FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2061034807> ?p ?o ?g. }

• W2061034807 endingPage "14837" @default.
• W2061034807 startingPage "14831" @default.
• W2061034807 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 inhibitorclasto-lactacystin-β-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. 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 inhibitorclasto-lactacystin-β-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. proteasome activating nucleotidase 7-amido-4-methylcoumarin dicyclohexylcarbodiimide 3,4-dichloroisocoumarin dimethylformamide dimethyl sulfoxide fluorescein isothiocyanate N-(9-fluorenyl)methoxycarbonyl insulin-like growth factor type 1 N-terminal nucleophile succinyl benzyloxycarbonyl 1,3-bis[tris(hydroxymethyl)methylamino]propane high pressure liquid chromatography The ubiquitin-proteasome system is the major pathway for degrading proteins in the cytosol and nucleus in eukaryotic cells (1.Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2186) Google Scholar, 2.Craiu A. Gaczynska M. Akopian T. Gramm C.F. Fenteany G. Goldberg A.L. Rock K.L. J. Biol. Chem. 1997; 272: 13437-13445Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). Proteins marked for degradation by an attachment of a polyubiquitin chain are hydrolyzed by the 26 S proteasome in an ATP-dependent manner (3.Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2226) Google Scholar, 4.Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6829) Google Scholar, 5.Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1453) Google Scholar). This complex consists of the 20 S core proteasome, in which proteolysis occurs, and two 19 S regulatory complexes (6.Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1585) Google Scholar, 7.Baumeister W. Walz J. Zühl F. Seemüller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1301) Google Scholar). 20 S proteasomes also exist in archaea and many eubacteria, which lack both 26 S proteasomes and ubiquitin (8.Zwickl P. Goldberg A.L. Baumeister W. Wolf D.H. Hilt W. Proteasomes in Prokaryotes. Proteasomes: The World of Regulatory Proteolysis. Landes Bioscience Publishing Co., Georgetown, TX1999: 8-20Google Scholar). The 20 S proteasome from Thermoplasma acidophilum has proven to be especially useful for studies of the proteasome's structure and catalytic mechanism (9.Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1372) Google Scholar, 10.Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Like the eukaryotic core particle, theT. acidophilum proteasome is a cylindrical complex consisting of four superimposed 7-membered rings (11.Wenzel T. Baumeister W. Nature Struct. Biol. 1995; 2: 199-204Crossref PubMed Scopus (187) Google Scholar). However, these archaeal particles are simpler and more symmetric in organization. The two outer rings are composed of identical α-subunits, and its inner rings are composed of identical β-subunits, each of which contains an active site. These 14 active sites are located on the inner surface of this particle. The narrow openings in the α-rings serve as sites of substrate entrance into the inner chamber of the cylinder where proteolysis occurs. Globular proteins are too large to traverse these openings and need to be unfolded to be translocated into the 20 S particle for degradation (11.Wenzel T. Baumeister W. Nature Struct. Biol. 1995; 2: 199-204Crossref PubMed Scopus (187) Google Scholar). Presumably, the ATPases in the 19 S particle catalyze this process (12.Larsen C.N. Finley D. Cell. 1997; 91: 431-434Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 13.Rubin D.M. Finley D. Curr. Biol. 1995; 5: 854-858Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Recently, a homologous ATPase complex, the proteasome-activating nucleotidase (PAN),1 was described in archaea. In the presence of ATP, this complex dramatically stimulates protein breakdown by 20 S proteasomes from T. acidophilum(14.Zwickl P. Ng D. Woo K.M. Klenk H.P. Goldberg A.L. J. Biol. Chem. 1999; 274: 26008-26014Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), and presumably, PAN was the evolutionary precursor of the 19 S complex. Both archaeal and eukaryotic 20 S and 26 S proteasomes degrade proteins in a highly processive manner; i.e. they cut polypeptides at multiple sites without release of intermediates and generate small peptides, 95% of which are shorter than 20 residues (10.Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 15.Kisselev A.F. Akopian T.N. Goldberg A.L. J. Biol. Chem. 1998; 273: 1982-1989Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 16.Kisselev A.F. Akopian T.N. Woo K.M. Goldberg A.L. J. Biol. Chem. 1999; 274: 3363-3371Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 17.Nussbaum A.K. Dick T.P. Keilholz W. Schirle M. Stevanovic S. Dietz K. Heinemeyer W. Groll M. Wolf D.H. Huber R. Rammensee H.G. Schild H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12504-12509Crossref PubMed Scopus (285) Google Scholar). The sequences and overall structural fold of the proteasomal subunits as well as their proteolytic mechanism differ from those of other classes of proteolytic enzymes (9.Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1372) Google Scholar, 18.Zwickl P. Grziwa A. Puhler G. Dahlmann B. Lottspeich F. Baumeister W. Biochemistry. 1992; 31: 964-972Crossref PubMed Scopus (200) Google Scholar). The tertiary structure of proteasome subunits is related to that of several other amidases, which all utilize the side chain of the N-terminal residue as the catalytic nucleophile. These enzymes, therefore, have been termed N-terminal nucleophile (Ntn) hydrolases (19.Brannigan J.A. Dodson G. Duggleby H.J. Moody P.C. Smith J.L. Tomchick D.R. Murzin A.G. Nature. 1995; 378: 416-419Crossref PubMed Scopus (542) Google Scholar). This N-terminal residue is cysteine in glutamine-5-phosphoribose-1-pyrophosphate amidotransferase (20.Smith J.L. Zaluzec E.J. Wery J.P. Niu L. Switzer R.L. Zalkin H. Satow Y. Science. 1994; 264: 1427-1433Crossref PubMed Scopus (226) Google Scholar), glucosamine-6-phosphate synthase (21.Isupov M.N. Obmolova G. Butterworth S. Badet-Denisot M.A. Badet B. Polikarpov I. Littlechild J.A. Teplyakov A. Structure. 1996; 4: 801-810Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and asparagine synthase (22.Larsen T.M. Boehlein S.K. Schuster S.M. Richards N.G.J. Thoden J.B. Holden H.M. Rayment I. Biochemistry. 1999; 38: 16146-16157Crossref PubMed Scopus (161) Google Scholar); serine in penicillin acylase (23.Choi K.S. Kim J.A. Kang H.S. J. Bacteriol. 1992; 174: 6270-6276Crossref PubMed Google Scholar, 24.Duggleby H.J. Tolley S.P. Hill C.P. Dodson E.J. Dodson G. Moody P.C. Nature. 1995; 373: 264-268Crossref PubMed Scopus (422) Google Scholar); and threonine in glycosylasparaginase (25.Tikkanen R. Riikonen A. Oinonen C. Rouvinen R. Peltonen L. EMBO J. 1996; 15: 2954-2960Crossref PubMed Scopus (82) Google Scholar, 26.Oinonen C. Tikkanen R. Rouvinen J. Peltonen L. Nature Struct. Biol. 1995; 2: 1102-1108Crossref PubMed Scopus (158) Google Scholar), all proteasomes, and their bacterial homologue HslVU (27.Rohrwild M. Coux O. Huang H.C. Moerschell R.P. Yoo S.J. Seol J.H. Chung C.H. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5808-5813Crossref PubMed Scopus (213) Google Scholar). A variety of observations indicate that proteasomes indeed 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.Seemüller E. Lupas A. Stock D. Lowe J. Huber R. Baumeister W. Science. 1995; 268: 579-582Crossref PubMed Scopus (583) Google Scholar) and yeast (29.Heinemeyer W. Fischer M. Krimmer T. Stachon U. Wolf D.H. J. Biol. Chem. 1997; 272: 25200-25209Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 30.Chen P. Hochstrasser M. Cell. 1996; 86: 961-972Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 31.Arendt C.S. Hochstrasser M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7156-7161Crossref PubMed Scopus (246) Google Scholar, 32.Dick T.P. Nussbaum A.K. Deeg M. Heinemeyer W. Groll M. Schirle M. Keilholz W. Stevanovic S. Wolf D.H. Huber R. Rammensee H.G. Schild H. J. Biol. Chem. 1998; 273: 25637-25646Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) proteasomes abolishes their proteolytic activity; (ii) the hydroxyl group of this threonine is modified by irreversible inhibitors lactacystin (33.Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1496) Google Scholar, 34.Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H. Huber R. Nature. 1997; 386: 463-471Crossref PubMed Scopus (1937) Google Scholar), 3,4-dichloroisocoumarin (10.Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 35.Orlowski M. Cardozo C. Eleuteri A.M. Kohanski R. Kam C.M. Powers J.C. Biochemistry. 1997; 36: 13946-13953Crossref PubMed Scopus (40) Google Scholar), and vinyl sulfone (36.Bogyo M. McMaster J.S. Gaczynska M. Tortorella D. Goldberg A.L. Ploegh H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6629-6634Crossref PubMed Scopus (405) Google Scholar); and (iii) it has been shown by x-ray diffraction to form a hemiacetal bond with the peptide aldehyde inhibitor (9.Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1372) Google Scholar, 34.Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H. Huber R. Nature. 1997; 386: 463-471Crossref PubMed Scopus (1937) Google Scholar). The catalytic β-subunits are synthesized as inactive precursors containing N-terminal extensions, autocatalytically cleaved off during particle assembly (37.Schmidt M. Kloetzel P.M. FASEB J. 1997; 11: 1235-1243Crossref PubMed Scopus (42) Google Scholar, 38.Zwickl P. Kleinz J. Baumeister W. Nature Struct. Biol. 1994; 1: 765-770Crossref PubMed Scopus (170) Google Scholar), that expose the catalytic N-terminal threonine. In addition to preventing catalysis before assembly, these propeptides protect the N termini from acetylation in eukaryotic cells (39.Groll M. Heinemeyer W. Jager S. Ullrich T. Bochtler M. Wolf D.H. Huber R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10976-10983Crossref PubMed Scopus (240) Google Scholar), which also abolishes catalytic activity (40.Arendt C.S. Hochstrasser M. EMBO J. 1999; 18: 3575-3585Crossref PubMed Scopus (129) Google Scholar, 41.Jager S. Groll M. Huber R. Wolf D.H. Heinemeyer W. J. Mol. Biol. 1999; 291: 997-1013Crossref PubMed Scopus (109) Google Scholar). These observations indicate an essential role of free N-terminal primary amine in catalysis, which presumably promotes nucleophilicity of the hydroxyl group (9.Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1372) Google Scholar, 42.Lupas A. Zwickl P. Wenzel T. Seemüller E. Baumeister W. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 515-524Crossref PubMed Scopus (31) Google Scholar). Surprisingly, mutation of catalytic threonine into a serine in archaeal proteasomes was not found to change the rate of cleavage of the fluorogenic peptide Suc-LLVY-amc, which is routinely used to assay proteasome activity (28.Seemüller E. Lupas A. Stock D. Lowe J. Huber R. Baumeister W. Science. 1995; 268: 579-582Crossref PubMed Scopus (583) Google Scholar). By contrast, in serine proteases, the replacement of the active site serine by a threonine reduces proteolytic activity by several orders of magnitude (43.Corey D.R. Craik C.S. J. Am. Chem. Soc. 1992; 114: 1784-1790Crossref Scopus (141) Google Scholar). Moreover, in another Ntn family member, glycosylasparaginase, the replacement of the catalytic threonine by serine also reduced its activity by more than 10-fold (44.Liu Y. Guan C. Aronson Jr., N.N. J. Biol. Chem. 1998; 273: 9688-9694Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). These findings raise an important question of why proteasomes have evolved with only threonine and not serine in their active sites. If threonine and serine were in fact functionally interchangeable, random mutations would have replaced threonine by serine in many, if not most, proteasomes, because threonine has four codons (ACX), whereas serine has six (UCX and AG(U/C)). These experiments were undertaken to investigate more thoroughly the catalytic properties of proteasomes containing N-terminal a threonine or serine residue and thus to enhance our understanding of the proteasome's novel proteolytic mechanism. We therefore compared activities of the wild type and the T1S mutant of proteasomes from archaea T. acidophilum against a variety of peptide and protein substrates as well as their susceptibilities to the inactivation by various irreversible inhibitors. Wild type T. acidophilum proteasomes and the T1S, T1A, and T1C mutants in the β-subunit were expressed inEscherichia coli without propeptides (28.Seemüller E. Lupas A. Stock D. Lowe J. Huber R. Baumeister W. Science. 1995; 268: 579-582Crossref PubMed Scopus (583) Google Scholar, 45.Seemüller E. Lupas A. Baumeister W. Nature. 