1fun
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SUPEROXIDE DISMUTASE MUTANT WITH LYS 136 REPLACED BY GLU, CYS 6 REPLACED BY ALA AND CYS 111 REPLACED BY SER (K136E, C6A, C111S)
OverviewOverview
Key charged residues in Cu,Zn superoxide dismutase (Cu,Zn SOD) promote, electrostatic steering of the superoxide substrate to the active site Cu, ion, resulting in dismutation of superoxide to oxygen and hydrogen, peroxide, Lys-136, along with the adjacent residues Glu-132 and Glu-133, forms a proposed electrostatic triad contributing to substrate, recognition. Human Cu,Zn SODs with single-site replacements of Lys-136 by, Arg,Ala, Gln, or Glu or with a triple-site substitution (Glu-132 and, Glu-133 to Gln and Lys-136 to Ala) were made to test hypotheses regarding, contributions of these residues to Cu,Zn SOD activity. The structural, effects of these mutations were modeled computationally and validated by, the X-ray crystallographic structure determination of Cu,Zn SOD having the, Lys-136-to-Glu replacement. Brownian dynamics simulations and, multiple-site titration calculations predicted mutant reaction rates as, well as ionic strength and pH effects measured by pulse-radiolytic, experiments. Lys-136-to-Glu charge reversal decreased dismutation activity, 50% from 2.2 x 10(9) to 1.2 x 10(9) M-1 s-1 due to repulsion of negatively, charged superoxide, whereas charge-neutralizing substitutions (Lys-136 to, Gln or Ala) had a less dramatic influence. In contrast, the triple-mutant, Cu,Zn SOD (all three charges in the electrostatic triad neutralized), surprisingly doubled the reaction rate compared with wild-type enzyme but, introduced phosphate inhibition. Computational and experimental reaction, rates decreased with increasing ionic strength in all of the Lys-136, mutants, with charge reversal having a more pronounced effect than charge, neutralization, implying that local electrostatic effects still govern the, dismutation rates. Multiple-site titration analysis showed that, deprotonation events throughout the enzyme are likely responsible for the, gradual decrease in SOD activity above pH 9.5 and predicted a pKa value of, 11.7 for Lys-136. Overall, Lys-136 and Glu-132 make comparable, contributions to substrate recognition but are less critical to enzyme, function than Arg-143, which is both mechanistically and electrostatically, essential. Thus, the sequence-conserved residues of this electrostatic, triad are evidently important solely for their electrostatic properties, which maintain the high catalytic rate and turnover of Cu,Zn SOD while, simultaneously providing specificity by selecting against binding by other, anions.
DiseaseDisease
Known disease associated with this structure: Amyotrophic lateral sclerosis, due to SOD1 deficiency OMIM:[147450]
About this StructureAbout this Structure
1FUN is a Single protein structure of sequence from Homo sapiens with CU, ZN and SO4 as ligands. Active as Superoxide dismutase, with EC number 1.15.1.1 Full crystallographic information is available from OCA.
ReferenceReference
Computational, pulse-radiolytic, and structural investigations of lysine-136 and its role in the electrostatic triad of human Cu,Zn superoxide dismutase., Fisher CL, Cabelli DE, Hallewell RA, Beroza P, Lo TP, Getzoff ED, Tainer JA, Proteins. 1997 Sep;29(1):103-12. PMID:9294870
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