REACTION OF PROTEASOMES WITH PEPTIDYLCHLOROMETHANES AND PEPTIDYLDIAZOMETHANES

The multicatalytic endopeptidase complex (proteasome) has multiple dis tinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptid e hydrolase' activities according to the type of residue in the P1 pos ition, although it is now clear that mammalian proteasomes have at lea st five distinct catalytic sites. In the present study, potential affi nity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes , a, peptidyl-fluoromethane and peptidylsulphonium salts) containing h ydrophobic, basic or acidic amino acid residues in the P1 position hav e been tested for inhibition of the different activities of the rat li ver proteinase complex. The results show that individual peptidase act ivities of proteasomes can be inhibited by a variety of peptidylchloro methanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and p eptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1 -10 M-1 . s-1) compared with the reaction of similar compounds with so me other proteinases, the results provide useful information concernin g the specificity of the distinct catalytic centres of proteasomes, an d some selective affinity-labelling reagents have been identified. Tyr -Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-l ike activity. Inhibition of the other peptidase activities was often i ncomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example , Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit 'chymotrypsin-lik e' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or ucci nyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibito rs of 'peptidylglutamyl-peptide hydrolase' activities (assayed with be nzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazometh anes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading , because the residue in the P1 position is not the only determinant o f specificity.