DUAL DIVALENT-CATION REQUIREMENT OF THE MUTT DGTPASE - KINETIC AND MAGNETIC-RESONANCE STUDIES OF THE METAL AND SUBSTRATE COMPLEXES

Kinetic analyses of both the Mn2+- and Mg2+-activated hydrolysis of dG TP by MutT show the requirement for two divalent cations. Whereas Mn2 supports a 20-fold lower k(cat) (0.19 s-1) than Mg2+ (4.0 s-1), the K (m) of Mn2+.dGTP (6.3 mum) is 45-fold lower than that of Me2+.dGTP (28 4 mum). Adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPCPP) is a linear competitive inhibitor with respect to dGTP with a K(i) for Mn2.AMPCPP (42 muM) which is 57-fold lower than the K(i) of Mg2+.AMPCPP ( 2.4 mm). Such tightening suggests that a metal-bridge E.M2+.NTP.M2+ co mplex is the catalytically active species. The 12 dissociation constan ts describing the quaternary MutT.M2+.AMPCPP.M2+ complex were evaluate d for both Mn2+ and Mg2+, using EPR and NMR methods. MutT binds a sing le Mn2+ with a K(d) of 130 +/- 40 muM in reasonable agreement with the kinetically determined activator constant of Mn2+ of 230 +/- 72 muM. The MutT.AMPCPP complex binds two Mn2+ ions, the weaker of which has a K(d) of 16 +/- 2 muM in agreement with the kinetically determined K(m )Mn2+ of 26 +/- 10 muM. MutT.Mn2+ binds Mn2+.AMPCPP with K(d) of 16 +/ - 4 muM, whereas MutT alone binds Mn2+.AMPCPP with a K(d) of 135 +/- 3 0 muM. The 17-fold enhanced paramagnetic effect of Mn2+ on the longitu dinal 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 sit e with a K(d) of 7.5 mM, and MutT.AMPCPP binds Mg2+ at two sites, the weaker of which has a K(d) of 0.9 mm. These values are comparable to t he kinetically determined K(a)Mg of 15 +/- 7 mM and K(m)Mg of 1.7 +/- 0.7 mM, respectively. Studies with the racemic, substitution-inert bet a,gamma-bidentate tetraamminecobalt (III)-beta,gamma-phosphate-ATP (Co 3+(NH3)4ATP) complex show that MutT slowly hydrolyzes only the A stere oisomer but requires Mg2+ or Mn2+ to do so, confirming a dual metal io n requirement.