scholarly journals Inhibition of glutathione S-transferase 3-3 by glutathione derivatives that bind covalently to the active site

1991 ◽  
Vol 278 (1) ◽  
pp. 63-68 ◽  
Author(s):  
A E P Adang ◽  
W J Moree ◽  
J Brussee ◽  
G J Mulder ◽  
A van der Gen
Keyword(s):  
Active Site ◽  
Current Knowledge ◽  
Control Level ◽  
Gel Filtration ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
British Library ◽  
Glutathione S Transferase ◽  
Enzyme Active Site ◽  
Active Site Cysteine

In all, 13 GSH derivatives have been synthesized and tested for their potency to inhibit glutathione S-transferase (GST) 3-3. All of these derivatives contained a reactive group that could potentially react with the enzyme active site. Best results were obtained with the phenylthiosulphonate derivative of GSH, GSSO2Ph. Preincubation of GST 3-3 with a 100 microM concentration of this inhibitor resulted in a time-dependent loss of activity: after 30 min at pH 6.5 and 25 degrees C, 51% of the activity was lost. At more alkaline pH, the activity is more rapidly inhibited: at pH 8.0 the 90%-inhibition level is already reached after 10 min preincubation. Separation of enzyme and excess unbound GSSO2Ph after preincubation by gel-filtration chromatography did not result in a reappearance of enzyme activity. If 100 microM-GSH was added to the preincubation mixture at pH 7.4, inhibition was almost completely prevented. Addition of S-(hexyl)glutathione (20 microM) could delay the inhibition but, ultimately, not prevent it. The inhibited enzyme could be re-activated by addition of 10 mM-2-mercaptoethanol: 60 min after this thiol was added, the inhibited GST-3- activity was bacxk to the control level. GSH at the same concentration could not re-activate the enzyme. On the basis of these results, on the known reactivity of thiosulphonate compounds, and on current knowledge about the amino acid residues involved in GST catalysis, a covalent modification of an active-site cysteine residue by mixed-disulphide formation between enzyme and the cosubstrate GSH is postulated. Information on the synthesis and characterization of the GSH derivatives is given in Supplementary Publication SUP 50166 (5 pages) which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

scholarly journals Characterization and sequencing of an active-site cysteine-containing peptide from the xylanase of a thermotolerant Streptomyces

1992 ◽  
Vol 281 (3) ◽  
pp. 601-605 ◽  
Author(s):  
S S Keskar ◽  
M B Rao ◽  
V V Deshpande
Keyword(s):  
Gas Phase ◽  
Active Site ◽  
Peptide Mapping ◽  
Gel Filtration ◽  
Cysteine Residue ◽  
Peptide Sequence ◽  
Aspartic Acid Residue ◽  
Active Site Cysteine ◽  
Complete Inactivation ◽  
Kinetics Of

The kinetics of chemical modification of the xylanase from a thermotolerant Streptomyces T7 indicated the involvement of 1 mol of cysteine residue/mol of enzyme [Keskar, Srinivasan & Deshpande (1989) Biochem. J. 261, 49-55]. The chromophoric reagent N-(2,4-dinitroanilino)maleimide (DAM) reacts covalently with thiol groups of xylanase with complete inactivation. Protection against inactivation was provided by the substrate (xylan). The purified xylanase that had been modified with DAM was digested with pepsin and the peptides were purified by gel filtration followed by peptide mapping. The active-site peptide was distinguished from the other thiol-containing peptides by comparison of the peptides generated by labelling the enzyme in the presence and in the absence of the substrate. The peptide mapping of the modified enzyme in the absence of xylan showed three yellow peptides, whereas in the presence of xylan only two yellow peptides were detected. The active-site peptide protected by the substrate failed to form the complex with DAM. The modified active-site peptide was isolated and sequenced. Gas-phase sequencing provided the following sequence: Ser-Val-Ile-Met-Xaa-Ile-Asp-His-Ile-Arg-Phe. This is the first report on the isolation and sequencing of the active-site peptide from a xylanase. The comparison of reactive cysteine-containing peptide sequence with the catalytic regions of other glucanases revealed the presence of a conserved aspartic acid residue.

Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules

2014 ◽  
Vol 70 (5) ◽  
pp. 1311-1320 ◽  
Author(s):  
Antje Schäfer ◽  
Monika Kuhn ◽  
Hermann Schindelin
Keyword(s):  
Active Site ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
Covalent Attachment ◽  
Adenylation Domain ◽  
Active Site Cysteine ◽  
The Status ◽  
Catalytic Cysteine ◽  
Covalently Linked ◽  
First Time

The activation of ubiquitin by the ubiquitin-activating enzyme Uba1 (E1) constitutes the first step in the covalent modification of target proteins with ubiquitin. This activation is a three-step process in which ubiquitin is adenylated at its C-terminal glycine, followed by the covalent attachment of ubiquitin to a catalytic cysteine residue of Uba1 and the subsequent adenylation of a second ubiquitin. Here, a ubiquitin E1 structure loaded with two ubiquitin molecules is presented for the first time. While one ubiquitin is bound in its adenylated form to the active adenylation domain of E1, the second ubiquitin represents the status after transfer and is covalently linked to the active-site cysteine. The covalently linked ubiquitin enables binding of the E2 enzyme without further modification of the ternary Uba1–ubiquitin2arrangement. This doubly loaded E1 structure constitutes a missing link in the structural analysis of the ubiquitin-transfer cascade.

