Amino acid substitution at the dimeric interface of human manganese superoxide dismutase.
The side chains of His30 and Tyr166 from adjacent subunits in the homotetramer human manganese superoxide dismutase (Mn-SOD) form a hydrogen bond across the dimer interface and participate in a hydrogen-bonded network that extends to the active site. Compared with wild-type Mn-SOD, the site-specific mutants H30N, Y166F, and the corresponding double mutant showed 10-fold decreases in steady-state constants for catalysis measured by pulse radiolysis. The observation of no additional effect upon the second mutation is an example of cooperatively interacting residues. A similar effect was observed in the thermal stability of these enzymes; the double mutant did not reduce the major unfolding transition to an extent greater than either single mutant. The crystal structures of these site-specific mutants each have unique conformational changes, but each has lost the hydrogen bond across the dimer interface, which results in a decrease in catalysis. These same mutations caused an enhancement of the dissociation of the product-inhibited complex. That is, His30 and Tyr166 in wild-type Mn-SOD act to prolong the lifetime of the inhibited complex. This would have a selective advantage in blocking a cellular overproduction of toxic H2O2.
Hearn
Amy S
AS
Departments of Pharmacology and Neuroscience, University of Florida, Gainesville, Florida 32610-0267, USA.
Fan
Li
L
Lepock
James R
JR
Luba
James P
JP
Greenleaf
William B
WB
Cabelli
Diane E
DE
Tainer
John A
JA
Nick
Harry S
HS
Silverman
David N
DN
D000596:Amino Acids / Q000737:chemistry*; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D017510:Protein Folding; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000737:chemistry*; D013997:Time Factors; D014961:X-Ray Diffraction
D000596:Amino Acids; D006639:Histidine; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase