Elementary steps of the (Na+ + K+)-ATPase mechanism, studied with formycin nucleotides.
1. Formycin triphosphate (FTP), a fluorescent analogue of ATP, is a substrate for (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3), with properties similar to those of ATP. 2. FTP and formycin diphosphate (FDP) bind to the enzyme with high affinity and, on binding, the nucleotide fluorescence is enhanced 3-4-fold. It is therefore possible, with a stopped-flow fluorimeter, to measure the rates of binding and release of FTP and FDP under conditions in which turnover does not occur. 3. When the enzyme-FTP complex is exposed to conditions permitting turnover (Mg2+, Na+ +/- K+), changes in fluorescence occur which can be explained by supposing that they reflect the interconversion of states with or without bound nucleotides. A rapid fall in fluorescence, that we attribute to the rapid release of FDP from newly phosphorylated enzyme, is followed by a steady state in which low fluorescence suggests that little nucleotide is bound. Eventually, exhaustion of FTP allows rebinding of FDP to the enzyme, which is signalled by a rise in fluorescence. 4. The estimated rate of FDP release from newly formed phosphoenzyme is unaffected by the presence of K+ (0-2 mM) or the concentration of FTP (1-20 micron). 5. Experiments with [gamma-32P]FTP show that about 1 mol of 32P is incorporated per mol of enzyme. The rate of phosphorylation of the enzyme by [gamma-32P]FTP has been measured with a rapid-mixing-and-quenching apparatus. 6. Kinetic data from the fluorescence and phosphorylation experiments show that the behaviour of the enzyme, at least at the low nucleotide concentrations employed, is consistent with the Albers-Post model, and is difficult to reconcile with models in which K+ acts at or before the step in which FDP is released during turnover.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D001665:Binding Sites; D005573:Formycins / Q000378:metabolism*; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D010766:Phosphorylation; D011485:Protein Binding; D012265:Ribonucleotides / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D000903:Antibiotics, Antineoplastic; D005573:Formycins; D012265:Ribonucleotides; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium