Mitochondrial-catalyzed ATP hydrolysis in highly enriched [ 18O]H 2O. : Frequency distributions of 18O-labelled P i species

Abstract

1. 1. The 2,4-dinitrophenol-resistant intermediate phosphate ⇌ water oxygen exchange reaction which accompanies the hydrolysis of ATP catalyzed by heart submitochondrial vesicles was studied in highly enriched 18O-labelled water. The phosphate formed was converted into the tris(trimethylsilyl) phosphate derivative which was analyzed by chemical ionization mass spectrometry. The amount of exchange relative to that of ATP hydrolysis, and the ATP dependence of the exchange on the ATP concentration were measured. The results were in good agreement with previous studies carried out on water of low enrichment. However, the new procedure enabled the determination of the relative frequency distributions of phosphate species with 1, 2, 3, or 4 oxygen atoms derived from water. Oxygen from water was not found only in singly or quadruply 18O-labelled species. This finding further helps to exclude the possibility that the vesicles contain two separate ATPases, one non-exchanging, the other highly exchanging. 2. 2. The frequency distributions of 18O-labelled P i species were measured at high (6 mM) and low (0.01 mM) ATP, as well as at three intermediate ATP concentrations. Regression analysis was carried out to see if the frequency distributions found with the intermediate ATP concentrations were simple composites of the patterns obtained at the high and low ATP levels. The analysis did not support the idea that the phosphate formed at the intermediate ATP levels was a binary mixture of phosphate produced by the hydrolysis of ATP at a high and a low affinity ATP site. This finding excluded models involving two independent Michaelis-Menten ATPases, as well as simple two-site negatively cooperative models. 3. 3. Oxygen from water was incorporated into all four oxygens of phosphate, and the extra oxygen (i.e., that incorporated over and above the single oxygen needed for the hydrolytic step) was distributed approximately binomially. This implies that the four oxygen atoms of P i were equivalent during the time enzyme-bound P i underwent exchange. Thus a pseudorotation mechanism in which one oxygen was strongly liganded to a group on the enzyme does not appear likely. The exchange may thus reflect the dynamic reaction, ATP + H 2O ⇌ ADP + P i on the enzyme surface. The effect of ATP concentration upon exchange may thus reflect the extent of hydrolysis and reesterification which occurs prior to P i release from the enzyme.