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

Abstract

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 μM, 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 μM, 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 In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP.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 Ap5A. 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.