Final answer:
To hypothesize a substrate for your enzyme, consider its natural interactions or structural similarities. Design an activity assay by selecting the appropriate buffer, and substrate concentration, and including consistent reagent amounts. Factor in the active site's amino acids and consider environmental effects on enzyme kinetics.
Step-by-step explanation:
Based on previous research, you would hypothesize a substrate for your protein of interest that it naturally interacts with or one that resembles the natural substrate structurally and chemically. When designing an activity assay for this substrate, begin by selecting an appropriate assay buffer and determining the assay volume.
Next, choose a substrate concentration that will allow you to measure the enzyme activity accurately, usually starting with a range that includes Km, the substrate concentration at half-maximal velocity. Calculate the stock substrate concentration required for the largest volume and concentration needed in your assays.
Once you have the stock concentration, determine the volume of substrate needed for each assay condition. Use consistent amounts of the enzyme and other reagents across all assays to ensure comparability. Incorporate a color-forming reagent if you plan to measure the activity spectrophotometrically.
Consider the active site of the enzyme; you may hypothesize an amino acid within the active site that participates in the reaction based on the type of interaction with the substrate. For instance, if you expect hydrogen bonding, an amino acid-like serine could be involved.
To conduct the enzyme kinetics experiment, prepare a chart to track the volumes of buffer, substrate, color-forming reagent, and enzyme that you will use in each assay. Remember to factor in variables such as pH and temperature, which could influence the enzyme's activity, and plan for controls that will account for these variables.
If studying enzyme inhibitors, evaluate the effect they have on activity using a specific substrate probe cocktail in an environment like human liver microsomes. Should you use a substrate analog like NAF, understand that it might bind permanently and label the active site, offering insights into the enzyme's mechanism.