Evidence for the Existence of a Non-catalytic Modifier Site of Peptide Hydrolysis by the 20 S Proteasome

Gunter Schmidtke,Sabine Emch,Marcus Groettrup,Hermann-Georg Holzhütter

doi:10.1074/jbc.m002513200

Abstract

The 20 S proteasome is an endoprotease complex that preferentially cleaves peptides C-terminal of hydrophobic, basic, and acidic residues. Recently, we showed that these specific activities, classified as chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide-hydrolyzing (PGPH) activity, are differently affected by Ritonavir, an inhibitor of human immunodeficiency virus-1 protease. Ritonavir competitively inhibited the chymotrypsin-like activity, whereas the trypsin-like activity was enhanced. Here we demonstrate that the Ritonavir-mediated up-regulation of the trypsin-like activity is not affected by specific active site inhibitors of the chymo-trypsin-like and PGPH activity. Moreover, we show that the mutual regulation of chymotrypsin-like and PGPH activities by their substrates as described previously by a "cyclical bite-chew" model is not affected by selective inhibitors of the respective active sites. These data challenge the bite-chew model and suggest that effectors of proteasome activity can act by binding to non-catalytic sites. Accordingly, we propose a kinetic "two-site modifier" model that assumes that the substrate (or effector) may bind to an active site as well as to a second non-catalytic modifier site. This model appears to be valid as it describes the complex kinetic effects of Ritonavir very well. Since Ritonavir partially inhibits major histocompatibility complex class I restricted antigen presentation, the postulated modifier site may be required to coordinate the active centers of the proteasome for the production of class I peptide ligands.

Highlights

The 20 S proteasome was purified 20 years ago as a cationsensitive neutral endopeptidase from bovine pituitary tissue and was originally named multicatalytic proteinase complex (MCP) [1,2,3]
A satisfactory quantitative description of the rather complex and seemingly erratic kinetic data on the effect of Ritonavir of the various specific activities of the 20 S proteasome can be obtained by using a two-site modifier model, which assumes that Ritonavir may bind to both an active site and to a second, non-catalytic modifier site
The chymotrypsin-like activity of the proteasome, as measured by the hydrolysis of the substrate Suc-LLVY-MCA, was inhibited by Ritonavir with an IC50 value of 3 ␮M, the trypsin-like activity measured with the substrate Bz-VGR-MCA was enhanced in a dose-dependent fashion [21]

Summary

Introduction

The 20 S proteasome was purified 20 years ago as a cationsensitive neutral endopeptidase from bovine pituitary tissue and was originally named multicatalytic proteinase complex (MCP) [1,2,3]. We present data that are difficult to rationalize by allosteric interactions among the various active sites and, require an alternative mechanistic explanation These experiments are based on a previously published observation that an inhibitor of the human immunodeficiency virus-1 protease, named Ritonavir, modulated proteasome activity in that the chymotrypsin-like activity was inhibited, whereas the trypsin-like activity was enhanced [20, 21]. A satisfactory quantitative description of the rather complex and seemingly erratic kinetic data on the effect of Ritonavir of the various specific activities of the 20 S proteasome can be obtained by using a two-site modifier model, which assumes that Ritonavir may bind to both an active site and to a second, non-catalytic modifier site. Our data challenge the bite-chew model and suggest that the kinetics of peptide hydrolysis by the 20 S proteasome is brought about by a modulation of the various specific activities by a so-far unidentified modifier site

Methods

Results

Conclusion