Cleavage of Pro-X and Glu-X bonds catalyzed by the branched chain amino acid preferring activity of the bovine pituitary multicatalytic proteinase complex (20S proteasome).
The multicatalytic proteinase complex or 20S proteasome is involved in the extralysosomal degradation of both long- and short-lived proteins. The eukaryotic enzyme is composed of 14 nonidentical subunits arranged as a complex dimer of the composition (alpha7beta7)2. Recent studies identify N-terminal threonines present on some beta-subunits as the active-site residues. It has been proposed that the molecule contains three or four proteolytically active subunits [Seemuller et al., Science 268, 579-582 (1995)]. Studies with synthetic substrates, activators, and inhibitors, however, have identified at least five distinct catalytic activities. To further characterize the specificity of the previously defined "peptidyl glutamyl peptide bond hydrolyzing activity," N-benzyloxycarbonyl-Leucyl-Leucyl-Glutamal was synthesized as a potential inhibitor. Surprisingly, this aldehyde most potently inhibited the "branched chain amino acid preferring activity" (BrAAP). To further explore BrAAP specificity, novel substrates containing internal prolyl and glutamyl residues were synthesized. Their use established that the BrAAP activity catalyzed both a postproline and a postglutamate cleavage and therefore has a broader specificity than previously recognized. These results help explain earlier observations on treatment of the multicatalytic proteinase complex with 3,4-dichloroisocoumarin. This reagent activates both the BrAAP activity and the degradation of beta-casein and inhibits the other catalytic activities.
Cardozo
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Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, New York 10029, USA.
Chen
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Wilk
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D000447:Aldehydes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D000597:Amino Acids, Branched-Chain / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D066298:In Vitro Techniques; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010902:Pituitary Gland / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity
D000447:Aldehydes; D000597:Amino Acids, Branched-Chain; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; C102027:N-benzyloxycarbonyl-leucyl-leucyl-glutamal; D009842:Oligopeptides; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex