Premium
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
Author(s) -
Randak Christoph,
Neth Peter,
Auerswald Ennes A,
Eckerskorn Christoph,
Assfalg-Machleidt Irmgard,
Machleidt Werner
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00574-7
Subject(s) - adenylate kinase , cystic fibrosis transmembrane conductance regulator , chemistry , gtp' , atp hydrolysis , walker motifs , biochemistry , nucleotide , atpase , enzyme , biophysics , biology , gene
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 In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP.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 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.