- The enzymes of dNTP synthesis in T4 infection associate to form a multienzyme complex, the T4 dNTP synthetase complex, facilitating the flow of metabolites en route to dNTPs, and their subsequent flow into DNA. Study of protein-protein interactions helps one to understand how the enzymes in the complex are organized and coordinated to function efficiently. By use of several approaches, namely, IAsys optical biosensing, fluorescence spectroscopy and analytical ultracentrifugation, a large number of direct protein-protein interactions among the proteins in the complex were detected. Those associations involved not only T4-encoded proteins but also two host-encoded enzymes, namely, E. coli NDP kinase and adenylate kinase. Quantitative analysis of some of those associations show that their equilibrium dissociation constants fall into the micromolar range, which is comparable to the estimated intracellular protein concentrations, suggesting that those interactions are significant in vivo. In addition, direct interactions between T4 single-strand DNA binding protein (gp32) and several proteins in the complex suggest a linkage between the dNTP synthetase complex and DNA replisome. We also found that some nucleotides, especially ATP, enhanced most of the direct protein-protein interactions. Quantitative analysis shows that, in the presence of 1 mM ATP, the dissociation constants were an order of magnitude lower than that in the absence of ATP. The intracellular concentration of ATP was determined in millimolar range, suggesting that in vivo the associations are even more significant.
IAsys analysis shows the self-association of E. coli NDP kinase and its enhancement by ATP. Equilibrium sedimentation indicates that, in the absence of ATP, the dissociation constant between dimers and tetramers was 0.8 uM. However, in the presence of 0.5 mM ATP, NDP kinase appeared completely in tetramer, suggesting that ATP might exert its effect through influence upon the quaternary structure of NDP kinase.
A mixture of purified T4 enzymes was assayed for activity of a three-step sequence (dCTP->dCMP->dUMP->dTMP), sequentially catalyzed by dCTPase/dUTPase, dCMP deaminase and thymidylate synthase. Kinetic coupling behavior was observed. Other proteins in the complex that are not catalytically involved in this pathway enhanced the kinetic coupling, suggesting positive cooperativity among interactions stabilizing the complex.