Abstract
Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine.
Highlights
The ubiquitin-proteasome system is the major pathway for degrading proteins in the cytosol and nucleus in eukaryotic cells [1, 2]
A variety of observations indicate that proteasomes cleave peptide bonds by this unusual mechanism, in which a hydroxyl group of the N-terminal threonine serves as the catalytic nucleophile: (i) replacement of this threonine by an alanine in archaeal [28] and yeast (29 –32) proteasomes abolishes their proteolytic activity; (ii) the hydroxyl group of this threonine is modified by irreversible inhibitors lactacystin [33, 34], 3,4-dichloroisocoumarin [10, 35], and vinyl sulfone [36]; and (iii) it has been shown by x-ray diffraction to form a hemiacetal bond with the peptide aldehyde
The present findings argue strongly that threonine residues and not serines are found in the active sites of proteasomes, because threonine provides a greater capacity to cleave peptides and degrade proteins (Tables II and III)
Summary
Wild type T. acidophilum proteasomes and the T1S, T1A, and T1C mutants in the -subunit were expressed in Escherichia coli without propeptides [28, 45] and purified to homogeneity as described previously [15]. A mixture of peptide standards, whose concentrations were determined by amino acid analysis, was used to calibrate the assay [10]. Values for kobs for 4-hydroxyl-3-iodo-3-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone and epoxomycin were obtained using nonlinear least square fit of the reaction to the equation: fluorescence ϭ vft ϩ [(vo Ϫ vs)kobs][1 Ϫ exp(Ϫkobst)], where vo and vs are initial and final velocities, respectively [52]. Such a fit was impossible for -lactone due to the reversibility of reaction (see Fig. 3). Rate constants for this inhibitor were determined as kobs ϭ [ln(vo/vt)]/t, where vt is the reaction velocity at the inflection point, which was reached 4 –13 min after the addition of -lactone (see Fig. 3)
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