Detection of the conformational change in the catalytic site of adenosine triphosphatase from beef liver mitochondria by affinity labeling with the dialdehyde derivative of ethenoadenosine triphosphate.
Beef liver mitochondrial F1ATPase was inactivated by the 2',3'-dialdehyde derivative of ethenoATP (epsilon ATP) in a pseudo-first order reaction. The kinetics of protection of the enzyme against inactivation by various nucleoside triphosphates (NTPs) revealed that the dial-epsilon ATP was bound to the catalytic site as an affinity label. Certain anions (sulfate or bicarbonate) were ineffective for protection. In the early phase of the reaction, inactivation was due to the binding of 1 mol dial-epsilon ATP per mol enzyme. In this phase, dial-epsilon ATP bound exclusively to the subunit beta of the enzyme, indicating that the catalytic site is in this subunit. The fluorescence of the ethenoadenosine moiety, bound exclusively to the subunit beta of the enzyme, was measured as a conformational probe of the catalytic site region. Addition of ATP or CTP to the labeled enzyme resulted in a decrease in the fluorescence intensity. GTP and other NTPs were less effective than ATP or CTP. The anions (sulfate of bicarbonate) suppressed the ability of ATP to decrease the fluorescence in a competitive manner. Quantitative analysis of these fluorescence changes suggested that they might originate from the binding of the NTP to the regulatory site of the enzyme. These findings are in good agreement with the two-site model proposed by us (Wakagi, T. & Ohta, T. (1981) J. Biochem. 89, 1205) which was deduced from the steady state kinetics of the NTPase reactions catalyzed by the F1ATPase.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004985:Ethenoadenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008930:Mitochondria, Liver / Q000201:enzymology*; D011487:Protein Conformation
D000345:Affinity Labels; D004985:Ethenoadenosine Triphosphate; D046911:Macromolecular Substances; C036830:ethenoadenosine triphosphate-2',3'-dialdehyde; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases