Rat mitochondrial manganese superoxide dismutase: amino acid positions involved in covalent modifications, activity, and heat stability.
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.
Castellano
Immacolata
I
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy.
Cecere
Francesca
F
De Vendittis
Alberto
A
Cotugno
Roberta
R
Chambery
Angela
A
Di Maro
Antimo
A
Michniewicz
Andzelika
A
Parlato
Giuseppe
G
Masullo
Mariorosario
M
Avvedimento
Enrico Vittorio
EV
De Vendittis
Emmanuele
E
Ruocco
Maria Rosaria
MR
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D004795:Enzyme Stability; D005973:Glutamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000099156:Superoxide Dismutase 2
D000596:Amino Acids; D005973:Glutamine; D008345:Manganese; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D012694:Serine; D013438:Sulfhydryl Compounds; D013482:Superoxide Dismutase; D014443:Tyrosine; D000099156:Superoxide Dismutase 2