Cofactor interaction screening

In the previous studies using inhibitors and additives, it became clear that AMDase requires no cofactors, such as biotin, coenzyme A and ATP. It is also suggested that at least one of four cysteine residues plays an essential role in asymmetric decarboxylation. One possibility is that the free SH group of a cysteine residue activates the substrate in place of coenzyme A. Aiming at an approach to the mechanism of the new reaction, an active site-directed inhibitor was screened and its mode of interaction was studied. Also, site-directed mutagenesis of the gene coding the enzyme was performed in order to determine which Cys is located in the active site. 

Sometimes it is necessary to use different substrates, cofactors and mediators in order to increase the sensitivity. Due to the irreversible nature of many analyte-enzyme interactions, sensing elements must either be reactivated or should be disposable elements. An example is the determination of diazinon and dichlorvos that uses tyrosinase immobilized on screen printed electrodes, and a redox mediator (l,2-naphtaquinone-4-sulfonate) (Everett and Rechnitz, 1998). For other systems a further drawback is the lack of selectivity of some of the enzymes involved. 

To rapidly identify novel interactors, genome-wide screens for binding partners have been carried out in yeast and mammalian-based two-hybrid systems. As mentioned above, over 200 human NR cofactors have been identified. These interactors are important in the era of NR modulator discovery since each new cofactor carries the potential to recapitulate a particular cellular interaction and thus provides the basis for a molecular screen for molecules that uniquely affect the interaction. 

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