TY - JOUR
T1 - Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates
AU - Brautigam, Chad A
AU - Steitz, Thomas A.
N1 - Funding Information:
The authors thank C. Joyce for expert advice on protein purification and handling, and helpful discussions, J. Jäger for initial help with refinement, and N. Ban, S. Bellon, C. Correll, D. Jerzalmi, S. Kamtekar, P. Klosterman, L. Min, K. Rodgers, Y. Shamoo and L. Silvian for assistance with data collection. In addition, we thank C. Correll, Y. Shamoo, P. Sigler, N. Grindly and F. Eckstein for helpful suggestions on the manuscript. This work was supported by NIH grant GM39546 to T.A.S.
PY - 1998/3/27
Y1 - 1998/3/27
N2 - A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 Å resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the R(p) isomer of phosphorothioate DNA, refined at 2.2 Å resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the S(p) phosphorothioate at 2.3 Å resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the R(p) isomer was reduced only about 15-fold, while no enzyme activity could be detected with the S(p) phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the S(p) phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.
AB - A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 Å resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the R(p) isomer of phosphorothioate DNA, refined at 2.2 Å resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the S(p) phosphorothioate at 2.3 Å resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the R(p) isomer was reduced only about 15-fold, while no enzyme activity could be detected with the S(p) phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the S(p) phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.
KW - 3'-5' exonuclease
KW - Mn rescue
KW - Phosphorothioates
KW - Two-metal-ion mechanism
KW - X-ray crystallography
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U2 - 10.1006/jmbi.1997.1586
DO - 10.1006/jmbi.1997.1586
M3 - Article
C2 - 9514742
AN - SCOPUS:0032571245
SN - 0022-2836
VL - 277
SP - 363
EP - 377
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -