Steady-State Kinetic Analysis of Human Ubiquitin-Activating Enzyme (E1) Using a Fluorescently Labeled Ubiquitin Substrate

Wee, Kevin; Lai, Zhihong; Auger, Kurt R.; Ma, Jun; Horiuchi, Kazuo; Dowling, Randine L.; Dougherty, Cristy S.; Corman, Jeanne I.; Wynn, Richard; Copeland, Robert A.

doi:10.1023/a:1026501515450

Cited by 24 publications

(19 citation statements)

References 19 publications

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“…The rate of Ub activation increased linearly with E1 concentration (Figure S4) but did not depend on Ub concentration in the 25 μM-200 μM range, consistent with the published K m values (0.2-2 μM). 34 The initial rates of Ub activation reaction are shown in Table S1. Since at saturation the initial rate equals k cat [E1] 0 , we determined the turnover rate for Ub to be k cat = 4.28 ± 0.90 min -1 , in good agreement with the published value (4.3 ± 1.2 min -1 ).…”

Section: Methodsmentioning

confidence: 99%

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1

et al. 2017

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Protein ubiquitination plays a role in essentially every process in eukaryotic cells. The attachment of ubiquitin (Ub) or Ub-like (UBL) proteins to target proteins is achieved by parallel but distinct cascades of enzymatic reactions involving three enzymes: E1, E2, and E3. The E1 enzyme functions at the apex of this pathway and plays a critical role in activating the C-terminus of ubiquitin or UBL, which is an essential step that triggers subsequent downstream transfer to their cognate E2s resulting in the fidelity of the Ub/UBL conjugation machinery. Despite the central role of the E1 enzyme in protein modification, a quantitative method to measure Ub/UBL activation by E1 is lacking. Here, we present a mass spectrometry-based assay to accurately measure the activation of Ub/UBL by E1 independent of the E2/E3 enzymes. Our method does not require radiolabeling of any components and therefore can be used in any biochemical laboratory having access to a mass spectrometer. This method allowed us to dissect the concerted process of E1-E2-catalyzed Ub conjugation in order to separately characterize the process of Ub activation and how it is affected by select mutations and other factors. We found that the hydrophobic patch of Ub is important for the optimal activation of Ub by E1. We further show that the blockers of the Ub-proteasome system such as ubistatin and fullerenol inhibit Ub activation by E1. Interestingly, our data indicate that the phosphorylation of Ub at the S65 position augments its activation by the E1 enzyme.

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Section: Methodsmentioning

confidence: 99%

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1

et al. 2017

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“…Protein Expression, Purification, and Modification-E1 and Ubc4 (UbcH5B (18)) were expressed and purified as previously described (19). Ubiquitin (Sigma-Aldrich) was chemically modified with Oregon Green (og) succinimidyl ester as described by the manufacturer (Molecular Probes) and purified as described previously (19).…”

Section: Methodsmentioning

confidence: 99%

“…Ubiquitin (Sigma-Aldrich) was chemically modified with Oregon Green (og) succinimidyl ester as described by the manufacturer (Molecular Probes) and purified as described previously (19).…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescence intensity was converted to molar units of og-Ub by reference to an og-Ub calibration curve and assuming that the quantum yield for og-Ub fluorescence was not significantly affected by formation of the og-Ub⅐p53 complex. Little or no change in the quantum yield for Oregon Green was detected after conjugating to Ub (19). Steady-state Analysis-The majority of steady-state experiments were run at a single time point of 20 min, using p53 (1 M), og-Ub⅐Ubc4 (5 M), and mdm2 (3 nM).…”

Section: Methodsmentioning

confidence: 99%

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Human mdm2 Mediates Multiple Mono-ubiquitination of p53 by a Mechanism Requiring Enzyme Isomerization

Lai

Ferry

Diamond

et al. 2001

Journal of Biological Chemistry

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145

104

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The mdm2 gene product is an important regulator of p53 function and stability. mdm2 is an E3 ubiquitin ligase for p53 and the RING finger domain of mdm2 is critical for ligase activity. Ubiquitin (Ub) conjugation is a general targeting modification and poly-ubiquitin chains specifically target proteins to the proteasome for degradation. In this report, we show that the multistep cascade of mdm2-mediated p53 ubiquitination can be reduced to three purified recombinant proteins: ubiquitin-conjugated E2, mdm2, and p53. This simplification allows enzymatic analysis of the isolated ligase reaction. The simplified reaction recapitulates the ubiquitination of p53 observed with individual components and the p53-Ub (n) is qualitatively similar to p53-Ub (n) detected in lactacystin-treated cells. Surprisingly, we find that p53 is modified with multiple mono-ubiquitin moieties as opposed to a poly-ubiquitin chain. Finally, kinetic analysis indicates the transfer reaction proceeds either through a modified Ping Pong mechanism involving requisite enzyme isomerization steps, or through a Rapid Equilibrium Random Bi Bi mechanism involving very large anti-cooperative interactions between the two substrate binding pockets on the enzyme, mediated through allosteric changes in enzyme structure.The p53 gene product is an important tumor suppressor and is inactivated by deletion or mutation in approximately 50% of all human cancer. The p53 protein functions as a transcription factor that binds DNA and induces the expression of a number of genes involved in cell growth arrest, DNA repair, and apoptosis. p53 is maintained at low steady-state levels in the cell and is induced and activated post-translationally by various signaling pathways that respond to cellular stress (1, 2). Cellular insults that initiate the stress response and activate p53 include DNA-damaging agents (chemical, UV, and ionizing radiation), oxygen stress, or the inappropriate activation of oncogenes. Activated p53 induces either growth arrest or apoptosis depending on the extent and duration of signals generated from the damage (3, 4). The post-translational modifications involved in p53 activation and increased steady-state levels of the protein include phosphorylation, dephosphorylation, acetylation, sumoylation, and ubiquitination. The stability and half-life of p53 are tightly regulated by mdm2 and the ubiquitin-proteasome pathway (5-7). Recent evidence suggests that mdm2 is an E3 1 ubiquitin ligase for p53 (8, 9). A number of critical regulatory proteins in the cell are modified by ubiquitin (Ub) conjugation. Proteasomal degradation of key regulatory proteins control biological events involving the cell cycle, differentiation, immune responses, DNA repair, chromatin structure, and apoptosis (10). The initial step in the Ub cascade is the activation of Ub by the ubiquitin-activating enzyme (E1). E1 hydrolyzes ATP to AMP and pyrophosphate to generate a thioester bond between the active site Cys of E1 and the carboxylterminal Gly of Ub. The activated Ub is transf...

