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+Pi by three different assays, monitoring ATP turnover, formation of ADP and release of Pi (Km 86 microM, 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 (Km 40 microM, 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 In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP.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.
Randak
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C
Kinderklinik, Dr. von Haunerschen Kinderspital, Ludwig-Maximilians-Universität München, Germany. randak@clinbio.med.uni-muenchen.de
Neth
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P
Auerswald
E A
EA
Eckerskorn
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C
Assfalg-Machleidt
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I
Machleidt
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W
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D004791:Enzyme Inhibitors; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D015226:Dinucleoside Phosphates; D004791:Enzyme Inhibitors; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000249:Adenosine Monophosphate; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases