PMID:8294428
| Citation |
Frick, DN, Weber, DJ, Gillespie, JR, Bessman, MJ and Mildvan, AS (1994) Dual divalent cation requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the metal and substrate complexes. J. Biol. Chem. 269:1794-803 |
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| Abstract |
Kinetic analyses of both the Mn(2+)- and Mg(2+)-activated hydrolysis of dGTP by MutT show the requirement for two divalent cations. Whereas Mn2+ supports a 20-fold lower kcat (0.19 s-1) than Mg2+ (4.0 s-1), the Km of Mn2+.dGTP (6.3 microM) is 45-fold lower than that of Mg2+.dGTP (284 microM). Adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPCPP) is a linear competitive inhibitor with respect to dGTP with a Ki for Mg2+.AMPCPP (42 microM) which is 57-fold lower than the Ki of Mg2+.AMPCPP (2.4 mM). Such tightening suggests that a metal-bridge E.M2+.NTP.M2+ complex is the catalytically active species. The 12 dissociation constants describing the quaternary MutT.M2+.AMPCPP.M2+ complex were evaluated for both Mn2+ and Mg2+, using EPR and NMR methods. MutT binds a single Mn2+ with a Kd of 130 +/- 40 microM in reasonable agreement with the kinetically determined activator constant of Mn2+ of 230 +/- 72 microM. The MutT.AMPCPP complex binds two Mn2+ ions, the weaker of which has a Kd of 16 +/- 2 microM in agreement with the kinetically determined KmMn2+ of 26 +/- 10 microM. MutT.Mn2+ binds Mn2+.AMPCPP with Kd of 16 +/- 4 microM, whereas MutT alone binds Mn2+.AMPCPP with a Kd of 135 +/- 30 microM. The 17-fold enhanced paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons found with the binary MutT.Mn2+ complex decreases to 4.7-fold upon binding of AMPCPP and to 8.7-fold upon binding of Mn2+.AMPCPP, further supporting a metal-bridge MutT.M2+.NTP.M2+ complex. By competition with Mn2+ MutT binds Mg2+ at one site with a Kd of 7.5 mM, and MutT.AMPCPP binds Mg2+ at two sites, the weaker of which has a Kd of 0.9 mM. These values are comparable to the kinetically determined KaMg of 15 +/- 7 mM and KmMg of 1.7 +/- 0.7 mM, respectively. Studies with the racemic, substitution-inert beta, gamma-bidentate tetraamminecobalt (III)-beta,gamma-phosphate-ATP (Co3+(NH3)4ATP) complex show that MutT slowly hydrolyzes only the lambda stereoisomer but requires Mg2+ or Mn2+ to do so, confirming a dual metal ion requirement. |
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| Keywords |
Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/metabolism; Adenosine Triphosphate/pharmacology; Bacterial Proteins/biosynthesis; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Binding, Competitive; Cations, Divalent/pharmacology; Cloning, Molecular; Electron Spin Resonance Spectroscopy; Enzyme Activation; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Genes, Bacterial; Kinetics; Magnesium/pharmacology; Magnetic Resonance Spectroscopy; Manganese/pharmacology; Phosphoric Monoester Hydrolases/biosynthesis; Phosphoric Monoester Hydrolases/isolation & purification; Phosphoric Monoester Hydrolases/metabolism; Protein Binding; Pyrophosphatases; Recombinant Proteins/biosynthesis; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism |
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