Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex.
The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H2O by D2O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000201:enzymology; D003903:Deuterium; D005573:Formycins / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism*; D012440:Saccharomyces / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013725:Terbium / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D005573:Formycins; D009711:Nucleotides; D012265:Ribonucleotides; D013725:Terbium; C051617:formycin triphosphate-terbium complex; D000255:Adenosine Triphosphate; D003903:Deuterium; D006180:Proton-Translocating ATPases; D008274:Magnesium