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.

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 Conformational changes in Apolipoprotein N-acyltransferase (Lnt)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin Wiseman ◽  
Martin Högbom
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Cell Envelope ◽  
Covalent Modification ◽  
Covalent Attachment ◽  
Gram Negative Bacteria ◽  
Cellular Functions ◽  
Crystal Forms ◽  
Active Site Cysteine ◽  
Open Conformation

AbstractLipoproteins are important components of the cell envelope and are responsible for many essential cellular functions. They are produced by the post-translational covalent attachment of lipids that occurs via a sequential 3-step process controlled by three integral membrane enzymes. The last step of this process, unique to Gram-negative bacteria, is the N-acylation of the terminal cysteine by Apolipoprotein N-acyltransferase (Lnt) to form the final mature lipoprotein. Here we report 2 crystal forms of Lnt from Escherichia coli. In one form we observe a highly dynamic arm that is able to restrict access to the active site as well as a covalent modification to the active site cysteine consistent with the thioester acyl-intermediate. In the second form, the enzyme crystallized in an open conformation exposing the active site to the environment. In total we observe 3 unique Lnt molecules that when taken together suggest the movement of essential loops and residues are triggered by substrate binding that could control the interaction between Lnt and the incoming substrate apolipoprotein. The results provide a dynamic context for residues shown to be central for Lnt function and provide further insights into its mechanism.

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.

scholarly journals Active site alanine substitutions can convert deubiquitinating enzymes into avid ubiquitin-binding domains

2017 ◽  
Author(s):  
Marie Morrow ◽  
Michael Morgan ◽  
Marcello Clerici ◽  
Katerina Growkova ◽  
Ming Yan ◽  
...  
Keyword(s):  
Active Site ◽  
Tight Binding ◽  
Dominant Negative ◽  
Structural Basis ◽  
Deubiquitinating Enzymes ◽  
Active Site Cysteine ◽  
Binding Domains ◽  
Common Strategy ◽  
Catalytic Cysteine

ABSTRACTA common strategy for studying the biological role of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

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