The question
Name: ______________________ Date: ____________ Adding a disulfide bridge to an enzyme (9 points)
Data table
Adding a disulfide bridge to an enzyme
Researchers studied an enzyme from a soil bacterium. They made an engineered version in which two amino acids that sit close together in the folded enzyme were each replaced with cysteine, so that the engineered enzyme has one extra disulfide bridge. Samples of the normal and engineered enzymes were held for 10 minutes at one of seven temperatures, then cooled to 25 °C, and the activity of each sample was measured at 25 °C. Activity is given as a percent of the activity of a normal enzyme sample that was never heated. Each value is the average of four separate samples.
| Holding temperature (°C) | Normal enzyme (%) | Engineered enzyme (%) |
|---|---|---|
| 25 | 100 | 99 |
| 45 | 98 | 99 |
| 55 | 85 | 96 |
| 60 | 52 | 90 |
| 65 | 18 | 71 |
| 70 | 4 | 33 |
| 80 | 0 | 2 |
(a) Describe what a disulfide bridge is and which level of protein structure it stabilizes. Explain why a protein that loses its folded shape stops working. [2 points]
(b) Identify the independent variable in this experiment. Justify the researchers' decision to cool every sample to 25 °C before measuring its activity. [2 points]
(c) Describe how the effect of holding temperature differs between the normal and engineered enzymes. Calculate the difference, in percentage points, between the engineered and normal enzymes' remaining activity at 65 °C. [2 points]
(d) Before heating, a sample of the engineered enzyme is treated with a reducing agent, which breaks disulfide bridges, and the reducing agent is then removed while the bridges are kept from re-forming. Predict the activity this sample would keep after 10 minutes at 65 °C, and justify your prediction. Then explain why the engineered and normal enzymes have almost the same activity after 10 minutes at 25 °C. [3 points]