The mechanism of the phosphodiesterase reaction catalyzed by staphylococcal nuclease is believed to involve concerted general acid-base catalysis by Arg-87 and Glu-43. The mutual interactions of Arg-87 and Glu-43 were investigated by comparing kinetic and thermodynamic properties of the single mutant enzymes E43S (Glu-43 to Ser) and R87G (Arg-87 to Gly) with those of the double mutant, E43S + R87G, in which both the basic and acidic functions have been inactivated. Denaturation studies with guanidinium chloride, CD, and 600-MHz ID and 2D proton NMR spectra, indicate all enzyme forms to be predominantly folded in absence of the denaturant and reveal small antagonistic effects of the E43S and R87G mutations on the stability and structure of the wild-type enzyme. The free energies of binding of the divalent cation activator Ca2+, the inhibitor Mn2+, and the substrate analogue 3′,5′-pdTp show simple additive effects of the two mutations in the double mutant, indicating that Arg-87 and Glu-43 act independently to facilitate the binding of divalent cations and of 3′,5′-pdTp by the wild-type enzyme. The free energies of binding of the substrate, 5′-pdTdA, both in binary E-S and in active ternary E-Ca2+-S complexes, show synergistic effects of the two mutations, suggesting that Arg-87 and Glu-43 interact anticooperatively in binding the substrate, possibly straining the substrate by 1.6 kcal/mol in the wild-type enzyme. The large free energy barriers to introduced by the R87G mutation (ΔG1* = 6.5 kcal/mol) and by the E43S mutation (ΔG2* = 5.0 kcal/mol) are partially additive in the double mutant (ΔG*1+2 = 8.1 kcal/mol). These partially additive effects on Vmax are most simply explained by a cooperative component to transition state binding by Arg-87 and Glu-43 of -3.4 kcal/mol. The combination of anticooperative, cooperative, and noncooperative effects of Arg-87 and Glu-43 together lower the kinetic barrier to catalysis by 8.1 kcal/mol.
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