title: Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates.
abstract: 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 A 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 Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A 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 Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Brautigam
authors.forename: T A
authors.initials: TA
authors.lastname: Steitz
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D010100:Oxygen / Q000737:chemistry; D013379:Substrate Specificity; D013873:Thionucleotides / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D013873:Thionucleotides; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D010100:Oxygen