Abstract
BackgroundHuman extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thøgersen IB, Højrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24):13875–80). One variant is enzymatically active (aEC-SOD) while the other is inactive (iEC-SOD). The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (aa, ai or ii) with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist.ResultsThe analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii) and a hetero-dimer (ai). Because EC-SOD is a tetramer the dimers may combine to generate 5 different mature EC-SOD molecules where the specific activity of each molecule is determined by the ratio of aEC-SOD and iEC-SOD subunits.ConclusionThis finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity. This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity.
Highlights
Human extracellular superoxide dismutase (EC-Superoxide dismutase enzymes (SOD)) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space
This result shows that the extracellular superoxide dismutase (EC-SOD) dimer exists in three distinct populations which can be separated by SDS-PAGE following deglycosylation
The level of SOD activity in the extracellular space can be regulated by varying the ratio of aEC-SOD and iEC-SOD subunits in the tetramer
Summary
Human extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. The N-terminal region of EC-SOD is involved in hydrophobic inter-subunit interactions stabilizing the ECSOD tetramer [12,13] The importance of these interactions has been emphasized by the finding that the dimeric rat EC-SOD [14], is converted to a tetramer by substituting a hydrophilic residue (Asp) within the N-terminal region for a hydrophobic one (Val) [15]. That this region can be proteolytically removed before secretion in a two-step process involving both an endoproteinase and a carboxypeptidase [20,21,22] This cleavage event changes the affinity for extracellular matrix (ECM) components and affects the biodistribution of the protein. When mature EC-SOD is analyzed under denaturing conditions, cleaved subunits will appear as monomers while intact subunits will be dimers [23]
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