Abstract
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 complex were purified from rabbit reticulocytes for characterization of their nucleotidase properties. Both particles hydrolyze ATP, CTP, GTP, and UTP to the corresponding nucleoside diphosphate and inorganic phosphate. The Km values for hydrolysis of specific nucleotides by the 26 S proteasome are 15 microM for ATP and CTP, 50 microM for GTP, and 100 microM for UTP; Km values for nucleotide hydrolysis by the regulatory complex are 2-4-fold higher for each nucleotide. Several ATPase inhibitors (erythro-9-[3-(2-hydroxynonyl)]adenine, oligomycin, ouabain, and thapsigargin) had no effect on ATP hydrolysis by either complex whereas known inhibitors of proteolysis by the 26 S enzyme (hemin, N-ethylmaleimide (NEM), and vanadate) significantly reduced ATP hydrolysis by both particles. Hydrolysis of all nucleotides was inhibited by 5 mM NEM but only GTP and UTP hydrolysis was significantly reduced at 50 microM NEM. The 15 microM Km for ATP hydrolysis by the 26 S proteasome is virtually identical to the observed Km of 12 microM ATP for Ub-conjugate degradation. Although nucleotide hydrolysis is required for protein degradation by the 26 S proteasome, nucleotide hydrolysis and peptide bond cleavage are not strictly coupled. Substrate specificity constants (kcat/Km) are similar for hydrolysis of each nucleotide, yet GTP and UTP barely supported Ub-conjugate degradation. Further evidence that nucleotide hydrolysis is not coupled to peptide bond cleavage was obtained using N-acetyl-leucyl-leucyl-norleucinal (LLnL). This compound inhibited peptide hydrolysis by the multicatalytic protease and Ub-conjugate degradation by the 26 S proteasome, but it had little effect on ATP or UTP hydrolysis by the 26 S enzyme.
Highlights
The abbreviations used are: MCP, multicatalytic protease; ATA, aurintricarboxylic acid; DTT, dithiothreitol; EHNA, erythro-9-[3-(2-hydroxynonyl)]adenine; LLnL, N-acetyl-leucyl-leucyl-norleucinal, calpain inhibitor I; NEM, N-ethylmaleimide; RC, regulatory complex of the 26 S proteasome; sLLVY-MCA, succinimidyl-Leu-Leu-Val-Tyr-MCA; TEMED, N,N,NЈ,NЈ-tetramethylethylenediamine
We have characterized the nucleotidase activities of the RC and the 26 S proteasome with regards to kinetic constants, inhibitors, effect of MCP, and we have evaluated the role of nucleotide hydrolysis in the degradation of ubiquitin-lysozyme conjugates
Enzymatic Activities of Isolated 26 S Proteasome and RC— The ATP-dependent 26 S proteasome and its RC were purified from rabbit reticulocytes and identified by their sedimentation characteristics, their migration through nondenaturing gels, their subunit composition, and their peptidase or ATPase activities (Fig. 1)
Summary
Materials—ATP, CTP, GTP, UTP, and dATP were obtained from Pharmacia Biotech, Inc. LLnL was obtained from Boehringer Mannheim. The standard ATPase assay included 2 g of protein (26 S proteasome or RC) and 200 M ATP (in 20 mM HEPES, pH 7.2, 5 mM MgCl2, 1 mM DTT, 100 l volume) incubated for 60 min at 37 °C; 900 l of 0.034% Malachite Green, 1.1% ammonium molybdate, 1 M HCl, and 0.04% Triton X-100 was added to stop the reaction. For inhibition of nucleotide hydrolysis by LLnL, ATP or UTP was added to 500 M final concentration and incubated with the LLnL/protein mixture for 1 h at 37 °C, the phosphate product was determined by the Malachite Green assay. The 26 S proteasome (13 g of rabbit reticulocyte DEAE-purified protein) was preincubated with nucleotide for 2 min at 37 °C (100 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 50 l volume) to bring the reaction up to temperature. Molecular weights of 700,000 daltons for RC and 1,400,000 daltons for the 26 S proteasome (assuming 1 RC at 700 kDa and 1 MCP at 700 kDa make the 26 S proteasome at 1,400 kDa) were used to calculate turnover numbers
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