The catalytic sites of 20S proteasomes and their role in subunit maturation:: A mutational and crystallographic study

We present a biochemical and crystallographic characterization of active site mutants of the yeast 20S proteasome with the aim to characterize substrate cleavage specificity, subunit intermediate processing, and maturation. beta 1(Pre3), beta 2(Pup1), and beta 5(Pre2) are responsible for the postacidic, tryptic, and chymotryptic activity, respectively. The maturation of active subunits is independent of the presence of other active subunits and occurs by intrasubunit autolysis. The propeptides of beta 6(Pre7) and beta 7(Pre4) are intermediately processed to their final forms by beta 2(Pup1) in the wild-type enzyme and by beta 5(Pre2) and beta 1(Pre3) in the beta 2(Pup1) inactive mutants. A role of the propeptide of beta 1(Pre3) is to prevent acetylation and thereby inactivation. A gallery of proteasome mutants that contain active site residues in the context of the inactive subunits beta 3(Pup3), beta 6(Pre7), and beta 7(Pre4) show that the presence of Gly-1, Thr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity.