A RECOMBINANT POLYPEPTIDE MODEL OF THE 2ND NUCLEOTIDE-BINDING FOLD OFTHE 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 p rotein was shown to catalyse the reaction ATP --> ADP + P-i by three d ifferent 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 produc t ADP inhibits this ATPase activity, In a similar manner the hydrolysi s 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 supersede d by the formation of two ADP from ATP and AMP, As typical for adenyla te 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 thre e enzymatic activities were inhibited by the symmetric double-substrat e-mimicking inhibitor Ap(5)A, As NBF-2 plays a central role in CFTR ch annel opening and closing the results reported here are fundamental in understanding mechanisms of CFTR channel activity regulation. (C) 199 7 Federation of European Biochemical Societies.