Role of divalent cations in the 3',5'-exonuclease reaction of DNA polymerase I.
X-ray studies of the proofreading 3',5'-exonuclease site of the large (Klenow) fragment of DNA polymerase I have detected a binuclear metal complex consisting of a pentacoordinate metal (site A) which shares a ligand, Asp-355, with an octahedral metal (site B) [Freemont, P. S., Friedman, J. M., Beese, L. S., Sanderson, M. R., & Steitz, T. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8924-8928; Beese, L. S., & Steitz, T. A. (1991) EMBO J. 10, 25-33]. Kinetic studies of the activation of the 3',5'-exonuclease reaction by Co2+, Mn2+, or Mg2+, at low concentrations of DNA, reveal sigmoidal activation curves for the three metal ions with Hill coefficients of 2.3-2.4 and K0.5 values of 16.6 microM, 4.2 microM, and 343 microM, respectively. The binding of Co2+ to the enzyme results in the appearance of an intense visible absorption spectrum of the metal ion with maxima at 633, 570, and 524 nm and extinction coefficients of 190, 194, and 150 M-1 cm-1, respectively, suggesting the formation of a pentacoordinate Co2+ complex. Optical titration with Co2+ yields a sigmoidal titration curve which is best fit by assuming the cooperative binding of three Co2+ ions with a K0.5 of 39.9 microM, comparable to the value of 16.6 microM obtained kinetically. Displacement of Co2+ by 1 equiv of Zn2+, which binds tightly to the A site of the 3',5'-exonuclease, shifts the optical spectrum to 524 nm and lowers the extinction coefficient to 30 -1 cm-1, indicative of octahedral coordination.2+ the formation of the binuclear complex.
Han
H
H
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Rifkind
J M
JM
Mildvan
A S
AS
D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002413:Cations, Divalent; D003035:Cobalt; D004256:DNA Polymerase I / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D007700:Kinetics; D014961:X-Ray Diffraction; D015032:Zinc
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D003035:Cobalt; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D015032:Zinc