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 si te mutants of the yeast 20S proteasome with the aim to characterize substra te cleavage specificity, subunit intermediate processing, and maturation. b eta 1(Pre3), beta 2(Pup1), and beta 5(Pre2) are responsible for the postaci dic, tryptic, and chymotryptic activity, respectively. The maturation of ac tive subunits is independent of the presence of other active subunits and o ccurs 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(Pu p1) inactive mutants. A role of the propeptide of beta 1(Pre3) is to preven t acetylation and thereby inactivation. A gallery of proteasome mutants tha t 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, T hr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity.