Lysine 188 substitutions convert the pattern of proteasome activation by REGγ to that of REGs α and β

11S REGs (PA28s) are multimeric rings that bind proteasomes and stimulate peptide hydrolysis. Whereas REGα activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REGγ only activates cleavage after basic residues. We have isolated REGγ mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome β subunits. The most robust REGγ specificity mutants involve substitution of Glu or Asp for Lys188. REGγ (K188E/D) variants are virtually identical to REGα in proteasome activation but assemble into less stable heptamers/hexamers. Based on the REGα crystal structure, Lys188 of REGγ faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsin-like subunits by (125)I-YL3-VS demonstrates that REGγ (K188E)’s activation of all three proteasome active sites is not due to relaxed gating. We propose that decreased stability of REGγ (K188E) heptamers allows them to change conformation upon proteasome binding, thus relieving inhibition of the CT and PGPH sites normally imposed by the wild-type REGγ molecule.