EVIDENCE THAT THE NATURE OF AMINO-ACID-RESIDUES IN THE P-3 POSITION DIRECTS SUBSTRATES TO DISTINCT CATALYTIC SITES OF THE PITUITARY MULTICATALYTIC PROTEINASE COMPLEX (PROTEASOME)

Cleavage of bonds after neutral amino acids by the multicatalytic proteinase complex (MPC) has been recently shown to be catalyzed by at least three distinct components [Orlowski, M., Cardozo, C., & Michaud, C. (1993) Biochemistry 32, 1563-1572]. One component, designated as chymotrypsin-like (ChT-L), cleaves peptide bonds on the carboxyl side of hydrophobic residues and is also active toward peptidyl-arylamide bonds. A second component, designated as branched-chain amino acid preferring (BrAAP), and a third component, designated as small neutral amino acid preferring (SNAAP), cleave preferentially bonds bn the carboxyl side of branched-chain amino acids and between small neutral amino acids, respectively. Evidence indicates that the BrAAP component is a major factor responsible for degradation of protein by the MPC. The purpose of the present study was to identify the structural requirements that determine the involvement of these components in cleavage of peptides after different neutral amino acids. A series of substrates was synthesized with the aim of probing the role of residues beyond those flanking the scissile bond in directing substrates to defined catalytic sites. The data indicate that a proline or glycine residue in the P-3 position directs the substrate to the catalytic site of the BrAAP component provided that a branched-chain amino acid is present in the P-1 position. A proline residue in P-3 is also important for involvement of the SNAAP component in substrate degradation. The presence of this residue interferes with substrate binding to the catalytic site of the ChT-L activity, even in the presence of a phenylalanine residue in the P-1 position. Substrates with a proline in P-3 and a phenylalanine in P-1 positions are poorly cleaved by both the BrAAP and ChT-L components, further supporting the preference of the former for branched-chain amino acid residues in the P-1 position. Replacement of proline by hydrophobic residues shifts the activity to a catalytic site with properties of the ChT-L component. Substrates with a glycine in the P-3 position can be cleaved by either the BrAAP or the ChT-L component, depending on the nature of the residue in the P-1 position. A substrate with glycine residues in both the P-3 and P-4 position, and a leucine residue in P-1 (Cbz-Gly-Gly-Ala-Leu down arrow-Ala-pAB), was cleaved by the BrAAP component with sigmoidal kinetics and an unusually high V-max, suggesting positive cooperativity between two or more active sites of the MPC, and possible involvement of both overt and latent activities of the MPC in substrate hydrolysis.