A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase

CFTR-NBF-2 expressed and purified in fusion with the maltose-binding protein was shown to catalyse the reaction ATP --> ADP + P-i by three different assays, monitoring ATP turnover, formation of ADP and release of P-i (K-m 86 mu M, rate constant 0.37 min(-1)), The reaction product ADP inhibits this ATPase activity, In a similar manner the hydrolysis of GTP to GDP and P-i was demonstrated (K-m 40 mu M, rate constant 0.29 min(-1)), In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP, As typical for adenylate kinases a distinct AMP-binding site could be verified for CFTR-NBF-2 by the inability of TNP-ATP and AMP to compete for binding, All three enzymatic activities were inhibited by the symmetric double-substrate-mimicking inhibitor Ap(5)A, As NBF-2 plays a central role in CFTR channel opening and closing the results reported here are fundamental in understanding mechanisms of CFTR channel activity regulation. (C) 1997 Federation of European Biochemical Societies.