1996; 382: 468-470Crossref PubMed Scopus (182) Google Scholar) and purified to homogeneity as described previously (15.Kisselev A.F. Akopian T.N. Goldberg A.L. J. Biol. Chem. 1998; 273: 1982-1989Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The proteasome-activating nucleotidase, PAN, from Methanococcus jannaschii was kindly provided by Dr. A. Navon. A mutant, M74A, of PAN was used because it allowed purification of the homogeneous PAN (i.e. it abolished the production of some truncated PAN subunits that result from an internal initiation of translation 2D. Ng, P. Zwickl, and A. L. Goldberg, submitted for publication.). Proteasome and PAN concentrations were determined using the Bradford assay (46.Bradford M.M. Analyt. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214515) Google Scholar). All protein substrates were prepared as described by Akopian et al. (10.Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Reduced and carboxymethylated bovine α-lactalbumin and recombinant human insulin-like growth factor type 1 (IGF) were reductively methylated. All 12 amino groups in bovine β-casein were either reductively methylated or modified with fluorescein isothiocyanate (FITC). Concentrations of substrate proteins were determined using UV absorption (47.Gill S.C. von Hippel P.H. Analyt. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5035) Google Scholar), except FITC-casein, which was measured with the Bradford assay because of the high absorbance of FITC at 280 nm. Synthetic peptide amides were purchased from Bachem (King of Prussia, PA), except Z-LLE-amc, which was a kind gift from Leukocyte, Inc. (Cambridge, MA). Amino acid derivatives for peptide synthesis were from Calbiochem/Novabiochem (San Diego, CA). Peptide libraries Biotin-LLVYX 4QQ that is homologous to the substrate Suc-LLVY-amc and Biotin-X 8QQ (whereX is any amino acid residue except Cys) were synthesized as described (48.Songyang Z. Cantley L.C. Methods Mol. Biol. 1998; 87: 87-98PubMed Google Scholar). Z-GGL was synthesized on a solid phase using standard Fmoc protocol (49.Wellings D.A. Atherton E. Methods Enzymol. 1997; 289: 44-67Crossref PubMed Scopus (238) Google Scholar), starting with Fmoc-Leu-Wang resin and using Z-Gly at the last step. It was purified with HPLC after cleaving from the resin. 10 mg (27 μmol) of Z-GGL were dissolved in 24 μl of dimethylformamide (DMF), which was used as the solvent for this synthesis, and mixed with 35 μl of 1m 2-naphtol. After addition of 20 μl of 2 mdicyclohexylcarbodiimide (DCC), the reaction mixture was placed on ice for 1 h and then at room temperature for 15 h. Precipitated dicyclohexyl urea was removed by centrifugation and thoroughly washed with DMF. Because HPLC analysis demonstrated that the reaction was not complete at this stage, the reaction mixture was concentrated to 50 μl, and, after the addition of 10 μl of 2 m DCC, was left at room temperature for additional 50 h. The excess DCC was destroyed by the addition of water, and the dicyclohexylurea precipitate was removed by centrifugation. Reaction mixture (100 μl) was diluted with 4 ml of 40% acetonitrile in 20 mmammonium acetate, and the reaction product was purified using HPLC. Structure of Z-GGL-2-naphtyl ester was confirmed using mass-spectrometry and amino acid analysis. Cleavage of fluorogenic peptide amides and ester was assayed in 50 mm Tris-HCl, pH 7.5, by continuously monitoring the release of fluorogenic products, 7-amino-4-methylcoumarin (excitation, 380 nm; emission, 460 nm), 2-naphtylamine (excitation, 340 nm; emission, 410 nm), or 2-naphtol (excitation, 329 nm; emission, 410 nm) (50.Kisselev A.F. Akopian T.N. Castillo V. Goldberg A.L. Mol. Cell. 1999; 4: 395-402Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). The reaction velocities were determined from the slopes of the initial linear portions of the reaction progress curves. Peptide libraries and proteins were incubated with proteasomes in 50 mm bis-tris propane, pH 7.5. At different times, aliquots were withdrawn, mixed with an equal volume of 1.2% SDS to stop the enzymatic reaction, and assayed for free amino groups with fluorescamine assay (10.Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). A mixture of peptide standards, whose concentrations were determined by amino acid analysis, was used to calibrate the assay (10.Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). All incubations were performed at 54 °C. All K m and V maxvalues were determined from direct linear plots (51.Henderson P.F.J. Eisenthal R. Danson M.J. Enzyme Assays. IRL Press, Oxford1991: 277-316Google Scholar).k cat calculations were based on the assumption that all 14 active sites in all molecules of the enzyme preparation were catalytically active. Cleavage of Suc-LLVY-amc (100 μm) by proteasomes was allowed to proceed for 5–10 min without the inhibitor (see Fig. 3). The inhibitor was added, and the incubation continued for up to 3 h. Fluorescence of the reaction product (amc) was followed continuously during the entire incubation. Not more than 1% of the substrate was consumed during the incubation, and the reaction progress curves were linear in the control experiments, where Me2SO carrier was used instead of inhibitors. Values fork obs 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 =v f t + [(v o −v s)k obs][1 − exp(−k obs t)], wherev o and v s are initial and final velocities, respectively (52.Morrison J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar). Such a fit was impossible for β-lactone due to the reversibility of reaction (see Fig. 3). Rate constants for this inhibitor were determined as kobs = [ln(v o/v t)]/t, where v t is the reaction velocity at the inflection point, which was reached 4–13 min after the addition of β-lactone (see Fig. 3). The wild type and T1S mutant were found to hydrolyze the fluorogenic peptide amide Suc-LLVY-amc at similar rates, in accord with findings by Seemüller et al. (28.Seemüller E. Lupas A. Stock D. Lowe J. Huber R. Baumeister W. Science. 1995; 268: 579-582Crossref PubMed Scopus (583) Google Scholar). Also, there was no significant difference in the rates of hydrolysis of several different peptide amides (Table I) by the two forms, except that the T1S cleaved Z-LLE-2-naphtylamide about 40% slower than the wild type (Table I). In all these substrates, the scissile bond is an amide bond between an amino acid residue and an aromatic amine (amc). Amide bonds differ in several structural features from true peptide bonds, and they are not hydrolyzed by numerous proteolytic enzymes, such as metalloproteases and aspartic proteases. To compare the abilities of the wild type and T1S proteasomes to cleave true peptide bonds, the fluorogenic leaving group in these substrates was replaced by a sequence of natural amino acids. Moreover, to exclude the possibility that different sub-site preferences of the two forms might affect the results and to compare their activities toward the maximal possible array of peptide substrates, we constructed two peptide libraries with the general sequences, LLVYX 4QQ and X 8QQ with N-terminal amino groups blocked by biotinylation. This N-terminal modification greatly reduced the background in the fluorescamine assay, which we used to measure the rates of cleavage of peptide bonds in these libraries. Fluorescamine forms a fluorescent adduct with primary amines (53.Udenfriend S. Stein S. Bohlen P. Dairman W. Leimgruber W. Weigele M. Science. 1972; 178: 871-872Crossref PubMed Scopus (2194) Google Scholar) and was used, as in our prior studies (10.Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 15.Kisselev A.F. Akopian T.N. Goldberg A.L. J. Biol. Chem. 1998; 273: 1982-1989Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), to measure the rates of appearance of free α-amino groups generated upon peptide bond hydrolysis by the two types of proteasomes.Table IThe wild type proteasome and its T1S mutant cleave fluorogenic peptide amides at similar ratesSubstrateActivityWild typeT1Snmol/min · mgSuc-LLVY-amc22 ± 217 ± 3Suc-AAF-amc5.9 ± 0.54.3 ± 0.8Z-GGL-amc27 ± 323 ± 2Z-LLE-2-naphtylamide6.9 ± 0.64.0 ± 0.3Values in the table are the means ± S.E. of three experiments. Substrates were at 100 μm. Open table in a new tab Values in the table are the means ± S.E. of three experiments. Substrates were at 100 μm. Peptide hydrolysis with both decapeptide libraries appeared to obey simple Michaelis-Menten kinetics (Fig.1). Surprisingly, although the T1S mutant hydrolyzed fluorogenic amides at similar rates as the wild type, it cleaved the peptide libraries with 10-fold lowerV max values than the wild type enzyme (TableII). Although the Ser substitution led to a surprising severalfold decrease in K m for peptides, the catalytic