Synthesis and properties of small molecules designed to covalently capture native and oxidized forms of protein tyrosine phosphatase 1B (PTP1B)

2016 ◽  
Author(s):  
◽  
Kasi Viswanatharaju Ruddraraju
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Active Site ◽  
Tyrosine Phosphatase ◽  
Covalent Modification ◽  
Affinity Labeling ◽  
Protein Tyrosine Phosphatase 1B ◽  
University Of Missouri ◽  
Protein Tyrosine ◽  
Enzyme Active Site ◽  
Carbon Nucleophiles

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Protein tyrosine phosphatase 1B (PTP1B) is a validated target for the treatment of type 2 diabetes and obesity. The discovery of selective inhibitors with drug-like properties has proven to be challenging because there are [about]80 PTP family members that share a similar and positively charged active site. To overcome these challenges, we have pursued two novel approaches for the covalent inactivation of PTP1B. Exo-affinity labeling agents exploit covalent reactions with amino acids outside the enzyme active site to gain both affinity and selectivity. We prepared several affinity labeling agents using a 12-step convergent synthesis. Enzyme assays revealed that some of these agents are capable of inactivating the enzyme by covalent modification. In another project, we prepared a low molecular weight mimic of the oxidized form of PTP1B that is generated in cells, during insulin signaling events. Seeking molecules capable of covalent capture of oxidized PTP1B, we treated this chemical model with several carbon nucleophiles, such as 1,3-diketones and sulfone-stabilized carbon anions. These carbon nucleophiles readily reacted with the model compound, under mild conditions to give stable adducts. Inactivation experiments revealed that 1,3-diketones are capable of inactivating the oxidized PTP1B at micromolar concentrations.

scholarly journals The location of the active-site histidine residue in the primary sequence of papain

1968 ◽  
Vol 108 (5) ◽  
pp. 861-866 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Amino Acid ◽  
Sodium Borohydride ◽  
Active Site ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Primary Sequence ◽  
Active Site Cysteine ◽  
Active Site Histidine ◽  
Active Site Cysteine Residue

Papain that had been irreversibly inhibited with 1,3-dibromo[2−14C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

scholarly journals Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum

Biochemistry ◽  
2018 ◽  
Vol 57 (38) ◽  
pp. 5513-5523 ◽  
Author(s):  
Matthias Fellner ◽  
Joel A. Rankin ◽  
Benoît Desguin ◽  
Jian Hu ◽  
Robert P. Hausinger
Keyword(s):  
Lactobacillus Plantarum ◽  
Active Site ◽  
Cysteine Residue ◽  
Active Site Cysteine ◽  
Active Site Cysteine Residue

scholarly journals Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols

2006 ◽  
Vol 398 (2) ◽  
pp. 197-206 ◽  
Author(s):  
Jingmin Zeng ◽  
Rachael A. Dunlop ◽  
Kenneth J. Rodgers ◽  
Michael J. Davies
Keyword(s):  
Active Site ◽  
Cysteine Proteases ◽  
Cysteine Residue ◽  
Dependent Manner ◽  
Cross Link ◽  
Concentration Dependent Manner ◽  
Active Site Cysteine ◽  
Reactive Aldehydes ◽  
Modified Proteins ◽  
Active Site Cysteine Residue

Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259–269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra-molecular active site cysteine–lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease.

Novel NADPH–cysteine covalent adduct found in the active site of an aldehyde dehydrogenase

2011 ◽  
Vol 439 (3) ◽  
pp. 443-455 ◽  
Author(s):  
Ángel G. Díaz-Sánchez ◽  
Lilian González-Segura ◽  
Enrique Rudiño-Piñera ◽  
Alfonso Lira-Rocha ◽  
Alfredo Torres-Larios ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
Enzyme Inactivation ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Oxidative Stresses ◽  
Covalent Adduct

PaBADH (Pseudomonas aeruginosa betaine aldehyde dehydrogenase) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to its corresponding acid, the osmoprotector glycine betaine. This reaction is involved in the catabolism of choline and in the response of this important pathogen to the osmotic and oxidative stresses prevalent in infection sites. The crystal structure of PaBADH in complex with NADPH showed a novel covalent adduct between the C2N of the pyridine ring and the sulfur atom of the catalytic cysteine residue, Cys286. This kind of adduct has not been reported previously either for a cysteine residue or for a low-molecular-mass thiol. The Michael addition of the cysteine thiolate in the ‘resting’ conformation to the double bond of the α,β-unsaturated nicotinamide is facilitated by the particular conformation of NADPH in the active site of PaBADH (also observed in the crystal structure of the Cys286Ala mutant) and by an ordered water molecule hydrogen bonded to the carboxamide group. Reversible formation of NAD(P)H–Cys286 adducts in solution causes reversible enzyme inactivation as well as the loss of Cys286 reactivity towards thiol-specific reagents. This novel covalent modification may provide a physiologically relevant regulatory mechanism of the irreversible PaBADH-catalysed reaction, preventing deleterious decreases in the intracellular NAD(P)+/NAD(P)H ratios.

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