Human mitochondrial manganese superoxide dismutase (MnSOD) catalyzes the dismutation of the superoxide radical anion 2O2(-) + 2H+ -> O2 + H2O2. This catalysis requires cycles of both oxidation and reduction at the metal followed by proton transfers from solution to the active site to release product peroxide. Reactive oxygen species are normally and continuously produced by numerous intracellular processes, including the mitochondrial electron transport chain, and MnSOD is extremely important as a main line of defense against oxidative damage associated with the inflammatory response and postischemic reperfusion of organs. The unifying goal of this proposal is to elucidate the role of active site residues in the catalytic mechanism of human MnSOD, and to emphasize ways to enhance the catalytic activity of MnSOD. The motivation to enhance the efficiency of MnSOD is both to understand the catalysis and to introduce clinical possibilities for gene therapy. The applicant plans structure-function studies using site-directed mutagenesis to alter residues near the active site. Stopped-flow spectrophotometry and pulse radiolysis will be used to evaluate changes in catalysis and x-ray crystallography will be used to detect structural changes. The crystal structure for human MnSOD is known. Investigators have already developed an expression system and prepared wild-type human MnSOD and two significant mutants. Activation will be achieved through several strategies some of which have been useful in other enzymes, particularly the Cu, ZnSOD and carbonic anhydrase: i) enhancement of the proton transfer mechanisms by providing intra- and intermolecular shuttle groups, ii) overcoming product (peroxide) inhibition by altering the active site region through mutagenesis, and iii) enhancement of electrostatic guidance of substrate by altering the charge character near the active site.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054903-02
Application #
2519077
Study Section
Biochemistry Study Section (BIO)
Project Start
1996-09-01
Project End
2000-08-31
Budget Start
1997-09-01
Budget End
1998-08-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Florida
Department
Pharmacology
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
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Quint, Patrick S; Domsic, John F; Cabelli, Diane E et al. (2008) Role of a glutamate bridge spanning the dimeric interface of human manganese superoxide dismutase. Biochemistry 47:4621-8
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Quint, Patrick; Reutzel, Robbie; Mikulski, Rose et al. (2006) Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation. Free Radic Biol Med 40:453-8
Chockalingam, Karuppiah; Luba, James; Nick, Harry S et al. (2006) Engineering and characterization of human manganese superoxide dismutase mutants with high activity and low product inhibition. FEBS J 273:4853-61
Quint, Patrick; Ayala, Idelisa; Busby, Scott A et al. (2006) Structural mobility in human manganese superoxide dismutase. Biochemistry 45:8209-15
Ayala, Idelisa; Perry, J Jefferson P; Szczepanski, Jan et al. (2005) Hydrogen bonding in human manganese superoxide dismutase containing 3-fluorotyrosine. Biophys J 89:4171-9
Abreu, Isabel A; Rodriguez, Jose A; Cabelli, Diane E (2005) Theoretical studies of manganese and iron superoxide dismutases: superoxide binding and superoxide oxidation. J Phys Chem B 109:24502-9

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