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

1999, Proceedings of the National Academy of Sciences

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. ␤1(Pre3), ␤2(Pup1), and ␤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 ␤6(Pre7) and ␤7(Pre4) are intermediately processed to their final forms by ␤2(Pup1) in the wild-type enzyme and by ␤5(Pre2) and ␤1(Pre3) in the ␤2(Pup1) inactive mutants. A role of the propeptide of ␤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 ␤3(Pup3), ␤6(Pre7), and ␤7(Pre4) show that the presence of Gly-1, Thr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity.

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