Binding of nucleotides by the mitochondrial ADP/ATP carrier as studied by 1H nuclear magnetic resonance spectroscopy.
Nucleotide binding to the cytosolic binding site of the mitochondrial ADP/ATP carrier (AAC) was studied by 1H-nuclear magnetic resonance spectroscopy. Binding (as opposed to translocation) could be identified as a result of the rapid ligand on/off kinetics, using the cytosolic side specific inhibitor carboxyatractyloside (CAT) for the distinction from nonspecific interactions. The off rate constant of the nonhydrolyzable ATP analogue AMP-PCP was more than 3 orders of magnitude larger than the transport rate. The nucleotides adopt an anti conformation in the carrier binding site as shown by measurements of the transferred nuclear overhauser effect (TRNOE). A thermal transition around 14 degreesC that had been previously detected in transport studies [Klingenberg, M., Grebe, K., and Appel, M. (1982) Eur. J. Biochem. 126, 263-269] was reflected by the inhibitor sensitive line broadening, indicating that this transition also affects nucleotide binding. Nucleotide monophosphates were employed to study the relation between nucleotide structure and affinity, using selective excitation, sample spinning with digital suppression of spinning sidebands, and line shape simulation. The binding of purines depends on the distribution of the electrical potential and on the position of ring substituents, while pyrimidines are barely recognized at all by the AAC. It is also shown that the photocleavable "caged" derivatives are more tightly bound than the original nucleotides. A two step model of carrier catalysis will be discussed on the basis of these results.
Huber
T
T
Institute of Physical Biochemistry, University of Munich, Germany.
Klingenberg
M
M
Beyer
K
K
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D001667:Binding, Competitive; D001865:Bongkrekic Acid / Q000494:pharmacology; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008929:Mitochondria, Heart / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D011522:Protons; D055672:Static Electricity; D013499:Surface Properties; D013696:Temperature
D004791:Enzyme Inhibitors; D011522:Protons; D001865:Bongkrekic Acid; D001278:Atractyloside; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; C003853:carboxyatractyloside