title: Degradation of oxidized insulin B chain by the multiproteinase complex macropain (proteasome).
abstract: The peptides generated from the degradation of the oxidized B chain of bovine insulin by the multiproteinase complex macropain (proteasome) have been analyzed by reverse-phase peptide mapping and identified by N-terminal amino acid sequencing and composition analysis. Six of the 29 peptide bonds in the insulin B chain were found to be rapidly cleaved by macropain. The catalytic center that cleaves the Gln4-His5 bond could be distinguished from the center or centers that cleave the other preferred bonds by its specific susceptibility to inhibition by leupeptin, antipain, chymostatin, and pentamidine, suggesting that macropain utilizes at least two distinct catalytic centers for the degradation of this model polypeptide. The same effectors simultaneously enhance the rate of cleavage at the other susceptible sites in insulin B. The quantitative characteristics of this effect indicate that different catalytic centers of the complex may be functionally coupled, possibly by an allosteric mechanism or possibly by a mechanism in which binding to the catalytic centers is preceded by a rate-limiting binding of the substrate to a site or sites on the enzyme distinct from the catalytic centers. The kinetics of insulin B chain degradation indicate that macropain can catalyze sequential hydrolysis of peptide bonds in a single substrate molecule via a reaction pathway that involves channeling of peptide intermediates between different catalytic centers within the multienzyme complex. This capacity for channeling may confer potential physiological advantages of increasing the efficiency of amino acid recycling and reducing the pool sizes of peptide intermediates that are generated during the degradation of polypeptides in the intracellular milieu.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
authors.forename: L R
authors.initials: LR
authors.lastname: Dick
authors.forename: C R
authors.initials: CR
authors.lastname: Moomaw
authors.forename: G N
authors.initials: GN
authors.lastname: DeMartino
authors.forename: C A
authors.initials: CA
authors.lastname: Slaughter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000976:Antipain / Q000494:pharmacology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010084:Oxidation-Reduction; D010419:Pentamidine / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex
chemical_list: D007328:Insulin; D007976:Leupeptins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D000976:Antipain; D010419:Pentamidine; C002101:chymostatin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C032854:leupeptin