Mutational, Kinetic, and NMR Studies of the Mechanism of E. coli GDP-Mannose Mannosyl Hydrolase, an Unusual Nudix Enzyme

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• 作者：Patricia M. Legler ; Michael A. Massiah ; and Albert S. Mildvan
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：September 3, 2002
• 年：2002
• 卷：41
• 期：35
• 页码：10834 - 10848
• 全文大小：265K
• 年卷期：v.41,no.35(September 3, 2002)
• ISSN：1520-4995

文摘

GDP-mannose mannosyl hydrolase (GDPMH) is an unusual Nudix family member, whichcatalyzes the hydrolysis of GDP-s/gifchars/alpha.gif" BORDER=0>-D-mannose to GDP and the s/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-sugar by nucleophilic substitution atcarbon 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 Nudixenzyme 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 whichdecreased k_cat 10^2.2-fold, increased the dissociation constant of Mn²⁺ from the ternary E-Mn²⁺-GDPcomplex 3-fold, increased the K_m^Mg2+ 20-fold, and decreased the paramagnetic effect of Mn²⁺ on 1/T₁ ofwater protons, indicating a change in the coordination sphere of Mn²⁺. In the E70Q mutant, Gln-70 wasshown to be very near the active site metal ion by large paramagnetic effects of Mn²⁺ on its side chain-NH₂ group. With wild-type GDPMH, the effect of pH on log(k_cat/K_m^GDPmann) at 37 s/entities/deg.gif">C showed an ascendinglimb of unit slope, followed by a plateau yielding a pK_a of 6.4, which increased to 6.7 ± 0.1 in the pHdependence of log(k_cat). The general base catalyst was identified as a neutral His residue by the s/gifchars/Delta.gif" BORDER=0 >H^ionization= 7.0 ± 0.7 kcal/mol, by the increase in pK_a with ionic strength, and by mutation of each of the fourhistidine residues of GDPMH to Gln. Only the H124Q mutant showed the loss of the ascending limb inthe pH versus log(k_cat) rate profile, which was replaced by a weak dependence of rate on hydroxideconcentration, as well as an overall 10^3.4-fold decrease in k_cat, 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 k_cat, a4.4-fold increase in K_m^GDPmann, and no change in the pH versus log(k_cat) rate profile, indicating an importantbut unidentified role of His-88 in catalysis. One and two-dimensional NMR studies permitted the sequencespecific assignments of the imidazole Hs/gifchars/delta.gif" BORDER=0 >C, Hs/gifchars/epsilon.gif" BORDER=0 >C, Ns/gifchars/delta.gif" BORDER=0 >, and Ns/gifchars/epsilon.gif" BORDER=0 > resonances of the four histidines and definedtheir protonation states. The pK_a of His-124 (6.94 ± 0.04) in the presence of saturating Mg²⁺ wascomparable to the kinetically determined pK_a at the same temperature (6.40 ± 0.20). The other threehistidines were neutral Ns/gifchars/epsilon.gif" BORDER=0 >H tautomers with pK_a values below 5.5. Arg-52 and Arg-65 were identified ascatalytic residues which interact electrostatically with the GDP leaving group by mutating these residuesto Gln and Lys. The R52Q mutant decreased k_cat 309-fold and increased K_m^GDPmann 40.6-fold, while theR52K mutant decreased k_cat by only 12-fold and increased K_m^GDPmann 81-fold. The partial rescue of k_cat,but not of K_m^GDPmann in the R52K mutant, suggests that Arg-52 is a bifunctional hydrogen bond donor tothe GDP leaving group in the ground state and a monofunctional hydrogen bond donor in the transitionstate. Opposite behavior was found with the Arg-65 mutants, suggesting this residue to be a monofunctionalhydrogen bond donor to the GDP leaving group in the ground state and a bifunctional hydrogen bonddonor in the transition state. From these observations, a mechanism for GDPMH is proposed involvinggeneral base catalysis and electrostatic stabilization of the leaving group.