Kinetic studies of the branched chain amino acid preferring peptidase activity 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 assay 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 kc/Km value of 13 000 M-1 s-1. Hydrolysis of this peptide is accompanied by an increase in fluorescence intensity (λex = 340 nm, λem = 415 nm) due to release of the internally quenched 2-aminobenzoyl fluorescence that accompanies diffusion apart of the hydrolysis products, Abz-Gly-Pro-Ala-Leu and Ala-Nba. Using this assay, we examined inhibition of the BrAAP activity of the proteasome by a series of tripeptide aldehydes, Z-Leu-Leu-Xaa-H. When Xaa = Phe, (p-Cl)Phe, and Trp we observe biphasic or partial inhibition of the BrAAP activity. In contrast, when Xaa = Nva and Leu, simple inhibition kinetics are observed and allow us to calculate Ki values of 120 ...