PROTEASOME FROM THERMOPLASMA-ACIDOPHILUM - A THREONINE PROTEASE

The catalytic mechanism of the 20S proteasome from the archaebacterium Thermoplasma acidophilum has been analyzed by site-directed mutagenes is of the beta subunit and by inhibitor studies. Deletion of the amino -terminal threonine or its mutation to alanine led to inactivation of the enzyme. Mutation of the residue to serine led to a fully active en zyme, which was over ten times more sensitive to the serine protease i nhibitor 3,4-dichloroisocoumarin. In combination with the crystal stru cture of a proteasome-inhibitor complex, the data show that the nucleo philic attack is mediated by the amino-terminal threonine of processed beta subunits. The conservation pattern of this residue in eukaryotic sequences suggests that at least three of the seven eukaryotic beta-t ype subunit branches should be proteolytically inactive.