Enzymes execute a wide variety of functions. For example, they regulate signal transduction via kinases and phosphatases. ATPases generate muscle contraction and active transport. Several enzymes can work together in a specific metabolic pathway (e.g., glycolysis). The common characteristic of enzymes is that they catalyze the chemical modification of specific substrates. Depending on their cellular sub-localization, enzymes can have various preferences toward the presence of reducing agents in biochemical assays. Redox regulation is, like phosphorylation, a covalent regulatory system that controls normal cell function. For example, mitochondria contain the most reducing compartment and are highly sensitive to oxidation. The secretory pathway contains oxidative systems which introduce disulfides into proteins for transport. As opposed to secretory mechanisms, uptake and activation through the endocytic pathway involve reduction of disulfides.
If it is necessary to keep the enzyme of interest under reducing conditions, the use of 1-5 mM GSH or DTT as Reducing Agents is recommended in TRIC assays. Enzyme function is often complex and requires a number of co-factors such as co-enzymes, specific ions, or nucleotides. It is therefore important to know the biochemical requirements for their interactions with binding partners.
Note that if the affinity of a substrate to an enzyme should be investigated the substrate might be processed by the enzyme before the TRIC measurement is complete. The use of substrate regenerating systems, non- catalytic enzyme mutants or non- catalyzable substrate analogues (e.g. AMP-PNP instead of ATP) is recommended in these cases.