The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates (NTP) to NMP and PP(i) by nucleophilic substitution at the rarely attacked β-phosphorus. The solution structure of the quaternary E-M2+-AMPCPP-M2+ complex indicated that conserved residues Glu-53, -56, -57, and -98 are at the active site near the bound divalent cation possibly serving as metal ligands, Lys-39 is positioned to promote departure of the NMP leaving group, and Glu-44 precedes helix I (residues 47-59) possibly stabilizing this helix which contributes four catalytic residues to the active site [Lin, J., Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211]. To test these proposed roles, the effects of mutations of each of these residues on the kinetic parameters and on the Mn2+, Mg2+, and substrate binding properties were examined. The largest decreases in k(cat) for the Mg2+-activated enzyme of 104.7- and 102.6-fold were observed for the E53Q and E53D mutants, respectively, while 97-, 48-, 25-, and 14-fold decreases were observed for the E44D, E56D, E56Q, and E44Q mutations, respectively. Smaller effects on k(cat) were observed for mutations of Glu-98 and Lys-39. For wild type MutT and its E53D and E44D mutants, plots of log(k(cat)) versus pH exhibited a limiting slope of 1 on the ascending limb and then a hump, i.e., a sharply defined maximum near pH 8 followed by a plateau, yielding apparent pK(a) values of 7.6 ± 0.3 and 8.4 ± 0.4 for an essential base and a nonessential acid catalyst, respectively, in the active quaternary MutT-Mg2+-dGTP-Mg2+ complex. The pK(a) of 7.6 is assigned to Glu-53, functioning as a base catalyst in the active quaternary complex, on the basis of the disappearance of the ascending limb of the pH-rate profile of the E53Q mutant, and its restoration in the E53D mutant with a 101.9-fold increase in (k(cat))(max). The pK(a) of 8.4 is assigned to Lys-39 on the basis of the disappearance of the descending limb of the pH-rate profile of the K39Q mutant, and the observation that removal of the positive charge of Lys-39, by either deprotonation or mutation, results in the same 8.7-fold decrease in k(cat). Values of k(cat) of both wild type MutT and the E53Q mutant were independent of solvent viscosity, indicating that a chemical step is likely to be rate-limiting with both. A liganding role for Glu-53 and Glu-56, but not Glu-98, in the binary E-M2+ complex is indicated by the observation that the E53Q, E53D, E56Q, and E56D mutants bound Mn2+ at the active site 36-, 27-, 4.7-, and 1.9-fold weaker, and exhibited 2.10-, 1.50-, 1.12-, and 1.24-fold lower enhanced paramagnetic effects of Mn2+, respectively, than the wild type enzyme as detected by 1/T1 values of water protons, consistent with the loss of a metal ligand. However, the K(m) values of Mg2+ and Mn2+ indicate that Glu-56, and to a lesser degree Glu-98, contribute to metal binding in the active quaternary complex. Mutations of the more distant but conserved residue Glu-44 had little effect on metal binding or enhancement factors in the binary E - M2+ complexes. Two-dimensional 1H-15N HSQC and three- dimensional 1H-15N NOESY-HSQC spectra of the kinetically damaged E53Q and E56Q mutants showed largely intact proteins with structural changes near the mutated residues. Structural changes in the kinetically more damaged E44D mutant detected in 1H - 15N HSQC spectra were largely limited to the loop I-helix I motif, suggesting that Glu-44 stabilizes the active site region. 1H-15N HSQC titrations of the E53Q, E56Q, and E44D mutants with dGTP showed changes in chemical shifts of residues lining the active site cleft, and revealed tighter nucleotide binding by these mutants, indicating an intact substrate binding site. A mechanism is proposed in which Glu-53 coordinates the metal in the binary MutT-M2+ complex, dissociates from the metal and orients and deprotonates the attacking water ligand in the quaternary MutT-M2+-dGTP-M2+ complex, and subsequently facilitates the displacement of PP(i)-M2+ from the MutT-M2+-PP(i)-M2+ product complex. From the effects of single mutations on k(cat), the 109-fold rate acceleration produced by wild type MutT can now be explained quantitatively by the cooperative effects of the enzyme- and nucleotide-bound divalent cations, with Glu53 activating the attacking water nucleophile, and Lys-39 promoting the departure of the dGMP leaving group.
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