ATP and ADP bind to cytochrome c oxidase and regulate its activity.
By equilibrium dialysis of cytochrome c oxidase from bovine heart with [35S]ATPalphaS and [35S]ADPalphaS, seven binding sites for ATP and ten for ADP were determined per monomer of the isolated enzyme. The binding of ATP occurs in a time-dependent manner, as shown by a filtration method, which is apparently due to slow exchange of bound cholate. In the crystallized enzyme 10 mol of cholate were determined and partly identified in the high resolution crystal structure. Binding of ADP leads to conformational changes of the Tween 20-solubilized enzyme, as shown by a 12% decrease of the gamma-band. The conformational change is specific for ADP, since CDP, GDP and UDP showed no effects. The spectral changes are not obtained with the dodecylmaltoside solubilized enzyme. The polarographically measured activity of cytochrome c oxidase is lower after preincubation with high ATP/ADP-ratios than with low, in the presence of Tween 20. This effect of nucleotides is due to interaction with subunit IV, because preincubation of the enzyme with a monoclonal antibody to subunit IV released the inhibition by ATP. In the presence of dodecylmaltoside the enzyme had a 2 to 3-fold higher total activity, but this activity was not influenced by preincubation with ATP or ADP.
Napiwotzki
J
J
Fachbereich Chemie, Philipps-Universität, Marburg, Germany.
Shinzawa-Itoh
K
K
Yoshikawa
S
S
Kadenbach
B
B
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002793:Cholic Acids / Q000032:analysis; D003460:Crystallization; D003956:Dialysis; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011048:Polarography; D011136:Polysorbates; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013462:Sulfur Radioisotopes; D013873:Thionucleotides / Q000737:chemistry* / Q000378:metabolism
D002793:Cholic Acids; D011136:Polysorbates; D013462:Sulfur Radioisotopes; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; C018444:adenosine 5'-O-(1-thiodiphosphate); D000244:Adenosine Diphosphate; D003576:Electron Transport Complex IV