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“…Dies zeigt, dass auch der K M -Wert für IV deutlich hçher ist als der für ATP [(4.7 AE 1.0) mm]. [12] Wegen der sperrigen Modifikationen an ATP ist dies jedoch nicht erstaunlich.…”

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Fluorogene ATP‐Analoga zur Detektion von ATP‐Verbrauch: Beobachtung der Aktivierung von Ubiquitin in Echtzeit

et al. 2013

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Die Konjugation von Ubiquitin an verschiedenste Proteine spielt eine entscheidende Rolle für die Regulation zahlloser zellulärer Vorgänge.[1] Die Fehlregulation dieses Vorgangs wurde mit verschiedenen humanen Erkrankungen, darunter Krebs und neurodegenerative Erkrankungen, in Verbindung gebracht.[2] Für die Ubiquitylierung wird Ubiquitin zunächst von einem ubiquitinaktivierenden Enzym (E1) auf Kosten von ATP (ATP = Adenosintriphosphat) aktiviert (Abbildung 1 a), wobei sich ein Thioester zwischen dem C-terminalen Glycin von Ubiquitin und einem Cystein von E1 bildet.[3] Darauffolgender Transfer auf einen Cysteinrest eines ubiquitinkonjugierenden Enzyms (E2) initiiert die Konjugation von Ubiquitin an ein Zielprotein (zumeist über eine Isopeptidbindung an einen Lysinrest). Dieser Prozess bençtigt in den meisten Fällen die Hilfe einer Ubiquitin-Ligase (E3).[3] Da UBA1 eines von nur zwei verschiedenen bekannten humanen E1-Enzymen für Ubiquitin ist, [4] kçnnte die Veränderung seiner Aktivität vorteilhaft für die Behandlung verschiedener Krankheiten sein. Daher sind Assays, die es ermçgli-chen, die Aktivierung von Ubiquitin durch UBA1 direkt und ohne den Einfluss weiterer Enzyme der Ubiquitylierungskaskade zu studieren, wichtige Hilfsmittel, um die Aktivität von UBA1 zu analysieren und Modulatoren seiner Aktivität zu identifizieren.Bisher wurden allerdings nur wenige Assays beschrieben, mit deren Hilfe sich die Aktivität von UBA1 direkt bestimmen lässt.[5] Diese Assays sind arbeitsaufwändig und nicht geeignet, die Aktivität von UBA1 kontinuierlich zu messen.Bis heute gibt es keinen Assay, der es ermçglicht, die Aktivierung von Ubiquitin in Echtzeit direkt zu untersuchen, und somit geeignet wäre, Effektoren von UBA1 auf einfache Weise zu identifizieren. Daher haben wir uns entschieden, einen konzeptionell neuen Assay auszuarbeiten: Echtzeitdetektion der Ubiquitinaktivierung durch die Beobachtung der Spaltung eines ATP-Analogons (Abbildung 1 b), das mit zwei Fluorophoren markiert ist, die Fçrster-Resonanzenergietransfer (FRET) eingehen. In diesem ATP-Analogon sollte die Anregung des Fluoreszenzdonors (D) zu einer Übertra-gung der Anregungsenergie auf den Fluoreszenzakzeptor führen, dessen Fluoreszenz beobachtet werden kann. Nach der Spaltung der a/b-anhydridischen Bindung dieses Analogons sollte FRET nicht mehr mçglich sein und daher eine direkte Emission des Donors beobachtet werden. Auf diese Art resultiert die Aktivität von UBA1 in einer großen ¾n-derung der Fluoreszenzeigenschaften des ATP-Analogons. ¾hnliche Ansätze wurden bereits verwendet, um die Aktivität anderer hydrolysierender Enzyme, z. B. Proteasen, zu studieren. [6] Für diesen zeitaufgelçsten ATPase-Sensorassay (timeresolved ATPase sensor assay, TRASE) müssen zwei Fluorophore an ATP angeknüpft werden. Die N6-Position wurde schon früher als Stelle, an der ATP ohne Verlust der Substrateigenschaften für UBA1 modifiziert werden kann, [7] identifiziert. Daher muss die zweite Modifikation an der Abbildung 1. a) Mechanismus der Aktivierung von Ubiquitin durch UB...

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Steady-State Kinetic Analysis of Human Ubiquitin-Activating Enzyme (E1) Using a Fluorescently Labeled Ubiquitin Substrate

Cited by 24 publications

References 19 publications

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1

Hydrophobic Patch of Ubiquitin is Important for its Optimal Activation by Ubiquitin Activating Enzyme E1

Human mdm2 Mediates Multiple Mono-ubiquitination of p53 by a Mechanism Requiring Enzyme Isomerization

Fluorogene ATP‐Analoga zur Detektion von ATP‐Verbrauch: Beobachtung der Aktivierung von Ubiquitin in Echtzeit

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