Putting enzymes on pause
Steady-state kinetics assay. The steady-state kinetic parameters K M and K cat for the phosphatase were determined by using the small molecule substrate p-nitrophenyl phosphate (pNPP). The concentrations of pNPP used were 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 40 mM, and 60 mM. An enzyme concentration of 0.1 μM was chosen based on initial activity assays and the p -nitrophenol standard curve. The reactions were incubated at room temperature for 15 minutes before being stopped by the addition of 40 µL of 5 mM NaOH to each well. Separate blank reactions for each substrate concentration were prepared using deionized water (ddH2O) and Cdc14. Substrate specificity assay. An assay was conducted using 24 distinct phosphopeptide substrates with known amino acid sequences. The concentrations of both substrate and each substrate were kept constant (100 μM and 0.1 μM, respectively). Each phosphopeptide reaction was run in tripli cate for 15 minutes before 160 μL of the reagent Bimol Green dye was added to stop the reaction. The data were then recorded by a plate reader operating at a wavelength of 405 nm. These absorption values were then converted into the concentration of substrate. Homologous protein modelling. The structure of S. cerevisiae Cdc14 was selected as a model due to its high similarity in conserved domains with the target A. alternata protein. Using this homologue enhances prediction accuracy, ensuring that our inhibitor design aligns closely with the functional characteristics of the A. alternata Cdc14 enzyme. Using the already identified genetic sequence for the Cdc14 protein in A. alternata, the amino acid sequence was generated. The Gene X, coding for Cdc14, was first translated into an amino acid sequence strand. Then, a homologous Cdc14 with a high similarity/identity score was identified in S. cerevisiae (PDB Code 5XW4) using the Basic Local Alignment Search Tool (BLAST). This structure was used as a template to model the potential structure of Cdc14 in A. alternata and to create a homology model using the Amber 14:EHT force field simulator . Then, the model was evaluated based on structural similarity with Cdc14, the presence of energetically unfavourable psi-phi angles, the presence of bond lengths exceeding 4 angstroms, and the prevalence of rotamer clashes above 3 kcal/mol. Protein docking. Next, the molecular structures of different possible inhibitors (figure 5) were docked using the docking feature in MOE, and the affinity of each molecule for the active site was tested. The molecules were then ranked based on (1) the S-score generated, (2) the length of the hydrogen bond formed with the catalytic cysteine and assisting aspartic acid, and (3) the presence of steric clashes. These docking simulations were used to predict the likelihood of successful inhibition and rank the inhibitors based on the probability of success. Inhibitor activity assay. To inform inhibitor design, ten different prefabricated inhibitors with known structures were included in wells containing both enzyme and pNPP, a known substrate of Cdc14. Firstly, a control assay was conducted to produce minimum and maximum values for substrate production. The maximum activity control contained 0.5 µM of Cdc14, 20 µL of reaction buffer to maintain a steady pH, and pNPP at a concentration of 10 mM. The minimum activity control contained 20µL of reaction buffer and pNPP at 10 mM. Each of the ten inhibitor functionality assays was conducted with three replicates of reactions and a blank, using 20 µL of reaction buffer, pNPP at 10 mM, and inhibitor at 100 µM. Each well that included an inhibitor contained the inhibitor at a concentration of 100 µM, Cdc14 at a concentration
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