Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase

Abstract

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TFoFl was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(l-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with K,,,(TF1) = 390 pM and K, (TFoF,) = 180 pM. The inhibition constants are for ADP Ki(TF1) = 20 pM and Ki(TFoF1) = 100 pM, for 3'-O-(l-naphthoyl)ATP Ki(TFI) = 150 pM and Ki(TFoF1) = 3 pM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TFo to TF1 the mechanism of ATP hydrolysis catalyzed by TFl is not changed qualitatively; however, the kinetic constants differ quantitatively.Membrane-bound ATPases of the FoFl type can catalyze proton-transport-coupled ATP synthesis/hydrolysis [ 1 -51. The hydrophilic F1 can be removed easily from the membrane and this soluble F1 has been used for studying the kinetics of ATP hydrolysis (see e. g. [6 -81). The kinetic mechanism of ATP hydrolysis, catalyzed by the soluble F1, can be regarded as a first step in the clarification of the mechanism of protontransport-coupled ATP hydrolysis catalyzed by FoFl. This implies that the binding of F1 to Fo at the membrane does not change the reaction mechanism.Using mitochondrial F1 it has been shown that under single-site conditions (i. e. ATP concentration is less than onethird of the F1 concentration) the kinetic constants for ATP binding, ATP dissociation, Pi dissociation, and ADP dissociation are similar for F1 and FoFl [9, 101. In this work the rate of ATP hydrolysis was measured as a function of the ATP concentration in the presence of the inhibitors 3'-O-(l-naphthoyl)adenosine triphosphate (N-ATP), ADP and Pi using F1 and reconstituted FoFl of the thermophilic bacterium PS3. Because of its remarkable stability [ll] this ATPase can be easily reconstituted into liposomes [12] without significant loss of enzymatic activity. The ATP analogue N-ATP has been shown earlier to be a competitive inhibitor of ATP synthesis and hydrolysis of mitochondrial [13] and chloroplast ATPase [14, 151. The results of this investigation show that the inhibition constants Correspondence to M. Rogner,

Kinetic analysis of proton translocation catalyzed by F0F1 ATPase

“…ANS fluorescence measurements were performed as described [4,5]. The assay mixture contained 50 mM Tris-SOA (pH 8.0), 2 mM MgS04, 7 mM KCN, 5 mM phosphoenolpyruvate, 3 pM ANS and 29 /g of pyruvate kinase in 2 ml.…”

“…SMPs were prepared from beef heart mitochondria suspended in 0.25 M sucrose, 10 mM Tris-Cl (pH 7.5), 5 mM MgS04, by sonication followed by centrifugation [6]. ANS fluorescence measurements were performed as described [4,5]. The assay mixture contained 50 mM Tris-SOA (pH 8.0), 2 mM MgS04, 7 mM KCN, 5 mM phosphoenolpyruvate, 3 pM ANS and 29 /g of pyruvate kinase in 2 ml.…”