Inhibition of ATPase, GTPase and adenylate kinase activities of the secondnucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator by genistein

In the presence of ATP, genistein, like the ATP analogue adenosine 5'-[beta ,gamma-imido]triphosphate (pp[NH]pA), increases cystic fibrosis transmembra ne conductance regulator (CFTR) chloride currents by prolonging open times. As pp[NH]pA is thought to increase CFTR currents by interfering with ATP h ydrolysis 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 e xhibited ATPase, GTPase and adenylate kinase activities that were inhibited by genistein in a partial non-competitive manner with respect to ATP or GT P. K-i values for competitive and uncompetitive inhibition were respectivel y 20 mu M and 63 mu M for ATPase, 15 mu M and 54 mu M for GTPase, and 46 mu M and 142 mu M for adenylate kinase. For ATPase activity, genistein reduce d V-max by 29 %, and V-max/K-m by 77%. Additional evidence for complex-form ation between genistein and MBP-NBF-2 was obtained by the detection of geni stein-dependent alterations in the CD spectrum of MBP-NBF-2 that were consi stent with the formation of a higher-ordered state. Addition of MBP-NBF-2 i ncreased the fluorescence intensity of genistein, consistent with a change to a less polar environment. pp[NH]pA partially eliminated this enhanced fl uorescence of genistein. These observations provide the first direct bioche mical evidence that genistein interacts with CFTR, thus inhibiting NBF-2 ac tivity, and suggest a similar mechanism for genistein-dependent stimulation of CFTR chloride currents.