Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The multicatalytic proteinase complex (MPC) or proteasome is a multimeric, high-molecular-weight (700,000), extralysosomal proteolytic enzyme found in eukaryotes and in archaebacteria. Its multiple catalytic sites grant it a broad cleavage specificity toward short peptides and protein substrates. The pH optima of the catalytic activities of MPC are in the neutral or slightly alkaline range. We present here evidence for cryptic catalytic components of MPC optimally active at an acidic pH. Studies with a hydrophobic fluorescent probe provide direct evidence for conformational changes brought about by exposing the complex to an acidic environment. One of the newly described components, designated "acidic chymotrypsin-like activity," cleaves the Leu-2-naphthylamide bond in the substrate Boc-Val-Glu-Ala-Leu-2-naphythylamide. Compared with the classical "neutral" chymotrypsin-like activity defined by cleavage of the Leu-p-nitroanilide bond in Z-Gly-Gly-Leu-p-nitroanilide, the newly described component is not inhibited by monovalent cations and is less sensitive to the peptidyl aldehyde Z-Gly-Gly-leucinal, an inhibitor of the neutral chymotrypsin-like activity. In addition, we describe the properties of a novel potent peptidyl aldehyde, Z-Ile-Glu(OtBu)-Ala-leucinal, which is an inhibitor of both the acidic and neutral chymotrypsin-like activities of MPC, with IC50 values of 0.25 and 6.5 microM, respectively. In the presence of 65 microM of the newly synthesized peptidyl aldehyde, other MPC components such as the trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities were decreased only by 14 and 9%, respectively. The hydrophobicity, potency, and specificity of Z-Ile-Glu(OtBu)-Ala-leucinal toward the chymotrypsin-like activities of the complex make it a valuable pharmacological tool with which to investigate the physiological roles of MPC.
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PMID:A novel chymotrypsin-like component of the multicatalytic proteinase complex optimally active at acidic pH. 787 5

Evidence indicates that a component of the multicatalytic proteinase complex (MPC) that preferentially cleaves bonds after branched chain amino acids (BrAAP) is a major factor responsible for the protein-degrading activity of the MPC. We report here the synthesis of substrate-related peptidyl aldehydes that inhibit the activity of this component toward both synthetic peptide substrates and proteins. The most potent of the inhibitors, Cbz-Gly-Pro-Phe-leucinal (Cbz-GPFL-CHO) inhibits competitively with a Ki of 1.5 microM. The peptidyl aldehydes also inhibit the small neutral amino acid preferring and the peptidylglutamyl-peptide hydrolyzing activities of the MPC. The chymotrypsin-like activity is only weakly inhibited, and the trypsin-like activity is moderately activated. The importance of a Pro residue in the P3 position and a leucinal residue in the P1 position for inhibition of the BrAAP component is indicated by the finding that replacement of these residues by a glycine or phenylalaninal, respectively, markedly increases the Ki. Cbz-GPFL-CHO inhibited the BrAAP activity with the same Ki both before and after activation of this component by exposure of the MPC to 3,4-dichloroisocoumarin, suggesting that the peptidyl aldehyde is an effective inhibitor of both the overt and latent proteolytic activities of the MPC. Incubation of a human breast cancer cell line (MCF-7) in culture with the inhibitors of the BrAAP component led to an accumulation of ubiquitin-protein conjugates, indicating inhibition of the ubiquitin-dependent proteolytic pathway.
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PMID:Inhibition of the proteolytic activity of the multicatalytic proteinase complex (proteasome) by substrate-related peptidyl aldehydes. 796 80

The gene of subtilisin S41, an alkaline protease secreted by the psychrophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids residues. The nucleotide sequence and NH2- and COOH-terminal amino acid sequencing of the purified enzyme indicate that the mature subtilisin S41 is composed of 309 residues with a predicted M(r) = 31,224. Subtilisin S41 shares most of its properties with mesophilic subtilisins (structure of the precursor, 52% amino acid sequence identity, alkaline pH optimum, broad specificity, Ca2+ binding) but is characterized by a higher specific activity on macromolecular substrate, by a shift of the optimum of activity toward low temperatures, and by a low thermal stability. The enzyme also differs by an acidic pI (5.3) and the presence of one disulfide bond. It is proposed that the psychrophilic enzyme possesses a more flexible molecular structure when compared to mesophilic and thermophilic subtilases in order to compensate for the reduction of reaction rates at low temperatures. The model of subtilisin S41 indeed reveals several features able to induce a more flexible, heat-labile conformation: the occurrence of four extended surface loops, a very hydrophilic surface through 11 extra Asp residues, and the lack of several salt bridges and aromatic-aromatic interactions. The low affinity of the Ca1 calcium binding site (Kd(app) = 10(-6) M), resulting possibly from one chelating side chain substitution and the stacking of Gly residues, also reflect a less compact conformation. The difference of free energy of stabilization between subtilisin S41 and a mesophilic subtilisin suggests that the balance of exo- and endothermically formed weak bonds is critical for the enzyme flexibility.
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PMID:Cold adaptation of proteins. Purification, characterization, and sequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41. 802 Dec 48

Diastereomeric peptide-esters (Ala-Ala-AA2-Phe-OMe, AA2 = Gly, D- or L-Ala, Pro, Phe, Lys, and Glu) have been used as substrates, and the kinetic constants (Kcat and Km) of the three alkaline proteases, subtilisin Carlsberg, alcalase, and Nagarse (subtilisin BPN') catalyzed ester-hydrolysis, were measured to investigate the selectivity of the enzyme-catalyzed peptide esterhydrolysis. All three proteases preferred the substrate which had a small side-chain at the s-2 site. Thus, the substrates with a bulky side-chain at the p-2 site such as Phe, Pro, Glu, and Lys, hydrolyzed with a rate of about one-tenth that of Ala at the p-2 site, and the Kcat decreased as the size of the p-2 amino acid residue increased. The diastereoselectivity of the alkaline protease-catalyzed hydrolysis of each diastereomeric pair depended on the size of the amino acid residue at the p-2 position of the substrate. The substrates with a bulky side-chain at the p-2 site hydrolyzed with higher diastereoselectivity than did the substrates with a small side-chain at the p-2 site. Molecular modeling of the enzyme-substrate complex show that: for the enzyme complexed with a substrate which has L-L-L-L configuration, each residue of the L-L-L-L tetrapeptide filled in and was completely enclosed by the cleft of the four subsites of the enzyme. The side-chains of the residues were identically positioned within the pocket of the binding-site. For the complex of enzyme with substrate of L-L-D-L, the side-chain of the D-amino acid residue was far away from the s-2 subsite of the enzyme, and had close contact with the side-chains of Leu-126 and Ile-107 of the enzyme.
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PMID:Investigating the s-2 subsite selectivity of alkaline proteases in hydrolysis of diastereo-peptide esters and molecular-modeling interpretation. 808 66

The multicatalytic proteinase complex (MPC; proteasome) can be isolated in a latent form which then can be activated for protein hydrolysis by physiological and nonphysiological treatments, including high temperature. In this study, the temperature dependency profiles for the hydrolysis of Cbz-Gly-Gly-Leu-pNA and Cbz-Val-Gly-Arg-pNA by bovine lens MPC are found to be those expected for a thermostable enzyme, with single optima above 50 degrees C. In contrast, hydrolyses of Cbz-Leu-Leu-Glu-2NNp and alpha 2-crystallin, a lens structural protein, show two temperature transitions, indicating that hydrolysis of these substrates can be activated by elevated temperature. Temperature dependency profiles of peptidase activity in Tris-HCl compared to Hepes buffer suggest that Tris decreases the thermal stability of MPC. After 10 min preincubation in Tris-HCl at 53 degrees C, lens MPC activities are reduced by 50-60% and loss of the major MPC band can be seen on nondenaturing gels. The presence of alpha 2-crystallin during preincubation partially prevents the loss of activity. Although alpha-crystallin has been reported to function as a molecular chaperone, similar protection by other MPC substrates suggests that alpha 2-crystallin stabilized the MPC as a substrate. Our findings indicate both activation and inactivation of the enzyme at elevated temperatures. It is proposed therefore that high temperature activates the MPC but to a more labile form which can be partially stabilized by protein substrates.
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PMID:Thermal stability and activation of bovine lens multicatalytic proteinase complex (proteasome). 823 52

The fate of a mutant form of each of the two yeast vacuolar enzymes proteinase yscA (PrA) and carboxypeptidase yscY (CPY) has been investigated. Both mutant proteins are rapidly degraded after entering the secretory pathway. Mutant PrA is deleted in 37 amino acids spanning the processing site region of the PrA pro-peptide. The mutant enzyme shows no activity towards maturation of itself or other vacuolar hydrolases, a function of wild-type PrA. Mutant CPY carries an Arg instead of a Gly residue in a highly conserved region, two positions distant from the active-site Ser. In contrast to wild-type CPY, the mutant form was quickly degraded by trypsin in vitro, indicating an altered structure. Using antisera specific for alpha-1-->6 and alpha-1-->3 outer-chain mannose linkages, no Golgi-specific carbohydrate modification could be detected on either mutant protein. Subcellular fractionation studies located both mutant enzymes in the endoplasmic reticulum. Degradation kinetics of both proteins show the same characteristics, indicating similar degradation pathways. The degradation process was shown to be independent of a functional sec18 gene product and takes place before Golgi-specific carbohydrate modifications occur. The proteasome, the major proteolytic activity of the cytoplasm, is not involved in this degradation event. All degradation characteristics of the two mutant proteins are consistent with a degradation process within the endoplasmic reticulum ('ER degradation').
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PMID:Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. 826 47

The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptide hydrolase' activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit 'chymotrypsin-like' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of 'peptidylglutamyl-peptide hydrolase' activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.
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PMID:Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes. 828 57

