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
Open Access
- 30 June 1997
- journal article
- Published by Wiley in FEBS Letters
- Vol. 410 (2-3) , 180-186
- https://doi.org/10.1016/s0014-5793(97)00574-7
Abstract
CFTR–NBF‐2 expressed and purified in fusion with the maltose‐binding protein was shown to catalyse the reaction ATP→ADP+Pi by three different assays, monitoring ATP turnover, formation of ADP and release of Pi (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 Pi 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 In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP.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.Keywords
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