Abstract
Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ~12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) M obtained with dTTP for dTMP kinase is ~3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) M), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.
Highlights
Gene 1.7 of bacteriophage T7 encodes a nucleotide kinase
Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase
Gene 1.7 of bacteriophage T7 came to our attention when we found that mutations in gene 1.7 rendered T7 growth on Escherichia coli resistant to exogenous dideoxythymidine in the media (1)
Summary
Gene 1.7 of bacteriophage T7 encodes a nucleotide kinase. Results: gp1.7 dodecamers catalyze the reversible dTMP and dGMP kinase reactions. Significance: The unique nucleotide kinase of bacteriophage T7 supplies dTDP and dGDP for conversion to the nucleoside 5Ј-triphosphate and recycles the dTDP derived from helicase function to dTTP. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. Gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg2؉ in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction
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