Abstract
Bacteriophage T7 gp4A' protein is a hexameric helicase-primase protein that separates the strands of a duplex DNA in a reaction coupled to dTTP hydrolysis. Here we reexamine in more detail the kinetic mechanism of dTTP hydrolysis by a preassembled T7 helicase hexamer in the absence of DNA. Pre-steady state dTTP hydrolysis kinetics showed a distinct burst whose amplitude indicated that a preformed hexamer of T7 helicase hydrolyzes on an average one dTTP per hexamer. The pre-steady state chase-time experiments provided evidence for sequential hydrolysis of two dTTPs. The medium [(18)O]P(i) exchange experiments failed to detect dTTP synthesis, indicating that the less than six-site hydrolysis observed is not due to reversible dTTP hydrolysis on the helicase active site. The P(i)-release rate was measured directly using a stopped-flow fluorescence assay, and it was found that the rate of dTTP hydrolysis on the helicase active site is eight times faster than the P(i)-release rate, which in turn is three times faster than the dTDP release rate. Thus, the rate-limiting step in the pathway of helicase-catalyzed deoxythymidine triphosphatase (dTTPase) reaction is the release of dTDP. Chase-time dTTPase kinetics in the steady state phase provided evidence for two to three slowly hydrolyzing dTTPase sites on the hexamer. The results of this study are therefore consistent with those reported earlier (Hingorani, M. M., Washington, M. T., Moore, K. C., and Patel, S. S. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 5012-5017), and they support a model of dTTP hydrolysis by T7 helicase hexamer that is similar to the binding change mechanism of F(1)-ATPase with dTTP hydrolysis occurring sequentially at the catalytic sites.
Highlights
Helicases are motor proteins that translocate along nucleic acids using the energy of NTP1 hydrolysis
The minimal pathway of helicase-catalyzed dTTP hydrolysis consists of the following steps; 1) dTTP binding to T7 helicase hexamer, 2) dTTP hydrolysis to dTDP and Pi, 3) release of Pi, and 4) release of dTDP
The aim of this paper is to determine the rate of dTTP hydrolysis, its equilibrium constant on the enzyme, the rate of Pi release, and the rate of dTDP release from T7 helicase in the absence of ssDNA
Summary
Helicases are motor proteins that translocate along nucleic acids using the energy of NTP1 hydrolysis. Our previous studies indicate that the dTTPase mechanism of gp4AЈ shows striking similarity to the binding change mechanism of the F1-ATPase protein [11] despite there being no amino acid sequence homology between them These studies showed that two-three dTTPs bound tightly but did not get hydrolyzed at the steady state rate, and these were referred to as the noncatalytic sites. The authors raised several questions casting doubts on the studies with T7 helicase regarding hydrolysis of dTTP in a sequential manner They questioned whether the observed kinetics were influenced by hexamer assembly. The findings of this study are consistent with those reported previously [11] and show that dTTP hydrolysis in the absence of DNA is sequential and, in addition, show that dTTP hydrolysis is not reversible on the enzyme active site These studies support the previously proposed mechanism of dTTP hydrolysis by T7 helicase and lay the basis for future studies in the presence of DNA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
