Covalent modification of the active site threonine of proteasomal beta subunits and the Escherichia coli homolog HsIV 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-[L.sub.3]VS) 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-[L.sub.3]VS and a 125I-labeled nitrophenol derivative (125I-NIP-[L.sub.3]VS) 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 N[H.sub.2] terminus. 125I-NIP-[L.sub.3]VS covalently modifies the HslV subunit of the Escherichia coli protease complex HslV/HslU, a reaction that requires ATP, and supports a catalytic mechanism shared with that of the eukaryotic proteasome.