Escherichia coli primase zinc is sensitive to substrate and cofactor binding

The ligation state of the single zinc site in primase from Escherichia coli changes when various substrates and cofactors are added alone or in combin ation as determined by X-ray absorption spectroscopy. X-ray absorption spec troscopy (XAS) provides information about the local structure (similar to 5 Angstrom) of atoms surrounding the metal and has been widely used to chara cterize metalloproteins. The zinc site in native primase and in primase bou nd to low (30 mM) magnesium acetate was found to be tetrahedrally ligated b y three sulfurs at an average distance of 2.36 +/- 0.02 Angstrom and one hi stidine nitrogen located at a distance of 2.15 +/- 0.03 Angstrom. When ATP, ATP and (dT)(17), or ATP, low magnesium acetate and (dT)17 was added to pr imase, one (or two) additional nitrogen/oxygen ligands were coordinated to the zinc together with the histidine nitrogen at an average distance of 2.1 5 +/- 0.03 Angstrom. These additional ligands are likely from adjacent phos phates from ATP. Another structure was observed for the primase-(dT)(17) co mplex in which an additional nitrogen/oxygen ligand likely from the phospha te backbone together with the histidine nitrogen was located at a significa ntly shorter average distance of 2.05 +/- 0.03 Angstrom. High magnesium ace tate (300 mM) completely inactivates primase in a reversible manner such th at the region near the zinc ligands becomes accessible to proteolytic diges tion [Urlacher, T. M., and Griep, M. A. (1995) Biochemistry 34, 16708-16714 ]. In this inactive complex, additional oxygen/nitrogen ligands from acetat e as well as the histidine nitrogen are located at a distance of 2.20 +/- 0 .03 Angstrom from the zinc site. To test whether the catalytic magnesium wa s binding within similar to 5 Angstrom of the zinc, we incubated primase wi th high (300 mM) manganese acetate. The functional properties of magnesium and manganese are similar, but the larger atomic number of manganese enhanc es the X-ray backscattering, making it possible to identify. Since no signi ficant difference was observed from the manganese-incubated sample, the cat alytic metal-binding site is likely located >5 Angstrom from the zinc. Thes e studies clearly show that primase zinc ligation changes upon binding subs trate.