Fluoroaluminate complexes are bifunctional analogues of phosphate in sarcoplasmic reticulum Ca(2+)-ATPase.
The mechanism of inhibition of the sarcoplamc reticulum (SR) Ca(2+)-ATPase by the fluoroaluminate complexes was investigated. First, AlF4- was shown to bind to the Ca(2+)-free conformation of the enzyme by a slow quasi-irreversible process. The rate constants of the reaction are k+ = 16 x 10(3) M-1 s-1 and k- < 1.5 10(-3) s-1. We directly measured a stoichiometry of about 4.8 nmol of AlF4- bound/mg of protein. Mg2+ was a necessary cofactor for the reaction with a dissociation constant of 3 mM. It was demonstrated (Dupont, Y., and Pougeois, R. (1983) FEBS Lett. 156, 93-98) that phosphorylation by P(i) induced a dehydration of the catalytic site. The same process has been shown here to occur upon AlF4- binding either by the use of Me2SO or by demonstration of an increase of bound 2',3'-O-(2,4,6-trinitrocyclohexadienyldene)adenosine triphosphate fluorescence. Phosphorylation by P(i) is inhibited by the binding of AlF4-. Second, a fluoroaluminate complex, presumably AlF4-, was also shown to bind to the Ca(2+)-bound conformation of the Ca(2+)-ATPase in the presence of ADP and stabilize a E1.Ca2.ADP.AlFx complex. The dissociation constant of the nucleotidic site for ADP was shifted to the micromolar range. The Ca2+ ions bound on the external high affinity sites became occluded upon binding of (ADP + AlFx). We propose that AlF4- mimics P(i) binding to the Ca(2+)-free conformation of the ATPase and stabilizes an intermediate similar to the acyl-phosphate derivative; it also acts as an analogue of the gamma-phosphate of ATP and stabilizes an E1.[Ca2].ADP.AlF4 complex where the Ca2+ ions are occluded.
Troullier
A
A
Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, France.
Girardet
J L
JL
Dupont
Y
Y
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000535:Aluminum / Q000378:metabolism / Q000494:pharmacology*; D000077410:Aluminum Chloride; D017607:Aluminum Compounds; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002712:Chlorides / Q000494:pharmacology; D005459:Fluorides; D005461:Fluorine / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology*; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012969:Sodium Fluoride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D017607:Aluminum Compounds; D002712:Chlorides; D010710:Phosphates; C050992:tetrafluoroaluminate; D005461:Fluorine; D000077410:Aluminum Chloride; D000244:Adenosine Diphosphate; D012969:Sodium Fluoride; D000535:Aluminum; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D005459:Fluorides