efficiency (k cat/K m) for the serine mutant was still significantly lower than for the wild type (Table II). In other words, replacement of the active site threonine by a serine results in a severalfold decrease in peptidase activity without affecting the particle's amidase activity.Table IIProteasomes with catalytic serine (T1S) have similar amidase activity as wild type enzymes but cleave true peptide bonds much more slowlySubstrateConstantProteasome mutantwtT1ST1CT1ASuc-LLVY-amck cat, s−10.210.1700 (amide bond-cleaved)K m, mm0.030.03k cat/K m, s−1mm−17.05.7Biotin-LLVYXXXXQQk cat, s−14.00.400K m, mm0.40.06k cat/K m, s−1mm−1105.8Biotin-XXXXXXXXQQk cat, s−13.80.4200K m, mm0.80.2k cat/K m, s−1mm−14.82.1X = any amino acid except Cys. The concentration of libraries varied from 0.125 to 2 mg/ml, and incubations lasted for 30 min. Molar concentrations of libraries were calculated assuming that the average molecular mass of the amino acids in each divergent position is 110 Da. k cat is defined as a number of individual peptide bonds cleaved per second. All values are the averages of at least two experiments. Open table in a new tab X = any amino acid except Cys. The concentration of libraries varied from 0.125 to 2 mg/ml, and incubations lasted for 30 min. Molar concentrations of libraries were calculated assuming that the average molecular mass of the amino acids in each divergent position is 110 Da. k cat is defined as a number of individual peptide bonds cleaved per second. All values are the averages of at least two experiments. It is also noteworthy that at V max the wild type proteasome cleaved both peptide libraries at much greater rates (up to 20-fold faster) than Suc-LLVY-amc, which is the most widely used fluorogenic substrate of the proteasome. However, theK m for Suc-LLVY-amc was much lower, perhaps because the bulky fluorogenic amide (amc) downstream of the scissile bond allowed binding with higher affinity (Table II). It has been proposed that, aside from the N-terminal threonine, additional active sites may exist in proteasomes (34.Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H. Huber R. Nature. 1997; 386: 463-471Crossref PubMed Scopus (1937) Google Scholar, 54.Rechsteiner M. Peters J.-M. Harris J.R. Finley J. Ubiquitin and the Biology of the Cell. Plenum Press, New York1998: 147-189Crossref Google Scholar). Such active sites may be capable of cleaving true peptide bonds only, and they may have specificity different from the threonine sites. Therefore, they would not be evident with the standard fluorogenic substrate, Suc-LLVY-amc. However, two other active site mutants, T1A and T1C, were completely inactive against the peptide libraries. This lack of peptide hydrolysis in these mutants makes it very unlikely that proteasomes contain additional proteolytic sites, as had been proposed by some investigators (34.Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H. Huber R. Nature. 1997; 386: 463-471Crossref PubMed Scopus (1937) Google Scholar, 54.Rechsteiner M. Peters J.-M. Harris J.R. Finley J. Ubiquitin and the Biology of the Cell. Plenum Press, New York1998: 147-189Crossref Google Scholar). To test whether these findings with libraries of synthetic peptides can be extended to protein breakdown, we first established that the T1S mutant, like the wild type enzyme (10.Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 15.Kisselev A.F. Akopian T.N. Goldberg A.L. J. Biol. Chem. 1998; 273: 1982-1989Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), degraded proteins in a processive manner into peptides" @default.
• W2061034807 created "2016-06-24" @default.
• W2061034807 creator A5006962945 @default.
• W2061034807 creator A5042332987 @default.
• W2061034807 creator A5058343808 @default.
• W2061034807 date "2000-05-01" @default.
• W2061034807 modified "2023-10-18" @default.
• W2061034807 title "Why Does Threonine, and Not Serine, Function as the Active Site Nucleophile in Proteasomes?" @default.
• W2061034807 cites W1484582783 @default.
• W2061034807 cites W1518071796 @default.
• W2061034807 cites W1585963332 @default.
• W2061034807 cites W1594617739 @default.
• W2061034807 cites W1965790877 @default.
• W2061034807 cites W1968330653 @default.
• W2061034807 cites W1969894269 @default.
• W2061034807 cites W1970498190 @default.
