Inhibition of the chymotrypsin-like activity of the pituitary multicatalytic proteinase complex

Abstract

The multicatalytic proteinase complex (MPC), also referred to as proteasome, is a large molecular mass intracellular particle (approximately 700 kDa), which exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPH), all sensitive to inhibition by 3,4-dichloroisocoumarin (DCI). The presence of a component resistant to inhibition by DCI with an apparent preference toward bonds on the carboxyl side of branched-chain amino acids has also been recently established. Peptide aldehydes and peptide alpha-keto esters containing a hydrophobic residue in the P1 position have been tested as potential inhibitors of the chymotrypsin-like activity. Three peptide aldehydes (benzyloxycarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO), N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO), and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO) were found to be slow-binding reversible inhibitors with Ki values of 0.46, 5.7, and 33 microM, respectively. The simplest kinetic model for inhibition is consistent with a mechanism involving a slow and reversible association of the enzyme with the inhibitor to form a EI complex. The aldehyde inhibitors also inhibited the trypsin-like and PGPH activities of the complex albeit with much higher Ki values than those for chymotrypsin-like activity. Z-LLF-CHO, the most selective of the three aldehydes, did not inhibit the PGPH activity at concentrations of up to 200 microM and inhibited the trypsin-like activity with a Ki approximately 2 orders of magnitude higher than that for the chymotrypsin-like activity. The activity of the DCI-resistant component was not affected by Z-LLF-CHO.(ABSTRACT TRUNCATED AT 250 WORDS)