Mutational, kinetic, and NMR studies of the mechanism of E. coli GDP-mannose mannosyl hydrolase, an unusual Nudix enzyme.

GDP-mannose mannosyl hydrolase (GDPMH) is an unusual Nudix family member, which catalyzes the hydrolysis of GDP-R-D-mannose to GDP and the ‚-sugar by nucleophilic substitution at carbon rather than at phosphorus (Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Biochemistry 39, 8603-8608). Using the structure and mechanism of MutT, the prototypical Nudix enzyme as a guide, we detected six catalytic residues of GDPMH, three of which were unique to GDPMH, by the kinetic and structural effects of site-specific mutations. Glu-70 (corresponding to Glu-57 in MutT) provides a ligand to the essential divalent cation on the basis of the effects of the E70Q mutation which decreased kcat 10 2.2 -fold, increased the dissociation constant of Mn 2+ from the ternary E-Mn 2+ -GDP complex 3-fold, increased the Km Mg2+ 20-fold, and decreased the paramagnetic effect of Mn 2+ on 1/T1 of water protons, indicating a change in the coordination sphere of Mn 2+ . In the E70Q mutant, Gln-70 was shown to be very near the active site metal ion by large paramagnetic effects of Mn 2+ on its side chain -NH2 group. With wild-type GDPMH, the effect of pH on log(kcat/Km GDPmann )a t 37°C showed an ascending limb of unit slope, followed by a plateau yielding a pKa of 6.4, which increased to 6.7 ( 0.1 in the pH dependence of log(kcat). The general base catalyst was identified as a neutral His residue by the ¢H ionization ) 7.0 ( 0.7 kcal/mol, by the increase in pKa with ionic strength, and by mutation of each of the four histidine residues of GDPMH to Gln. Only the H124Q mutant showed the loss of the ascending limb in the pH versus log(kcat) rate profile, which was replaced by a weak dependence of rate on hydroxide concentration, as well as an overall 10 3.4 -fold decrease in kcat, indicating His-124 to be the general base, unlike MutT, which uses Glu-53 in this role. The H88Q mutant showed a 10 2.3 -fold decrease in kcat ,a 4.4-fold increase in Km GDPmann , and no change in the pH versus log(kcat) rate profile, indicating an important but unidentified role of His-88 in catalysis. One and two-dimensional NMR studies permitted the sequence specific assignments of the imidazole H‰C, HC, N‰, and N resonances of the four histidines and defined their protonation states. The pKa of His-124 (6.94 ( 0.04) in the presence of saturating Mg 2+ was comparable to the kinetically determined pKa at the same temperature (6.40 ( 0.20). The other three histidines were neutral NH tautomers with pKa values below 5.5. Arg-52 and Arg-65 were identified as catalytic residues which interact electrostatically with the GDP leaving group by mutating these residues to Gln and Lys. The R52Q mutant decreased kcat 309-fold and increased Km GDPmann 40.6-fold, while the R52K mutant decreased kcat by only 12-fold and increased Km GDPmann 81-fold. The partial rescue of kcat, but not of Km GDPmann in the R52K mutant, suggests that Arg-52 is a bifunctional hydrogen bond donor to the GDP leaving group in the ground state and a monofunctional hydrogen bond donor in the transition state. Opposite behavior was found with the Arg-65 mutants, suggesting this residue to be a monofunctional hydrogen bond donor to the GDP leaving group in the ground state and a bifunctional hydrogen bond donor in the transition state. From these observations, a mechanism for GDPMH is proposed involving general base catalysis and electrostatic stabilization of the leaving group.