Properties of binding sites for adenine nucleotides on ATPase from yeast mitochondria.
High pressure column chromatography was applied to estimate the bound nucleotides on F1-ATPase. In this way, various species of adenine nucleotide in 0.1 to 0.3 mg of enzyme protein were well separated and estimated with a precision of about 10 nmol within 10 min. ATPase in submitochondrial particles contained 2 mol of ATP and 1 to 2 mol of ADP. The nucleotide content and binding nature of the enzyme varied at different stages of purification, but a total of four binding sites were found in enzyme preparations at all steps. Highly purified enzyme had two tight binding sites for ATP and two loose binding sites, one for ATP and one for ADP. The tight binding of ADP observed in submitochondrial particles was reconstituted by continuous supply of ATP and Mg2+ to the purified enzyme. Removal and rebinding of the nucleotides did not affect ATP-hydrolyzing activity but caused conformational changes of the enzyme, as demonstrated by measuring cold-lability and trypsin digestion. An analog of ATP, AMPP(NH)P, was found to bind to loose binding sites of the purified enzyme with 2 mol of tightly bound ATP, and to inhibit ATP-hydrolyzing activity competitively. The analog also bound to the tight sites under special conditions, protecting the enzyme against cold inactivation. During enzymatic hydrolysis of [3H]ATP, labeled ATP and ADP were both bound at the loose sites, but only slight amounts of these nucleotides were bound to the tight sites. From these results it is inferred that the loose sites are catalytic, while the tight sites are not.
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D001665:Binding Sites; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D006180:Proton-Translocating ATPases; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship
D000227:Adenine Nucleotides; D008565:Membrane Proteins; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D008274:Magnesium