Isoprenoid synthases are a class of enzymes involved in the synthesis of several crucial metabolic intermediates which are used to form important biological compounds such as cholesterol, ubiquinone and prenylated proteins. Consequently, much effort has gone into developing inhibitors for this class of polypeptides.  In this work, we report on the development of a new, more sensitive assay for the activity of these enzymes, specifically farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase. The assay is a continuous fluorescence assay that monitors the synthesis and subsequent incorporation of the enzymatic product into fluorescently labeled substrates whose fluorescence quantum yield increases upon product incorporation.  We show how this method can quickly and specifically monitor enzyme activity in vitro and in complex cellular environments.  This new method offers a simple, sensitive and continuous method to assay isoprenoid synthase activity that should greatly facilitate the screening and development of inhibitors of these important targets.

Enzyme-coupled continuous fluorescence assay for isoprenoid synthases

243rd National Meeting (2012)

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