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

Engin H. Serpersu, David Shortle, Albert S. Mildvan

Research output: Contribution to journalArticlepeer-review

Abstract

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 kcatare 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 kcatof 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 Kcat 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 kcatby 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 Kcat 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+ (≥104,6); transition-state stabilization by Arg-87 (≥104,6); catalysis by approximation of the attacking water (≥103); and general base catalysis (~104).

Original languageEnglish (US)
Pages (from-to)1289-1300
Number of pages12
JournalBiochemistry
Volume26
Issue number5
DOIs
StatePublished - 1987

ASJC Scopus subject areas

  • Biochemistry

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