Covalent modification of the active site threonine of proteasomal beta subunits and the Escherichia coli homolog HslV by a new class of inhibitors

The proteasome is a multicatalytic protease complex that plays a key role in diverse cellular functions, The peptide vinyl sulfone, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) covalently inhibits the trypsin-like, chymotrypsin-like and, unlike lactacystin, also the peptidyl-glutamyl peptidase activity in isolated proteasomes, and blocks their function in living cells, Although described as a class of mechanism-based inhibitors for cysteine proteases, the peptide vinyl sulfone Z-L3VS and a I-125-labeled nitrophenol derivative (I-125-NIP-L3VS) covalently modify the active site threonine of the catalytic beta subunits of the proteasome, Modification of Thermoplasma proteasomes demonstrates the requirement for a hydroxyl amino acid (threonine, serine) as nucleophile at the beta subunit's NH2 terminus, I-125-NIP-L3VS covalently modifies the HsIV subunit of the Escherichia coli protease complex HsIV/HsIU, a reaction that requires ATP, and supports a catalytic mechanism shared with that of the eukaryotic proteasome.