Biochemistry, published online 28 November 2011, doi:10.1021/bi201313s

Identifying small-molecule effectors that affect enzyme activity often relies on functional enzyme assays as a readout, but this strategy can overlook metabolites that indirectly alter enzyme activity. To search for these species, Orsak et al. report a new method, mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS), that detects protein-mediated accumulation of small molecules. In particular, the authors tested a panel of 138 endogenous metabolites against 5 protein targets, including NAD kinase and L-glutamate dehydrogenase type III (GDH), and identified 16 known and 13 unknown interactions. Unknown interactions were further investigated using traditional assays, identifying competitive, noncompetitive and uncompetitive inhibitors as well as activators and those with no obvious effect on function. For example, glucose-6-phosphate's role as an activator of NAD kinase was identified, along with several new inhibitors; the authors speculate that these molecules might work in combination to switch the cell from a low-energy state, in which the reducing equivalents generated by the enzyme are not needed, to a high-energy state, supportive of biogenesis and growth. Similarly, the authors' discovery that trans-aconitate functions as an inhibitor of GDH suggests a new point of regulation within the citric acid cycle. Although the full biological significance of these interactions requires further study, this method should provide important starting points for understanding cellular metabolism.