Autocatalytic processing of the 20S proteasome

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Abstract
THE Ntn (N-terminal nucleophile) hydrolases are enzymes with an unusual four-layer α+β fold1–5. The amino-terminal residue (cysteine, serine or threonine) of the mature protein is the catalytic nucleophile6–10, and its side chain is activated for nucleophilic attack by transfer of its proton to the free N terminus2, although other active-site residues may also be involved4,8. The four currently known Ntn hydrolases (glutamine PRPP amidotransferase1,6, penicillin acylase2,7, the 20S proteasome3,8,9 and aspartylglucosaminidase4,10) are encoded as inactive precursors, and are activated by cleavage of the peptide bond preceding the catalytic residue. It has been suggested that auto-catalytic processing is a common feature of Ntn hydrolases, and proceeds by an intramolecular mechanism determined by their common fold5. Here we show that propeptide processing in the proteasome from Thermoplasma acidophilum is indeed autocatalytic, but is probably intermolecular. Processing is not required for assembly, is largely unaffected by propeptide length and sequence, and occurs before β-subunit folding is completed. Although serine is an acceptable active-site nucleophile for proteolysis, and cysteine for processing, only threonine is fully functional in both. This explains why threonine is universally conserved in active proteasome subunits.