Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes.
The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptide hydrolase' activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, 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-like' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of 'peptidylglutamyl-peptide hydrolase' activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes 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 of specificity.
Savory
P J
PJ
Department of Biochemistry, University of Leicester, U.K.
Djaballah
H
H
Angliker
H
H
Shaw
E
E
Rivett
A J
AJ
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003978:Diazomethane / Q000031:analogs & derivatives / Q000494:pharmacology*; D006843:Hydrocarbons, Chlorinated / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D017208:Rats, Wistar
D000345:Affinity Labels; D006843:Hydrocarbons, Chlorinated; D009097:Multienzyme Complexes; D010455:Peptides; D011480:Protease Inhibitors; D003978:Diazomethane; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex