Demonstration that 1-trans-epoxysuccinyl-<scp>l</scp>-leucylamido-(4-guanidino) butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis. Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-β-trypsin complex

Sreedharan, Suneal K.; Verma, Chandra; Caves, Leo S. D.; Brocklehurst, Simon M.; Gharbia, Saheer E.; Shah, Haroun N.; Brocklehurst, Keith

doi:10.1042/bj3160777

Cited by 53 publications

(30 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This supports our contention that Phe is not readily accepted in the P 1 -binding site (Tables I and III). Interestingly, OP-Tb was inhibited competitively by E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane), with a K i of 62.5 M. This contrasts with the widely held view that E-64 is a class-specific inhibitor of cysteine peptidases (52), although the inhibition of bovine ␤-trypsin by E-64 by a reversible competitive mechanism with a K i of 36 M has also been reported (53). Benzamidine, a low molecular mass inhibitor of trypsin-like peptidases, was a comparatively poor inhibitor of OP-Tb, with a K i of 254 M, compared with K i values of 36 M for bovine ␤-trypsin and 12 M for mast cell tryptase (54).…”

contrasting

confidence: 49%

Oligopeptidase B from Trypanosoma brucei, a New Member of an Emerging Subgroup of Serine Oligopeptidases

Morty

Lonsdale‐Eccles

Morehead

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

Trypanosoma brucei contains a soluble serine oligopeptidase (OP-Tb) that is released into the host bloodstream during infection, where it has been postulated to participate in the pathogenesis of African trypanosomiasis. Here, we report the identification of a single copy gene encoding the T. brucei oligopeptidase and a homologue from the related trypanosomatid pathogen Leishmania major. The enzymes encoded by these genes belong to an emerging subgroup of the prolyl oligopeptidase family of serine hydrolases, referred to as oligopeptidase B. The trypanosomatid oligopeptidases share 70% amino acid sequence identity with oligopeptidase B from the intracellular pathogen Trypanosoma cruzi, which has a demonstrated role in mammalian host cell signaling and invasion. OP-Tb exhibited no activity toward the prolyl oligopeptidase substrate H-Gly-Pro-7-amido-4-methylcoumarin. Instead, it had activity toward substrates of trypsin-like enzymes, particularly those that have basic amino acids in both P 1 and P 2 (e.g. benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin k cat /K m ‫؍‬ 529 s ؊1 M ؊1). The activity of OP-Tb was enhanced by reducing agents and by polyamines, suggesting that these agents may act as in vivo regulators of OP-Tb activity. This study provides the basis of the characterization of a novel subgroup of serine oligopeptidases from kinetoplastid protozoa with potential roles in pathogenesis.

show abstract

contrasting

confidence: 49%

Oligopeptidase B from Trypanosoma brucei, a New Member of an Emerging Subgroup of Serine Oligopeptidases

Morty

Lonsdale‐Eccles

Morehead

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…E-64, originally discovered as an inhibitor of papain-like cysteine proteases, 18,29 was also shown to inhibit calpain, although with an order of magnitude lower rate constants of inactivation (k 2 /K 1 Z80 000/M/s versus k 2 /K 1 B8000/M/s; 8,30,31 ), and even a serine protease trypsin, although by a different, reversible mechanism with a K i value of 0.3 mM. 32 CA-074, a cathepsin B-specific E-64 analogue, 19 failed when it came to the specificity in in vivo studies. Namely, its esterified cell- Figure 3 Inhibition of cathepsins B, X, H and C in rat liver extracts by E-64, Z-DEVD-fmk, Z-YVAD-fmk and Z-VAD-fmk.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity

et al. 2003

View full text Add to dashboard Cite

We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 lM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 lM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.

show abstract

“…The trans-epoxysuccinyl group (active moiety) of E64 irreversibly binds to an active thiol group of many cysteine proteases such as papain, actinidase, and cathepsins B, H, and L (58) to form a thioether linkage. The mechanism of inhibition of some cysteine proteases including cathepsins B and L, and of trypsin, have been reported (58).…”

Section: Methodsmentioning

confidence: 99%

Extracellular proteasome in the human alveolar space: a new housekeeping enzyme?

