KINETIC-STUDIES OF THE BRANCHED-CHAIN AMINO-ACID PREFERRING PEPTIDASEACTIVITY OF THE 20S PROTEASOME - DEVELOPMENT OF A CONTINUOUS ASSAY AND INHIBITION BY TRIPEPTIDE ALDEHYDES AND CLASTO-LACTACYSTIN BETA-LACTONE

We have developed an assay to continuously monitor the branched amino acid preferring peptidase (BrAAP) activity of the proteasome. This ass ay is based on the hydrolysis of the flourogenic peptide, Abz-Gly-Pro- Ala-Leu-Ala-Nba (Abz is 2-aminobenzoyl and Nba is 4-nitrobenzylamide) which is cleaved exclusively at the Leu-Ala bond by the 20S proteasome with a k(c)/K-m value of 13000 M-1 s(-1) Hydrolysis of this peptide i s accompanied by an increase in fluorescence intensity (lambda(ex) = 3 40 nm, lambda(em) = 415 nm) due to release of the internally quenched 2-aminobenzoyl fluorescence that accompanies diffusion apart of the hy drolysis products, Abz-Gly-Pro-Ala-Leu and Ala-Nba. Using this assay, we examined inhibition of the BrAAP activity of the proteasome by a se ries of tripeptide aldehydes, Z-Leu-Leu-Xaa-H. When Xaa = Phe, (p-Cl)P he, and Trp we observe biphasic or partial inhibition of the BrAAP act ivity. In contrast, when Xaa = Nva and Leu, simple inhibition kinetics are observed and allow us to calculate K-i values of 120 nM and 12 nM , respectively. The inhibitors that exhibit simple inhibition kinetics for BrAAP activity are also approximately equipotent for inhibition o f the chymotrypsin-like (ChT-L) and peptidyl-glutamyl peptide hydrolyz ing (PGPH) activities, dissociation constants varying by less than 25- fold, whereas the inhibitors that exhibit biphasic inhibition kinetics for BrAAP activity are >300-fold more potent for inhibiting ChT-L act ivity than for PGPH activity. Inactivation of the BrAAP activity of th e proteasome by clasto-lactacystin beta-lactone is also biphasic. beta -Lactone inactivates approximately 60% of the BrAAP activity rapidly, with kinetics indistinguishable from its inactivation of the chymotryp sinlike activity. The remaining 40% of the BrAAP activity is inactivat ed by beta-lactone at a 50-fold slower rate, with kinetics indistingui shable from its inactivation of the PGPH activity. These results sugge st a mechanism in which hydrolysis of Abz-Gly-Pro-Ala-Leu-Ala-Nba (i.e ., BrAAP activity) occurs at two different active sites in the 20S pro teasome, and that these two active sites are the same ones that cataly ze the previously described ChT-L and PGPH activities.