EFFECTS OF NUCLEOTIDES ON ASSEMBLY OF THE 26S PROTEASOME AND DEGRADATION OF UBIQUITIN CONJUGATES

We have investigated three aspects of nucleotide usage by the 26S prot easome and its regulatory complex (RC). Both particles hydrolyze the f our major ribonucleotides, but ATP and CTP have substantially lower K( m)s for hydrolysis than do GTP and UTP. The K-m for ATP hydrolysis is 15 mu m for the 26S proteasome and 30 mu(m) for the regulatory complex . Formation of the 26S proteasome from the RC and the 20S proteasome r equires about 5 mu m ATP. Although measurable degradation of Ubiquitin (Ub)-lysozyme conjugates occurs in the presence of CTP, GTP, and UTP, the best nucleotide for Ub-conjugate degradation by the 26S proteasome is ATP, with an estimated K-m of 12 mu m In summary, our studies show that micromolar concentrations of ATP are sufficient for several 26S proteasome activities.