Kinetic pathway of dTTP hydrolysis by hexameric T7 helicase-primase in the absence of DNA.
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.
Jeong
Yong-Joo
YJ
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Kim
Dong-Eun
DE
Patel
Smita S
SS
D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D004247:DNA / Q000378:metabolism; D019915:DNA Primase / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D013942:Thymine Nucleotides / Q000378:metabolism*; D013997:Time Factors; D014867:Water / Q000378:metabolism
D010710:Phosphates; D013942:Thymine Nucleotides; D014867:Water; D004247:DNA; D019915:DNA Primase; C024157:thymidine 5'-triphosphate; D010100:Oxygen