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Enzyme Activity Measurement for Transferases



Transferases, designated as EC 2, are a class of enzymes that transfer a specific functional group from the donor molecule to the acceptor molecule. In some cases, both the donor and acceptor are substrates, while, in other cases, one of them could be the coenzyme. Transferases are extremely important to a vast range of physiological functions. They catalyze numerous biological reactions that are critical to a living system. These reactions include essential metabolism, genetic regulations, detoxification, and many others. The transferase enzymes can be sorted into the following sub-classes according to the functional groups that the enzymes act on:

Due to the versatile roles of transferases, they are popular targets in biomedical and biotechnical research. Malfunctioning transferases are the main causes of many diseases. For example, the Glutathione S-transferases (GSTs) catalyze the conjugation of glutathione (GSH) and toxic compounds to perform the cellular detoxification. However, this invaluable service hampers chemotherapy of cancers, as overexpression of GSTs is considered associated with multi-drug resistance of cancer cells. Another example is farnesyltransferase, which is related to the unregulated cancer cell activities in one-third of human cancers. The transferase catalyzes the post-translational modification of the Ras protein, which is essential to cancer cell activities. At molecular level, DNA methylation serves as the principal form of post-replicative epigenetic modification, which is extensively involved in gene regulation and silencing. DNA methylation and maintenance of methylation is controlled by three DNA methyltransferases. Understanding and modulating the action of DNA methyltransferases will lead to treatment of many related diseases. In chemical and environmental applications, several types of acyltransferases are gaining growing research interests for their potential roles in producing biofuels. These transferases transform the structures of long-chain fatty acids and their esters into precursors for the desired hydrocarbon mixtures. Therefore, studies on acyltransferases would improve the biofuel production efficiency. Because the transferases catalyze such a wide scope of reactions, their activity measurement could be quite challenging. First, some transferases act on two substrates with no significant spectroscopic characters, which complicates the detection of the reaction progresses using spectrophotometric, fluorometric, or even chromatographic assays. More advanced analytical methods need to be used to test these enzymes, including radiometric and calorimetric analysis. For some other enzymes, however, the choices of substrates and cofactors are abundant, and the identity of the substrate and cofactor could have huge impacts on the activity measurement. Therefore, the assay needs to be carefully designed around the target enzyme activity and the selected substrates. 

Enzyme Activity Measurement for Phosphorus Transferases Figure: The two domains in the subunit A of glutathione transferase, showing glutathione in the binding site (yellow stick mode).
Reference: Fyfe, P. K., Westrop, G. D., Silva, A. M., Coombs, G. H., & Hunter, W. N. Proceedings of the National Academy of Sciences, 2012, 109(29), 11693–11698.


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