ACTIVATION OF THE MULTICATALYTIC ENDOPEPTIDASE BY OXIDANTS - EFFECTS ON ENZYME STRUCTURE

It is well established that the functional properties of proteins can be compromised by oxidative damage and, in vivo, proteins modified by oxidants are rapidly degraded, It was hypothesized that oxidants may a lso affect the ability of proteases to hydrolyze peptides and proteins . We therefore examined the effect of oxidants on the endopeptidase ac tivities of the 650 kDa 20S proteasome or multicatalytic endopeptidase (MCP), which is thought to play a central role in nonlysosomal protei n breakdown. Treatment of the MCP with the oxidant system, FeSO4-EDTA- ascorbate, stimulated the peptidase activities of the MCP while H2O2 t reatment showed little or no stimulation, However, treatment of the MC P with FeSO4-EDTA-ascorbate or H2O2 stimulated proteinase activity by 480% and 730%, respectively. An endogenous activator of the MCP, PA28, stimulated the acidic, basic, and hydrophobic peptidase activities of the MCP, but had no effect on proteolytic activity. Treatment of PA28 with oxidants in the presence of MCP or alone did not greatly affect PA28's ability to activate the peptidase activities of the MCP, Using nondenaturing polyacrylamide gel electrophoresis, structural alteratio ns in the enzyme which may be responsible for the activation of peptid ase and protease activities following exposure to oxidants were invest igated. Treatment of the MCP with reagents that activate proteolysis, including H2O2, as well as the serine protease inhibitor 3,4-dichloroi socoumarin and the cysteine protease inhibitor p-(chloromercuri)benzen esulfonic acid, all caused dissociation of the 650 kDa MCP, However, e xposure to FeSO4-EDTA-ascorbate resulted in little or no dissociation of the complex. The MCP complex dissociated by p-(chloromercuri)benzen esulfonic rid could be reassociated upon treatment with the reducing a gent dithiothreitol, but dithiothreitol failed to completely reassocia te 3,4-dichloroisocoumarin- or H2O2-treated MCP, Therefore, chemical m odification of the MCP can cause activation with varying degrees of co mplex dissociation. These results suggest that metabolites, such as re active oxygen species, in addition to endogenous proteins, such as PA2 8, are capable of modulating MCP activity.