reaction at the noncatalytic sites was determined and was compared with the ATPase mechanism at the catalytic sites. A sequential mechanism for ATP binding or hydrolysis that was proposed for the catalytic sites was not observed at the noncatalytic sites. Pre-steady-state pulse-chase experiments showed that three ATPs were tightly bound to the noncatalytic sites and these were simultaneously hydrolyzed at a rate of 1.8 s(-1) at 18 degrees C. The apparent bimolecular rate constant for ATP binding was determined as 5.4 x 10(5) M(-1) s(-1) in the presence of poly(C) RNA. The ATP hydrolysis products dissociated from the noncatalytic sites at 0.02 s(-1). The hydrolysis of ATP at the noncatalytic sites was at least 130 times slower, and the overall ATPase turnover was 1500 times slower than that at the catalytic sites. These results from studies of the rho protein are likely to be general to hexameric helicases. We propose that the ATPase activity at the noncatalytic site is too slow to drive translocation of the protein on the nucleic acid or to provide energy for nucleic acid unwinding. |
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| Keywords |
Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/metabolism; Binding Sites; DNA Helicases/chemistry; Escherichia coli/enzymology; Kinetics; Poly C/chemistry; Protein Binding; Protein Conformation; Rho Factor/chemistry |
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