Processive degradation of proteins and other catalytic properties of the proteasome from Thermoplasma acidophilum.
Although the structure of the 20 S proteasome from Thermoplasma acidophilum has been elucidated, its enzymatic properties have not been explored in depth. Thermoplasma proteasomes, which contain one type of active site, exhibit not only "chymotrypsin-like" activity (as reported), but also some "post-glutamyl" and "trypsin-like" activities. Like eukaryotic proteasomes, its activity can be stimulated by SDS, Mg2+, and also guanidine HCl, but not urea. The enzyme was strongly inhibited by novel peptide aldehydes with hydrophobic P4 residues, and was rapidly inactivated by 3, 4-dichloroisocoumarin (DCI). DCI modified the N-terminal threonine of the catalytic beta-subunit, the presumed active site nucleophile. To define how proteins are degraded, casein was derivatized with fluorescein isothiocyanate to facilitate detection of released products by the proteasome. Many fluorescent peptides were generated, but the relative amounts of different peptides were independent of the duration of the reaction. The rate of disappearance of protein substrates paralleled the rate of appearance of small products. Unlike conventional proteases, proteasome degrades proteins processively without release of polypeptide intermediates. Upon activation by SDS, guanidine, heat (55 degrees C), or partial inhibition with DCI, proteasomes still functioned processively, but generated a different pattern of peptides under each condition. Thus, processivity is an inherent feature of the 20 S proteasome, not requiring all active sites or ATP hydrolysis.
Akopian
T N
TN
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Kisselev
A F
AF
Goldberg
A L
AL
D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D006868:Hydrolysis; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity; D013822:Thermoplasma / Q000201:enzymology*
D009097:Multienzyme Complexes; D010455:Peptides; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D016650:Fluorescein-5-isothiocyanate