The alpha 3 beta 3 complex, the catalytic core of F1-ATPase.

Abstract

The a3.83 complex was reconstituted from a and (3 subunits of the thermophilic bacterium PS3 F1-ATPase (TFI) and then isolated. It is less stable at high and low temperatures than TFi?, and the complex dissociates into subunits during native polyacrylamide gel electrophoresis. The a_3%3 complex has about 20% of the ATPase activity of TF1. Its enzymic properties are similar to those of the native TFI, exhibiting similar cooperative kinetics as a function of ATP concentration, similar substrate specificity for nucleotide triphosphates, and the presence of two peaks in its temperatureactivity proffle. Differing from TFI, the ATPase activity of the a3(33 complex is insensitive to Nj-inhibition, its divalent cation specificity is less stringent, and its optimum pH shifts to the alkaline side. The addition of the y subunit to the a3P3 complex leads to the formation ofthe a3_33y complex, indicating that the a3.83 complex is an intermediate in the process of assembly of the holoenzyme from each subunit. These results definitely show that the essential structure for eliciting the ATPase activity of Fj-ATPase is trimeric aj pairs and that the kinetic cooperativity of the FI-ATPase is an inherent property of this trimeric structure but is not due to the presence of single-copy subunits. In this sense, the a433 complex is the catalytic core of Fl-ATPase.In energy-transducing membranes of bacteria, mitochondria, and chloroplasts, ATP synthases catalyze ATP synthesis coupled with proton flow (1-3). These enzymes are composed of an integral membrane protein sector, F0, and a peripheral protein sector, F1. F1 has an ATP-hydrolyzing activity, and its molecular weight is about 380,000. The subunit structure of the Fj-ATPase is a3f33'y8e. The were expressed in the E. coli DK-8 strain, a mutant with a deletion of FoF1 genes, which was given to us by M. Futai, and were isolated individually as described (17, 18). The isolated subunits were stored in 75% ammonium sulfate suspension at 40C. Prior to use, the a and ( subunits were applied to an HPLC gel permeation column (G3000SWxl, Tosoh, Tokyo) and were eluted at room temperature with 50 mM Tris sulfate buffer (pH 7.0) containing 200 mM Na2SO4 at a flow rate 0.5 ml/min. The purified subunits were mixed, incubated at 30'C for 15 min in the same buffer, and the mixture was reapplied to the same gel permeation column.The a3,3 complex was eluted at 14.7 min and was prepared freshly for each experiment.Assays of ATP Hydrolytic Activity.

Assembled F1-(αβ) and Hybrid F1-α3β3γ-ATPases fromRhodospirillum rubrum α, Wild Type or Mutant β, and Chloroplast γ Subunits

“…This mutant complex was expressed and purified in large amounts and responded to the ␥ C thiol modulation, but even its DTT-reduced MgATPase activity reached at the most 5 units/mg. Furthermore, no CaATPase activity has been reported in this mutant TF 1 -␣ 3 ␤ 3 ␥ complex, probably because in the native TF 1 , unlike in RrF 1 (Table I) and CF 1 (30), the CaATPase activity is 10-fold lower than its MgATPase activity (43).…”

“…1). But in the TF 1 -␣ 3 ␤ 3 hexamers, which were assembled without AlF x , the CaATPase activity is 5-fold higher than their MgATPase, although the whole native TF 1 has a 10-fold lower Ca-than MgATPase activity (43). The specific inhibition of the RrF 1 -CaATPase by AlF x , which has not been tested on any other F 1 -CaATPase activity, provides an additional clear difference of functional properties of the RrF 1 -CaATPase and MgATPase activities.…”

The α3β3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 with the βT165S Substitution Does Not Entrap Inhibitory MgADP in a Catalytic Site during Turnover

“…The ␣ 3 ␤ 3 , ␣ 3 ␤ 3 ␥, and ␣ 3 ␤ 3 ␦ subcomplexes reconstituted from the isolated subunits of TF 1 1 are active as ATPases (3)(4)(5)(6). Both the ␣ 3 ␤ 3 and ␣ 3 ␤ 3 ␦ subcomplexes differ from TF 1 in that they are less specific for divalent cations and are insensitive to inhibition by azide (4,5). In contrast, the catalytic characteristics of the ␣ 3 ␤ 3 ␥ subcomplex are very similar to those of TF 1 (4,7,8).…”

“…When separated from F 0 as a soluble complex, F 1 is composed of five different subunits in a stoichiometry of ␣ 3 ␤ 3 ␥␦⑀ and functions as an ATPase (2). The ␣ 3 ␤ 3 , ␣ 3 ␤ 3 ␥, and ␣ 3 ␤ 3 ␦ subcomplexes reconstituted from the isolated subunits of TF 1 1 are active as ATPases (3)(4)(5)(6). Both the ␣ 3 ␤ 3 and ␣ 3 ␤ 3 ␦ subcomplexes differ from TF 1 in that they are less specific for divalent cations and are insensitive to inhibition by azide (4,5).…”

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F 1 -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

“…However, accumulating evidence has emerged to support the idea that the ␣ 3 ␤ 3 subcomplex can intrinsically catalyze cooperative ATP hydrolysis independently of ␥. Early studies have shown an assembled and catalytically active ␥-less complex in the thermophilic bacterium PS3 F 1 -ATPase (30,31). Genetic studies also supported the assembly of an active F 1 lacking ␥ in S. cerevisiae mitochondria (32).…”