Kinetic and Magnetic Resonance Studies of Active-Site Mutants of Staphylococcal Nuclease: Factors Contributing to Catalysis

To Determine the origin of the overall ~ 10₁₆-fold rate enhancement of DNA hydrolysis catalyzed by staphylococcal nuclease, the effects of single mutations that alter the amino acid residue at each of the essential positions Asp-21, Asp-40, Thr-41, Arg-35, and Arg-87 have been examined. Metal ion and substrate analogue binding were quantitated by EPR, by the paramagnetic effects of Mn₂₊ on 1 /T₁of water protons, and by fluorescence titrations, yielding the six dissociation constants of the ternary and complexes. The kinetic parameters were determined by monitoring the rate of DNA hydrolysis. By thermodynamic and kinetic criteria, Mn₂₊ binds tightly to the Ca₂₊ binding site of the enzyme but is at least 36 000-fold less effective than Ca₂₊ in activating the enzyme. Alterations of the liganding residues in the D40G, D40E, T41P, D21E, and D21Y mutants generally weaken the binding of Ca₂₊ ≤ 12.7-fold and of Mn₂₊ ≤ 5.4-fold, exert little effect on the or (≤3.2-fold), and raise the affinity of the enzyme and its metal complexes for 3_/,5_/-pdTp by factors ≤ 13.5-fold. Small changes in the ligand geometry are also reflected in the Mn₂₊ complexes of the liganding mutants (i.e., those in which the metal-liganding amino acids have been altered) by decreases in the electron-spin relaxation time of Mn₂₊. Inhibitory effects on k_catare noted in all of the liganding mutants with D40E, D40G, T41P, D21E, and D21Y showing 12-, 30-, 37-, 1500-, and ≥29 000-fold reductions, respectively. The ≥10₃-fold larger inhibitory effects on k_catof enlarging Asp-21 as compared to enlarging Asp-40 are ascribed to the displacement of the adjacent water molecule which attacks the phosphodiester. Mutations of each of the essential Arg residues to Gly (R35G and R87G) reduce K_cat by factors ≥35 000 but weaken metal binding ≤9-fold. While R35G and its metal complexes bind 3_/,5_/-pdTp only very weakly, consistent with Arg-35 functioning as a hydrogen-bond donor to the phosphodiester substrate, the identical alteration of Arg-87 does not significantly change the affinity of the enzyme or its metal complexes for the substrate analogue 3_/,5_/-pdTp, in contrast to its described role in binding either 3_/,5_/-pdTp or the phosphodiester substrate. A revised mechanism for staphylococcal nuclease is proposed in which Arg-87 interacts only with the trigonal bipyramidal transition state rather than with the ground state of the bound substrate. Since the active-site mutations studied reduce k_catby factors approaching 10₅, and yet alter metal ion and substrate analogue binding by at most 1 order of magnitude (except for R35G), we argue that these mutations do not greatly perturb the native conformation of the enzyme and that their effects on K_cat can therefore be interpreted quantitatively in terms of specific changes in the chemistry of the catalytic events. Hence, the 10₁₆-fold rate acceleration produced by staphylococcal nuclease may result from the product of the following factors: metal catalysis by Ca₂₊ (≥10_4,6); transition-state stabilization by Arg-87 (≥10_4,6); catalysis by approximation of the attacking water (≥10₃); and general base catalysis (~10₄).