The conserved residue tyrosine 34 is essential for maximal activity of iron-superoxide dismutase from Escherichia coli.
We have expressed, purified, and analyzed the iron-containing superoxide dismutase (FeSOD) of Escherichia coli with mutations directed at tyrosine position 34 to introduce phenylalanine (SODY34F), serine (SODY34S), or cysteine (SODY34C). FeSOD and mutant enzymes were purified from SOD-deficient cells using a GST-FeSOD fusion protein intermediate which was subsequently cleaved with thrombin and repurified. Specific activities were measured using the xanthine-xanthine oxidase method and gave 3148 u/mg for wild-type FeSOD. The SODY34S mutation virtually inactivates the enzyme (42 u/mg); mutation to cysteine greatly reduces activity (563 u/mg), but the SODY34F mutant retains nearly 40% of the activity of wild type (1205 u/mg). Fusion protein intermediates were also shown to be active and were demonstrated to protect SOD-deficient E. coli cells from the induced effects of oxidative stress, with growth rates directly proportional to the specific activities of the expressed mutant enzymes. SODY34F exhibited decreased thermal stability, reduced activity at high pH, and a pronounced increase in sensitivity to the inhibitor sodium azide compared with wild-type FeSOD. These results suggest that tyrosine at position 34 is multifunctional and plays a structural role (probably through hydrogen bonding to glutamine at position 69) in maintaining the integrity of the active site, a stabilizing role at high pH, and a steric role in obstructing access to the active site of both substrate and inhibitor molecules.
Hunter
T
T
Department of Physiology and Biochemistry, University of Malta, Msida.
Ikebukuro
K
K
Bannister
W H
WH
Bannister
J V
JV
Hunter
G J
GJ
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D015183:Restriction Mapping; D012694:Serine; D013329:Structure-Activity Relationship; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D013482:Superoxide Dismutase; D003545:Cysteine