Computational protocol: 6-Thioguanine is a noncompetitive and slow binding inhibitor of human deubiquitinating protease USP2

Similar protocols

Protocol publication

[…] Deubiquitination assays were performed in 20 mM phosphate buffer (pH 7.6) at 30 °C. The concentration of the fluorogenic substrate ubiquitin-7-amino-4-trifluoromethylcoumarin (Ub-AFC; Boston Biochem) was varied while the concentration of USP2 catalytic domain or its C276S mutant was held at 0.2 μM. Enzyme activity was measured by continuously monitoring the fluorescence emission increase at 485 nm upon substrate cleavage at excitation wavelength of 350 nm in a PerkinElmer LS 50B luminescence spectrometer. The increase in fluorescence was linear for the first 25 s. Kinetic parameters were obtained by fitting the initial velocities (vi) to the Michaelis-Menten equation. The program Sigmaplot 12 (Systat Software) was used for data analysis.For the inhibition assays, concentrations of the substrate Ub-AFC and an inhibitor, 6-mercaptopurine (6MP) or 6TG, were varied while the concentration of the USP2 catalytic domain was held at 0.2 μM. The inhibition data for the inhibitors was found to best fit a noncompetitive inhibition model according to Eq. ():1vi=Vmax[S]/((1+[I]/Kis)(KM+[S]))in which Vmax is the maximal initial velocity of the enzyme while [S] and [I] denote the concentrations of the substrate and inhibitor, respectively. KM is the Michaelis-Menten constant for the enzyme-substrate complex and Kis is the inhibition constant for the enzyme-inhibitor complex. [...] X-ray diffraction data were collected at 100 K on beamlines BL13C1 and BL15A1 at the National Synchrotron Radiation Research Center, Taiwan, ROC, using an ADSC Q315r or Rayonix MX300HE CCD detector (X-ray wavelength 0.976 Å). The image data was processed and scaled with the HKL-2000 package. Both kinds of crystals belong to space group C2; the cell dimensions of the USP2-Ub-6TG crystal were a = 103.0, b = 54.7, c = 72.7 Å, α = γ = 90° and β = 107.5°, while those of USP2 C276S-Ub were a = 102.5, b = 54.0, c = 74.8 Å, α = γ = 90° and β = 107.5°. The crystallographic asymmetric unit contained one USP2 catalytic domain and one Ub. The structures were solved by the molecular replacement method with Phaser using the structure of USP2-Ub (PDB code: 2hd5) as the model. Structural refinement was carried out using REFMAC and the atomic model was built using Coot. […]

Pipeline specifications

Software tools SigmaPlot, HKL-2000, Coot
Applications Miscellaneous, Small-angle scattering, Protein structure analysis
Organisms Homo sapiens, Dipturus trachyderma
Diseases Leukemia, Neoplasms, Ovarian Neoplasms, Prostatic Neoplasms
Chemicals Thioguanine