Kinetic characterization of the chymotryptic activity of the 20S proteasome.
In this paper, we report kinetic studies for the chymotryptic activity of the 20S proteasome. Major observations include the following: (1) Reaction progress curves that are recorded at concentrations of Suc-Leu-Leu-Val-Tyr-AMC greater than about 40 microM are biphasic and characterized by initial velocities that decay by a first-order process to final, steady-state velocities. (2) Also at [Suc-Leu-Leu-Val-Tyr-AMC] > 40 microM, initial and steady-state velocities are smaller than predicted from simple, Michaelis-Menten kinetics. (3) The first-order rate constant for the approach to steady-state has a complex dependence on substrate concentration and decreases sigmoidally as substrate concentration increases. These results indicate that the 20S proteasome is a hysteretic enzyme and is subject to substrate inhibition. To explain these observations we propose a minimal kinetic model with two critical mechanistic features: (1) the 20S proteasome has two cooperative active sites for Suc-Leu-Leu-Val-Tyr-AMC and (2) there are two interconvertible conformers of active 20S proteasome. To probe this mechanism in greater detail, we explored the kinetic mechanism of inhibition of the 20S proteasome-catalyzed hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC by the peptide aldehyde, Ac-Leu-Leu-Nle-H. Our studies reveal a nonlinear dependence of reciprocal steady-state velocity on inhibitor concentration (i.e., parabolic inhibition) as well as a nonlinear dependence of the apparent inhibitor dissociation constant on substrate concentration. Both of these observations are explained by binding of inhibitor at multiple sites on the enzyme. Taken together, the results of this study indicate that the 20S proteasome is a conformationally flexible protein that can adjust to the binding of ligands and that has multiple and cooperative active sites. These results support a view of the proteasome's substrate specificity in which (1) substrates are recognized and hydrolyzed by more than one active site; (2) each active site can bind substrates that possess a variety of P1 residues; and (3) the P1 residue plays a relatively minor role as a specificity determinant. Finally, we interpret the results of this study to suggest that, in vivo, the 20S proteasome requires conformational plasticity for its interactions with regulatory complexes and, after it has combined with appropriate regulatory complexes, to catalyze hydrolysis of proteins.
Stein
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ProScript, Inc., Cambridge, Massachusetts 02139, USA.
Melandri
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Dick
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D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology*; D009842:Oligopeptides; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D009842:Oligopeptides; D012967:Sodium Dodecyl Sulfate; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex