Evidence that the nature of amino acid residues in the P3 position directs substrates to distinct catalytic sites of the pituitary multicatalytic proteinase complex (proteasome).
Evidence that the nature of amino acid residues in the P3 position directs substrates to distinct catalytic sites of the pituitary multicatalytic proteinase complex (proteasome).
复制标题
有证据表明，P3 位点氨基酸残基的性质将底物引导至垂体多催化蛋白酶复合物（蛋白酶体）的不同催化位点。
DOI：
10.1021/bi00187a014
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Orlowski,M
中科院分区：
文献类型：
--
作者：
Cardozo,C;Vinitsky,A;Michaud,C;Orlowski,M
Revised Manuscript Received March 22, 1994® abstract: Cleavage of bonds after neutral amino acids by the multicatalytic proteinase complex (MPC) has been recently shownto 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 on the carboxyl side of branched-chain amino acids and between small neutral amino acids, respectively. Evidence indicates that the BrAAP component is a major factorresponsible for degradation of protein by the MPC. Thepurpose 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 P3 position directs the substrate to the catalytic site of the BrAAP component provided that a branched-chain amino acid is present in the Pi position. A proline residue in P3 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 Pi position. Substrates with a proline in P3 and a phenylalanine in Pi 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 Pi 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 P3 position can be cleavedby either the BrAAP or the ChT-L component, depending on the nature of the residue in the Pi position. A substrate with glycine residues in both the P3 and P4 position, and a leucine residue in Pi (Cbz-Gly-Gly-Ala-Leul-Ala-pAB), was cleaved by the BrAAP component with sigmoidal kinetics and an unusually high Kmax, 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.The multicatalytic proteinase complex (MPC1; multicata-lytic endopeptidase complex; EC 3.4. 99.46), also referred to as the proteasome, is a high molecular mass (~ 700 kDa; 19S) intracellular particle [for reviews see Orlowski,(1990, 1993), Rechsteiner et al.(1993) and Rivett (1993)] composed of 13-15 low molecular weight nonidentical subunits (21 000-34 000). It is organized into four stacked rings, each containing 6-8 subunits surrounding a water-filled tunnel (Kopp et al.,