Within 1 h after slaughter, two 10-g samples of longissimus muscle were obtained from four crossbred beef cattle. Samples were homogenized in three or six volumes of extraction solution that consisted of 50 mM Tris base, 10 mM EDTA, and 10 mM 2-mercaptoethanol, pH adjusted to 8.3 with 6 N HCl. After centrifugation the supernatant from the three-volume extract was fractionated by addition of solid (NH4) 2SO4. Proteins that precipitate between 40 and 65% (NH4) 2SO4 were dialyzed and then loaded onto a DEAE-Sephacel column and eluted with a continuous gradient of NaCl from 100 to 400 mM (125 mL of each; Method A). The six-volume extract was loaded onto a DEAE-Sephacel column and eluted with a continuous gradient of NaCl from 0 to 350 mM (250 mL of each; Method B). Total peptidase activity eluted from the column was determined using the synthetic peptide N-CBZ-Gly-Gly-Leu-p-nitroanilide. Method B yielded greater multicatalytic proteinase complex (MCP) activities (picomoles of p-nitroaniline released/hour-1) per gram of muscle (1,538.25 +/- 105.15) than did Method A (1,195.05 +/- 86.55; P < .05). In addition, Method B permitted the quantification of calpain activity from the same fractions eluted. The relationship between enzyme activity and assay time (up to 45 min) and protein concentration (up to 10 micrograms) in the assay was linear. Studies indicated that the optimum temperature is in the range of 50 to 60 degrees C and the optimum pH in the range of 7.5 to 8.5.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Technical note: quantification of multicatalytic proteinase complex (proteasome) activity by ion-exchange chromatography. 829 81

Initial studies on the specificity of the multicatalytic proteinase complex (MPC; EC 3.4.99.46) led to the identification of three distinct proteolytic components designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing, all sensitive to inactivation by 3,4-dichloroisocoumarin (DCI), a general serine proteinase inhibitor. The three components cleave the peptidyl-arylamide bonds in the model synthetic substrates, Z-(D)-Ala-Leu-Arg-2-naphthylamide, Z-Gly-Gly-Leu-p-nitroanilide, and Z-Leu-Leu-Glu-2-naphthylamide, respectively. We report here evidence for the presence in the MPC of two additional distinct components, neither of them capable of cleaving the three model substrates. One of these components cleaves the Leu-Gly and the Leu-Ala bonds in the substrates Cbz-Gly-Pro-Ala-Leu-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Leu-Ala-p-aminobenzoate, respectively, and is activated by treatment of the MPC with DCI, N-ethylmaleimide, Mg2+, Ca2+, and low concentrations of sodium dodecyl sulfate and fatty acids. This component is apparently identical with the previously identified DCI-resistant component of the MPC that cleaves preferentially bonds on the carboxyl side of branched chain amino acids in natural peptides including neurotensin and proinsulin [Cardozo, C., Vinitsky, A., Hidalgo, M. C., Michaud, C., & Orlowski, M. (1992) Biochemistry 31, 7373-7380]. It is probably also identical with the component proposed to be the main factor responsible for the caseinolytic activity [Pereira, M. E., Nguyen, T., Wagner, B. J., Margolis, J. W., Yu, B., & Wilk, S. (1992a) J. Biol. Chem. 267, 7949-7955]. The designation "branched chain amino acid preferring" (BrAAP) is proposed for this component. The second component cleaves peptide bonds between the small neutral amino acids Ala-Gly and Gly-Gly in the substrates Cbz-Gly-Pro-Ala-Ala-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Gly-Gly-p-aminobenzoate, respectively. This component is sensitive to inactivation by DCI, N-ethylmaleimide, and organic mercurials, but unlike the BrAAP it is significantly activated neither by Mg2+ or Ca2+ nor by fatty acids or sodium dodecyl sulfate. The designation "small neutral amino acid preferring" (SNAAP) is proposed for this component. Both components are sensitive to inhibition by the peptidyl-aldehydes N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO; calpain inhibitor I) and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO; calpain inhibitor II) but are resistant to inhibition by Z-LLF-CHO, a potent inhibitor of the chymotrypsin-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Evidence for the presence of five distinct proteolytic components in the pituitary multicatalytic proteinase complex. Properties of two components cleaving bonds on the carboxyl side of branched chain and small neutral amino acids. 843 36

Using specific substrates, benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide, benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide, cytosolic chymotrypsin-like, trypsin-like and cucumsin-like activities were determined, respectively, in rat epithelial tissues and differentiated human Caco-2 cells. The cytosolic fractions of rat colonic, rectal, nasal, and alveolar epithelial cells and differentiated human Caco-2 cells contained these three distinct enzyme activities. However, effects of enzyme inhibitors revealed that these three distinctive activities were not extensively involved in cytosolic or homogenate degradation of insulin and insulin-like growth factor I (IGF-I). It is concluded that proteasome-like activities may not significantly limit nonparenteral absorption of peptide and protein drugs such as insulin and IGF-I.
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PMID:The involvement of cytosolic chymotrypsin-like, trypsin-like, and cucumsin-like activities in degradation of insulin and insulin-like growth factor I by epithelial tissues. 858 71


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