• W2061034807 cites W1973963032 @default.
• W2061034807 cites W1975460230 @default.
• W2061034807 cites W1986397685 @default.
• W2061034807 cites W1991930087 @default.
• W2061034807 cites W1993201799 @default.
• W2061034807 cites W1994454656 @default.
• W2061034807 cites W1995163298 @default.
• W2061034807 cites W1996460413 @default.
• W2061034807 cites W1999119754 @default.
• W2061034807 cites W1999659926 @default.
• W2061034807 cites W2002527751 @default.
• W2061034807 cites W2003849992 @default.
• W2061034807 cites W2004699309 @default.
• W2061034807 cites W2005986251 @default.
• W2061034807 cites W2008744347 @default.
• W2061034807 cites W2010039708 @default.
• W2061034807 cites W2013339424 @default.
• W2061034807 cites W2021769945 @default.
• W2061034807 cites W2024460245 @default.
• W2061034807 cites W2037592320 @default.
• W2061034807 cites W2043358813 @default.
• W2061034807 cites W2044005280 @default.
• W2061034807 cites W2044430464 @default.
• W2061034807 cites W2048190963 @default.
• W2061034807 cites W2052448379 @default.
• W2061034807 cites W2053026504 @default.
• W2061034807 cites W2058106838 @default.
• W2061034807 cites W2062803375 @default.
• W2061034807 cites W2066284211 @default.
• W2061034807 cites W2077405632 @default.
• W2061034807 cites W2081604308 @default.
• W2061034807 cites W2085555089 @default.
• W2061034807 cites W2085760247 @default.
• W2061034807 cites W2086961233 @default.
• W2061034807 cites W2087298783 @default.
• W2061034807 cites W2088749908 @default.
• W2061034807 cites W2090736922 @default.
• W2061034807 cites W2091823485 @default.
• W2061034807 cites W2096063983 @default.
• W2061034807 cites W2106712618 @default.
• W2061034807 cites W2118617212 @default.
• W2061034807 cites W2121781216 @default.
• W2061034807 cites W2123781000 @default.
• W2061034807 cites W2146488386 @default.
• W2061034807 cites W2159831275 @default.
• W2061034807 cites W2160927584 @default.
• W2061034807 cites W2166987747 @default.
• W2061034807 cites W2178503716 @default.
• W2061034807 cites W4235721540 @default.
• W2061034807 cites W4293247451 @default.
• W2061034807 cites W4299297499 @default.
• W2061034807 doi "https://doi.org/10.1074/jbc.275.20.14831" @default.
• W2061034807 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10809725" @default.
• W2061034807 hasPublicationYear "2000" @default.
• W2061034807 type Work @default.
• W2061034807 sameAs 2061034807 @default.
• W2061034807 citedByCount "116" @default.
• W2061034807 countsByYear W20610348072012 @default.
• W2061034807 countsByYear W20610348072013 @default.
• W2061034807 countsByYear W20610348072014 @default.
• W2061034807 countsByYear W20610348072015 @default.
• W2061034807 countsByYear W20610348072016 @default.
• W2061034807 countsByYear W20610348072017 @default.
• W2061034807 countsByYear W20610348072018 @default.
• W2061034807 countsByYear W20610348072019 @default.
• W2061034807 countsByYear W20610348072020 @default.
• W2061034807 countsByYear W20610348072021 @default.
• W2061034807 countsByYear W20610348072022 @default.
• W2061034807 countsByYear W20610348072023 @default.
• W2061034807 crossrefType "journal-article" @default.
• W2061034807 hasAuthorship W2061034807A5006962945 @default.
• W2061034807 hasAuthorship W2061034807A5042332987 @default.
• W2061034807 hasAuthorship W2061034807A5058343808 @default.
• W2061034807 hasBestOaLocation W20610348071 @default.
• W2061034807 hasConcept C11960822 @default.
• W2061034807 hasConcept C14036430 @default.
• W2061034807 hasConcept C161790260 @default.
• W2061034807 hasConcept C178907741 @default.
• W2061034807 hasConcept C181199279 @default.
• W2061034807 hasConcept C185592680 @default.
• W2061034807 hasConcept C2775880066 @default.
• W2061034807 hasConcept C2776414213 @default.

• next