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(Received for publication, August 19, 1996)
From the Department of Biochemistry, University of Utah School of
Medicine, Salt Lake City, Utah 84132
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 µM for ATP and CTP, 50 µM for GTP, and 100 µM 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 µM NEM. The 15 µM Km for ATP hydrolysis by the 26 S
proteasome is virtually identical to the observed Km of 12 µM 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.
Volume 271, Number 51,
Issue of December 20, 1996
pp. 32538-32545
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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