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 REG alpha activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REG gamma only activates cleavage after basic residues. We have isolated REG gamma mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome beta subunits, The most robust REG gamma specificity mutants involve substitution of Glu or Asp for Lys188. REG gamma (K188E/D) variants are virtually identical to REG alpha in proteasome activation but assemble into less stable heptamers/hexamers. Based on the REG alpha crystal structure, Lys188 of REG gamma faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsinlike subunits by I-125-YL3-VS demonstrates that REG gamma (K188E)'s activation of all three proteasome active sites is not due to relaxed gating, We propose that decreased stability of REG gamma (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 REG gamma molecule.