Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase

The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates (NTP) to NMP and PPi by nucleophilic substitution at the rarely attacked beta-pho sphorus. The solution structure of the quaternary E-M2+-AMPCPP-M2+ complex indicated that conserved residues Glu-53, -56, -57, and -98 are at the acti ve site near the bound divalent cation possibly serving as metal ligands, L ys-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., Abeygunawa rdana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemi stry 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+, Mg2and substrate binding properties were examined. The largest decreases in k( cat) for the Mg2+-activated enzyme of 10(4.7)- and 10(2.6)-fold were observ ed for the E53Q and E53D mutants, respectively, while 97-, 48-, 25-, and 14 -fold decreases were observed far 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 p lateau, yielding apparent pK(a) values of 7.6 +/- 0.3 and 8.4 +/- 0.4 for a n essential base and a nonessential acid catalyst, respectively, in the act ive 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, o n the basis of the disappearance of the ascending limb of the pH-rate profi le of the E53Q mutant, and its restoration in the E53D mutant with a 10(1.9 )-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 pi-I-rate pro file of the K39Q mutant, and the observation that removal of the positive c harge 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 chemi cal 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 t he 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+, resp ectively, than the wild type enzyme as detected by 1/T-1 values of water pr otons, consistent with the loss of a metal ligand. However, the K-a values of Mg2+ and Mn2+ indicate that Glu-56, and to a lesser degree Glu-98, contr ibute to metal binding in the active quaternary complex. Mutations of the m ore distant but conserved residue Glu-44 had little effect on metal binding or enhancement factors in the binary E-M2+ complexes. Two-dimensional H-1- N-15 HSQC and three-dimensional H-1-N-15 NOESY-HSQC spectra of the kinetica lly damaged E53Q and E56Q mutants showed largely intact proteins with struc tural changes near the mutated residues. Structural changes in the kinetically more damaged E44D mutant detected in H-1-N-15 HSQC spectra were largely limited to the loop I-helix I motif, sug gesting that Glu-44 stabilizes the active site region. H-1-N-15 HSQC titrat ions of the E53Q, E56Q, and E44D mutants with dCTP showed changes in chemic al shifts of residues lining the active site cleft, and revealed tighter nu cleotide binding by these mutants, indicating an intact substrate binding s ite. A mechanism is proposed in which Glu-53 coordinates the metal in the b inary MutT-M2+ complex, dissociates from the metal and orients and deproton ates the attacking water ligand in the quaternary MutT-M2+-dGTP-M2+ complex , and subsequently facilitates the displacement of PPi-M2+ from the MutT-M2 +-PPi-M2+ product complex. From the effects of single mutations on k(cat) t he 10(9)-fold rate acceleration produced by wild type MutT can now be expla ined quantitatively by the cooperative effects of the enzyme- and nucleotid e-bound divalent cations, with Glu-53 activating the attacking water nucleo phile, and Lys-39 promoting the departure of the dGMP leaving group.