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-Le u-Leu-Val-Tyr-AMC greater than about 40 mu M are biphasic and characte rized by initial velocities that decay by a first-order process to fin al, steady-state velocities. (2) Also at [Suc-Leu-Leu-Val-Tyr-AMC] > 4 0 mu M, initial and steady-state velocities are smaller than predicted from simple, Michaelis-Menten kinetics. (3) The first-order rate cons tant for the approach to steady-state has a complex dependence on subs trate concentration and decreases sigmoidally as substrate concentrati on increases. These results indicate that the 20S proteasome is a hyst eretic enzyme and is subject to substrate inhibition. To explain these observations we propose a minimal kinetic model with two critical mec hanistic features: (1) the 20S proteasome has two cooperative active s ites for Suc-Leu-Leu-Val-Tyr-AMC and (3) there are two interconvertibl e conformers of active 20S proteasome. To probe this mechanism in grea ter detail, we explored the kinetic mechanism of inhibition of the 20S proteasome-catalyzed hydrolysis of Sue-Leu-Leu-Val-Tyr-AMC by the pep tide aldehyde, Ac-Leu-Leu-Nle-H. Our studies reveal a nonlinear depend ence of reciprocal steady-state velocity on inhibitor concentration (i .e,, parabolic inhibition) as well as a nonlinear dependence of the ap parent inhibitor dissociation constant on substrate concentration. Bot h of these observations are explained by binding of inhibitor at multi ple sites on the enzyme. Taken together, the results of this study Ind icate that the 20S proteasome is a conformationally flexible protein t hat can adjust to the binding of ligands and that has: multiple and co operative active sites. These results support a view of the proteasome 's substrate specificity in which (1) substrates are recognized and hy drolyzed by more than one active site; (2) each active site can bind s ubstrates that possess a variety of P-1 residues; and (3) the P-1 resi due plays a relatively minor role as a specificity determinant, Finall y, we interpret the results of this study to suggest that, in vivo, th e 20S proteasome requires conformational plasticity fur its interactio ns with regulatory complexes and, after it has combined with appropria te regulatory complexes, to catalyze hydrolysis of proteins.