Chapter 2 Cysteine proteinases

Brocklehurst, Keith; Willenbrock, Frances; Salih, Erdjan

doi:10.1016/s0167-7306(09)60016-8

Cited by 78 publications

(78 citation statements)

References 584 publications

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“…At pH Յ 6 the rate represents the burst phase only, whereas at pH Ͼ 6 the primary and secondary phases of the reaction could not be separated, and the fitted rate constant was a composite of the rates of the primary and secondary phases. These profiles are reminiscent of the reactivity of the active site cysteine of papain toward 2-PDS under acidic conditions, which gives a bell-shaped pH profile centered at pH 3.75 (20), as well as the pH profile for the reaction of DTNB with iodoacetamide-treated cytosolic phospholipase A 2 , which showed a maximum at pH 6.5 as well as high reactivity at pH 9.5 (21).…”

Section: Reaction Of Shp1⌬nsh2 With Dtnb and 2-pdsmentioning

confidence: 94%

Active Site Titration of the Tyrosine Phosphatases SHP-1 and PTP1B Using Aromatic Disulfides

Pregel

Storer

1997

Journal of Biological Chemistry

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Aromatic disulfides were found to inactivate truncated forms of the SHP-1 and PTP1B phosphatases by reaction with the essential active site cysteine residue. For truncated SHP-1 at pH 5.0, the reaction proceeded through an initial burst phase followed by a slower secondary phase. Our experiments demonstrated that the burst phase corresponded to the reaction of the aromatic disulfide with the active site cysteine. The magnitude of the burst phase was found to measure the active enzyme concentration, and the rate of the burst reflected the reactivity of the active site cysteine. The data were consistent with a mechanism in which an intramolecular disulfide is formed between the active site cysteine and a proximal cysteine during the burst reaction. Aromatic disulfides were found to react with the active site cysteines of full-length SHP-1 and truncated PTP1B also. Using vanadate to mask the active site cysteine, the active enzyme concentration could be assayed by comparing product yields for the reaction with aromatic disulfides in the presence and absence of vanadate at pH 8.0. These findings demonstrate the utility of aromatic disulfides as active site titrants and reactivity probes for tyrosine phosphatases.

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Section: Reaction Of Shp1⌬nsh2 With Dtnb and 2-pdsmentioning

confidence: 94%

Active Site Titration of the Tyrosine Phosphatases SHP-1 and PTP1B Using Aromatic Disulfides

Pregel

Storer

1997

Journal of Biological Chemistry

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“…Its structure and catalytic mechanism have been intensively studied [2,3], as the findings can be applied to other cysteine proteinases of medical importance. Examples of such proteinases include the caspases and cathepsins, as well as proteinases from viral and bacterial pathogens.…”

Section: Introductionmentioning

confidence: 99%

“…1 Present address : Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. 2 To whom correspondence should be addressed (e-mail timothy.skern!univie.ac.at). abrogated activity towards the hexapeptide.…”

Section: Introductionmentioning

confidence: 99%

Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme

Schlick

Kronovetr

Hampoelz

et al. 2002

Biochemical Journal

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The leader proteinase (Lpro) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp163, which orients the catalytic histidine residue, and Asp164; the equivalent residues in papain are Asn175 and Ser176. The importance of these residues for Lpro activity was examined by site-directed mutagenesis. Replacement of Asp163 with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of Lpro, eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp164 by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp164, and to a lesser extent that of Asp163, is extremely important for Lpro to attain full activity upon synthesis.

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“…However, because of the diversity of cysteine proteases and their contribution to normal physiological processes, cysteine protease inhibitors should preferably be enzyme selective in order to be considered for potential therapeutic applications. Designing inhibitors that are highly selective has proven to be a difficult task for the cathepsin class of cysteine proteases due to their broad substrate specificity [9]. Some success has been obtained using E-64 analogs developed for the specific inhibition of cathepsin B [10].…”

Section: Introductionmentioning

confidence: 99%

Delineating functionally important regions and residues in the cathepsin B propeptide for inhibitory activity

Chen¹,

Plouffe²,

Ménard³

et al. 1996

FEBS Letters

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Synthetic peptides derived from the proregion of rat cathepsin B were used to identify functionally important regions and residues for cathepsin B inhibition. Successive 5 amino acid deletions of a 56 amino acid propeptide from both the N-and Ctermini has allowed the identification of two regions important for inhibitory activity: the NTTWQ (residues 21p-25p) and CGTVL (42p--46p) regions. Alanine scanning of residues within these two regions indicates that Trp-24p and Cys-42p contribute strongly to inhibition, their replacement by Ala resulting in 160-and 140-fold increases in Ki, respectively.

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Chapter 2 Cysteine proteinases

Cited by 78 publications

References 584 publications

Active Site Titration of the Tyrosine Phosphatases SHP-1 and PTP1B Using Aromatic Disulfides

Active Site Titration of the Tyrosine Phosphatases SHP-1 and PTP1B Using Aromatic Disulfides

Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme

Delineating functionally important regions and residues in the cathepsin B propeptide for inhibitory activity

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