The Fpinoiely of rat liver iratochonclrial ATP synthase is capable of hydrolyzing ATP in the absence as well as m the presence of Mg++ ion. The rate of ATP hydrolysis increases several orders of magnitude when Mg++ ion is present in the reaction mixture. However, the chemical mechanism involving the divalent metal cation by which the F,-moiety of the ATP synthase hydrolyzes and synthesizes ATP remains unknown. For this reason, we have carried out studies with orthovaiiadate IV,), a phosphate analog which has the potential of ++trapping++ an ATPase, in its transition state (T.S.) and also the potential, in a photochemical reaction resulting in pepüde bond cleavage, of identifying an ainino acid very near the y-phosphate of ATP hi our earlier studies we have demonstrated that F; can form a MgADPW, complex and the y-phosphate of ATP is near the conserved alanine within the p-loop of the p-subumt(Ko et. al, J.B.C 272. 18875-18881, 1997) In this study we report that a MgV, complex is sufficient lo trap the enzyme in the T.S. in the absence of ADP The inhibition pattern and the photochemical cleavage upon irradiation of the MgV, complex with uv are very similar to those of the MgADPV, complex except the rates of inhibition and photo cleavage reactions are -1/2. This result suggests that in the T.S. the interaction of Mg++ ion with ATP is preferentially via the y-phosphate of ATI and the Mg++ at the active site helps the enzyme to form the trigonal bipyramidal T.S. (an associative mechanism) resulting in the acceleration of the enzymatic reaction In the absence of Mg++ ion the F, moiety still hydrolyzes ATP bul at a much slower rule However, the AI>PV, complex did not provide any photo cleavage products after the uv irradiation treatment. This suggests that in the absence of Mg++ ion, the hydrolysis reaction does not involve a trigonal bipyramidal T S., but rather a planar trigonal metaphosphate T.S. Hence, Vt, having the trigonal bipyramidal geometry, would not have much effect at the active site of the cii/vme in the absence of Mg++ ion in which the ATP hydrolysis is probably via a planar T.S. Hence, we propose that Mg++ binding near the y-pbosphate of ATP is an important process for F, to hyurolyze ATP at its maximal rate involving the trigonal bipyramidal T.S. suggesting a more associative mechanism.
|Original language||English (US)|
|State||Published - Dec 1 1998|
ASJC Scopus subject areas
- Molecular Biology