Sixt¹,

Beiderlinden²,

Jennissen³

et al. 2007

American Journal of Physiology-Lung Cellular and Molecular Phys

View full text Add to dashboard Cite

We hypothesized that 20S proteasome is present and functional in the extracellular alveolar space in humans. Proteasomal activity was measured in bronchoalveolar lavage (BAL) supernatant from eight humans using specific proteasomal fluorogenic substrates and I 125 -albumin with and without specific proteasome inhibitors. Furthermore, gelfiltration, Western blot technique, and mass spectrometry were applied for proteasome characterization. All proteasomal fluorogenic substrates were hydrolyzed by BAL supernatant, with hydrolysis inhibited by epoxomicin (P ϭ 0.024) and other proteasome inhibitors as well. E64, a lysosomal inhibitor, did not inhibit enzyme activity. The majority of proteolytic activity was detected in BAL supernatant rather than in the cell pellet. No correlation was found between proteasomal hydrolysis in BAL supernatant and lactate dehydrogenase activity, the total cell count in the cell pellet, and the fraction of avital cells in the cell pellet, ruling out cell lysis as a major source of proteasomal activity. Gelfiltration revealed hydrolyzing activity in the supernatant at 660 kDa and proteasome core proteins after analysis by ESI-QqTOF mass spectrometry. Furthermore, Western blots using a polyclonal antibody against proteasomal ␣-/␤-subunits detected proteasomal proteins in the typical 20-to 30-kDa range in BAL supernatant. Incubation of BAL supernatant with I 125 -albumin showed a high mean cleavage rate (101.8 g/ml ϫ h lavage Ϯ 46 SD) that was inhibited by epoxomicin (P ϭ 0.013) and was ATP and ubiquitin independent. We identified for the first time extracellular, biologically active, ATP-and ubiquitin-independent 20S proteasome in the human alveolar space, with a high albumin cleavage rate. Possibly, the proteasome assists in maintenance of a low intraalveolar oncotic pressure and/or alveolar protein degradation. albumin; bronchoalveolar lavage; circulating proteasome; fluorogenic peptides; alveolar protein degradation; lung proteins THE UBIQUITIN/PROTEASOME SYSTEM is a major pathway for selective intracellular non-lysosomal protein degradation in eukaryotic cells and plays an important role in numerous processes (3, 13, 53). The 20S proteasome is a multicatalytic proteinase complex with a cylinder-shaped structure arranged as four axially stacked heptameric rings composed of seven ␣-subunits (outer rings) and seven ␤-subunits (inner rings) (36), respectively. Its catalytic sites are exclusively associated with the ␤-subunits (40, 56) and are ATP and ubiquitin independent. The caspase-like activity of the ␤ 1 -subunit cleaves after acidic residues, the trypsin-like activity of the ␤ 2 -subunit cleaves after basic residues, and the chymotrypsin-like activity of the ␤ 5 -subunit cleaves after hydrophobic residues. Together, these three enzyme activities allow the proteasome to cleave many different substrates into diverse products. Within the cell, the 20S proteasome is associated with large ATP-and ubiquitindependent 19S regulatory cap-like complexes, together yielding a 26S complex. It is ...

show abstract

Demonstration that 1-trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis. Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-β-trypsin complex

Cited by 53 publications

References 48 publications

Oligopeptidase B from Trypanosoma brucei, a New Member of an Emerging Subgroup of Serine Oligopeptidases

Oligopeptidase B from Trypanosoma brucei, a New Member of an Emerging Subgroup of Serine Oligopeptidases

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity

Extracellular proteasome in the human alveolar space: a new housekeeping enzyme?

Contact Info

Product

Resources

About