A novel, arsenite-sensitive E2 of the ubiquitin pathway: purification and properties

In the multienzyme ubiquitin-dependent proteolytic pathway, conjugation of ubiquitin to target proteins serves as a signal for protein degradation. Rabbit reticulocytes possess a family of proteins, known as E2's, that form labile ubiquitin adducts by undergoing transthiolation with the ubiquitin thiol ester form of ubiquitin activating enzyme (E1). Only one E2 appears to function in ubiquitin-dependent protein degradation. The others have been postulated to function in regulatory ubiquitin conjugation. We have purified and characterized a previously undescribed E2 from rabbit reticulocytes. E2(230K) is an apparent monomer with a molecular mass of 230 kDa. The enzyme forms a labile ubiquitin adduct in the presence of E1, ubiquitin, and MgATP and catalyzes conjugation of ubiquitin to protein substrates. Exogenous protein substrates included yeast cytochrome c(Km = 125 mu M; kcat approximately 0.37 min-1) and histone H3 (Km less than 1.3 mu M; kcat approximately 0.18 min-1) as well as lysozyme, alpha-lactalbumin, and alpha-casein. E2(230K) did not efficiently reconstitute Ub-dependent degradation of substrates that it conjugated, either in the absence or in the presence of the ubiquitin-protein ligase that is involved in degradation. E2(230K) may thus be an enzyme that functions in regulatory Ub conjugation. Relative to other E2's, which are very iodoacetamide sensitive, E2(230K) was more slowly inactivated by iodoacetamide (k(obs) = 0.037 min-1 at 1.5 mM iodoacetamide; pH 7.0, 37 degrees C). E2(230K) was also unique among E2's in being subject to inactivation by inorganic arsenite (k(i)max = 0.12 min-1; K(0.5) = 3.3 mM; pH 7.0, 37 degrees C). Arsenite is considered to be a reagent specific for vicinal sulfhydryl sites in proteins, and inhibition is usually rapidly reversed upon addition of competitive dithiol compounds. Inactivation of E2(230K) by arsenite was not reversed within 10 min after addition of dithiothreitol at a concentration that blocked inactivation if it was premixed with arsenite; inactivation is therefore irreversible or very slowly reversible. We postulate that a conformation change of E2(230K) may be rate-limiting for interaction of enzyme thiol groups with arsenite.