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Rat mitochondrial manganese superoxide dismutase: Amino acid positions involved in covalent modifications, activity, and heat stability
Author(s) -
Castellano Immacolata,
Cecere Francesca,
De Vendittis Alberto,
Cotugno Roberta,
Chambery Angela,
Di Maro Antimo,
Michniewicz Andzelika,
Parlato Giuseppe,
Masullo Mariorosario,
Avvedimento Enrico Vittorio,
De Vendittis Emmanuele,
Ruocco Maria Rosaria
Publication year - 2009
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.21208
Subject(s) - chemistry , thermostability , biochemistry , superoxide dismutase , enzyme , mutagenesis , amino acid , cofactor , escherichia coli , glutathione , protein engineering , mutant , gene
Abstract The role of three amino acid residues (Q143, Y34, S82) of rat mitochondrial superoxide dismutase (ratSOD2) in the enzymatic activity, thermostability, and post‐translational modification of the enzyme was investigated through site‐directed mutagenesis studies. Six recombinant forms of the enzyme were produced, carrying the Q143 or H143 residue with or without the Y34F or S82A replacement. All proteins bound manganese as active cofactor and were organized as homotetramers. The greatest effect on the activity (sixfold reduction) was observed in ratSOD2 forms containing the H143 variant, whereas Y34F and S82A substitutions moderately reduced the enzymatic activity compared to the Q143 form. Heat inactivation studies showed the high thermo‐tolerance of ratSOD2 and allowed an evaluation of the related activation parameters of the heat inactivation process. Compared to Q143, the H143 variant was significantly less heat stable and displayed moderately lower enthalpic and entropic factors; the Y34F substitution caused a moderate reduction of heat stability, whereas the S82A replacement slightly improved the thermo‐tolerance of the Q143 variant; both substitutions significantly increased enthalpic and entropic factors of heat inactivation, the greatest effect being observed with S82A substitution. All recombinant forms of ratSOD2 were glutathionylated in Escherichia coli , a feature pointing to the high reactivity of ratSOD2 toward glutathione. Moreover, the S82 position of the enzyme was phosphorylated in an in vitro system containing human mitochondrial protein extracts as source of protein kinases. These data highlight the role played by some residues in ratSOD2 and suggest a fine regulation of the enzyme occurring in vivo. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1215–1226, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com