Thermostabilization of recombinant human and bovine CuZn superoxide dismutases by replacement of free cysteines.
Human CuZn superoxide dismutase (HSOD) has two free cysteines: a buried cysteine (Cys6) located in a beta-strand, and a solvent accessible cysteine (Cys111) located in a loop region. The highly homologous bovine enzyme (BSOD) has a single buried Cys6 residue. Cys6 residues in HSOD and BSOD were replaced by alanine and Cys111 residues in HSOD by serine. The mutant enzymes were expressed and purified from yeast and had normal specific activities. The relative resistance of the purified proteins to irreversible inactivation of enzymatic activity by heating at 70 degrees C was HSOD Ala6 Ser111 greater than BSOD Ala6 Ser109 greater than BSOD Cys6 Ser109 (wild type) greater than HSOD Ala6 Cys111 greater than HSOD Cys6 Ser111 greater than HSOD Cys111 (wild type). In all cases, removal of a free cysteine residue increased thermostability.
Hallewell
R A
RA
Chiron Corporation, Emeryville, CA 94608.
Imlay
K C
KC
Lee
P
P
Fong
N M
NM
Gallegos
C
C
Getzoff
E D
ED
Tainer
J A
JA
Cabelli
D E
DE
Tekamp-Olson
P
P
Mullenbach
G T
GT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004795:Enzyme Stability; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics
D011994:Recombinant Proteins; D004228:Dithionitrobenzoic Acid; D013482:Superoxide Dismutase; D003545:Cysteine