The arginine finger of bacteriophage T7 gene 4 helicase: role in energy coupling.
The DNA helicase encoded by gene 4 of bacteriophage T7 couples DNA unwinding to the hydrolysis of dTTP. The loss of coupling in the presence of orthovanadate (Vi) suggests that the gamma-phosphate of dTTP plays an important role in this mechanism. The crystal structure of the hexameric helicase shows Arg-522, located at the subunit interface, positioned to interact with the gamma-phosphate of bound nucleoside 5' triphosphate. In this respect, it is analogous to arginine fingers found in other nucleotide-hydrolyzing enzymes. When Arg-522 is replaced with alanine (gp4-R522A) or lysine (gp4-R522K), the rate of dTTP hydrolysis is significantly decreased. dTTPase activity of the altered proteins is not inhibited by Vi, suggesting the loss of an interaction between Vi and gene 4 protein. gp4-R522A cannot unwind DNA, whereas gp4-R522K does so, proportionate to its dTTPase activity. However, gp4-R522K cannot stimulate T7 polymerase activity on double-stranded DNA. These findings support the involvement of the Arg-522 residue in the energy coupling mechanism.
Crampton
Donald J
DJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Guo
Shenyuan
S
Johnson
Donald E
DE
Richardson
Charles C
CC
D001120:Arginine; D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000821:virology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013816:Thermodynamics; D013942:Thymine Nucleotides / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; D001120:Arginine; D019915:DNA Primase; C024157:thymidine 5'-triphosphate