In the presence of ATP, genistein, like the ATP analogue adenosine 5'-[beta,gamma-imido]triphosphate (pp[NH]pA), increases cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents by prolonging open times. As pp[NH]pA is thought to increase CFTR currents by interfering with ATP hydrolysis at the second nucleotide-binding fold (NBF-2), the present study was undertaken to investigate the effects of genistein on a fusion protein comprising maltose-binding protein (MBP) and NBF-2 (MBP-NBF-2). MBP-NBF-2 exhibited ATPase, GTPase and adenylate kinase activities that were inhibited by genistein in a partial non-competitive manner with respect to ATP or GTP. Ki values for competitive and uncompetitive inhibition were respectively 20 microM and 63 microM for ATPase, 15 microM and 54 microM for GTPase, and 46 microM and 142 microM for adenylate kinase. For ATPase activity, genistein reduced Vmax by 29% and Vmax/Km by 77%. Additional evidence for complex-formation between genistein and MBP-NBF-2 was obtained by the detection of genistein-dependent alterations in the CD spectrum of MBP-NBF-2 that were consistent with the formation of a higher-ordered state. Addition of MBP-NBF-2 increased the fluorescence intensity of genistein, consistent with a change to a less polar environment. pp[NH]pA partially eliminated this enhanced fluorescence of genistein. These observations provide the first direct biochemical evidence that genistein interacts with CFTR, thus inhibiting NBF-2 activity, and suggest a similar mechanism for genistein-dependent stimulation of CFTR chloride currents.