title: Kinetic and magnetic resonance studies of the glutamate-43 to serine mutant of staphylococcal nuclease.
abstract: The Glu-43 residue of staphylococcal nuclease has been proposed to function as a general base that facilitates the attack of water on the phosphodiester substrate [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555]. With DNA as substrate, Vmax in the glutamate-43--serine (E43S) mutant enzyme is decreased by 2700-fold at pH 7.4 but only 376-fold at pH 9.9. With the wild-type enzyme, Vmax increases with pH to pH 9.2, above which it becomes less sensitive to further increase in pH, leveling off at pH 9.8. In contrast, Vmax of the E43S mutant continues to rise, first order in [OH-], to pH 9.8. Above pH 10 both activities fall irreversible. Hence the hydroxyl ion can partially replace the effect of Glu-43 on kcat, in accord with the proposed role of Glu-43 as a general base. The inflection point in the curve relating pH to log Vmax of the wild-type enzyme at pH 9.4 may reflect the ionization of a Ca2+-bound water, or of a Lys or Tyr residue at the active site. The activator Ca2+ and the competitive inhibitor Mn2+ bind to the E43S mutant an order of magnitude more weakly than to the wild-type enzyme as detected by kinetics and by direct metal binding studies, and approximately one additional water ligand on Mn2+ is found in the binary Mn2+ complex of the E43S mutant (1.4 +/- 0.2) as compared to that of the wild-type enzyme (0.8 +/- 0.2). These data suggest that Glu-43 coordinates the divalent cation in the binary enzyme-metal complex but dissociates from the metal to create a water binding site and to function as a general base in the ternary enzyme-metal-DNA complex. While a 2-fold weaker binding of DNA to the Ca2+ complex of the E43S mutant than to the wild-type enzyme is found by kinetic studies, an order of magnitude tighter binding of the competitive inhibitor 3',5'-pdTp to the Mn2+ and Ca2+ complexes of E43S is found by direct binding studies. Distances from Co2+ to phosphorus in the ternary enzyme-Co2+-pdTp complexes reveal coordination of only the 5'-phosphate by Co2+ on the wild-type enzyme but coordination of both the 3'- and 5'-phosphates of pdTp on the E43S mutant.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: E H
authors.initials: EH
authors.lastname: Serpersu
authors.forename: D W
authors.initials: DW
authors.lastname: Hibler
authors.forename: J A
authors.initials: JA
authors.lastname: Gerlt
authors.forename: A S
authors.initials: AS
authors.lastname: Mildvan
mesh_terms: D002413:Cations, Divalent; D005971:Glutamates; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism*; D008962:Models, Theoretical; D009154:Mutation; D011485:Protein Binding; D012694:Serine
chemical_list: D002413:Cations, Divalent; D005971:Glutamates; D018698:Glutamic Acid; D012694:Serine; D008836:Micrococcal Nuclease