NUCLEOTIDASE ACTIVITIES OF THE 26 S PROTEASOME AND ITS REGULATORY COMPLEX

The 26 S proteasome can be assembled from the multicatalytic protease (or 20 S proteasome) and a large multisubunit regulatory complex in an ATP-dependent reaction. The 26 S proteasome and its regulatory comple x were purified from rabbit reticulocytes for characterization of thei r nucleotidase properties. Both particles hydrolyze ATP, CTP, GTP, and UTP to the corresponding nucleoside diphosphate and inorganic phospha te. The K-m values for hydrolysis of specific nucleotides by the 26 S proteasome are 15 mu M for ATP and CTP, 50 mu M for GTP, and 100 mu M for UTP; K-m values for nucleotide hydrolysis by the regulatory comple x are 2-4-fold higher for each nucleotide. Several ATPase inhibitors ( erythro-9-[3-(2-hydroxynonyl)] adenine, oligo-mycin, ouabain, and thap sigargin) had no effect on ATP hydrolysis by either complex whereas kn own inhibitors of proteolysis by the 26 S enzyme (hemin, N-ethylmale i mide (NEM), and vanadate) significantly reduced ATP hydrolysis by both particles. Hydrolysis of all nucleotides was inhibited by 5 mM NEM bu t only GTP and UTP hydrolysis was significantly reduced at 50 mu M NEM , The 15 mu M K-m for ATP hydrolysis by the 26 S proteasome is virtual ly identical to the observed K-m of 12 mu M ATP for Ub-conjugate degra dation, Although nucleotide hydrolysis is required for protein degrada tion by the 26 S proteasome, nucleotide hydrolysis and peptide bond cl eavage are not strictly coupled, Substrate specificity constants (k(ca t)/K-m) are similar for hydrolysis of each nucleotide, yet GTP and UTP barely supported Ub-conjugate degradation, Further evidence that nucl eotide hydrolysis is not coupled to peptide bond cleavage was obtained using N-acetyl-leucyl-leucyl-norleucinal (LLnL). This compound inhibi ted peptide hydrolysis by the multicatalytic protease and Ub-conjugate degradation by the 26 S proteasome, but it had little effect on ATP o r UTP hydrolysis by the 26 S enzyme.