Putting enzymes on pause
Figure 8: The product production over time graph was examined to identify an enzyme concentration that yielded a consistently linear range over time. The concentration of 0.62 uM was the strongest candidate for the Steady-state assay, given its high linearity and peak just after 5 minutes. This value was used in the production of the Steady-State Enzyme Assay, which was used to produce the Michaelis-Menten graph. The calculated K M from
this graph was 9.72 µM (close to the literature value of 10.6 µM seen in S. Cerevisiae ) (Wang et al., 2004). This relatively low Km represents the high affinity that Cdc14 has for its substrate. Next, the assay of 24 different phosphopeptides with known amino acid sequences was used to determine the specificity of the Cdc14 active site, thereby assisting in the design of inhibitors (Figure 6). After the assay, the absorbance data were converted to phosphate concentration using the standard curve generated with sodium phosphate. These concentrations were used to calculate the initial velocity of the reaction for each phosphopeptide. Several of the phosphopeptides had initial velocities close to zero, showing the enzyme's poor affinity for that substrate. Eleven, however, had Kcat/Km values above 4 μM/min, indicating a high affinity. The homology model was first mapped to the Cdc14 model in S. Cerevisiae and evaluated using structural similarity (Figure 5). Cdc14 is highly conserved across most fungi, meaning that a homology model for Cdc14 closely resembles the structure of Cdc14 in S. Cerevisiae. Afterwards, the homology model was evaluated using a Ramachandran Plot in order to identify psi-phi angles that were energetically unfavourable (figure 6). The majority of the psi-phi angles fell within the expected values, while only two amino acids, Val 367 and Val 321, had energetically unfavourable positions. The model contained no bonds exceeding 4 angstroms and only 7 different clashes, with energies exceeding 3 kcal. The inhibitor docking helped with trialling inhibitors before the in vitro assay. Inhibitors had docking scores between -4 and -7 kcal/mol (with a lower score showing a higher affinity). The strongest candidates from the docking simulation were I1 and I2, due to their high S-values and their ability to form hydrogen bonds with both cysteine and Asparagine in the active site.
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