Modulation of Charge in the Phosphate Binding Site of Escherichia coli ATP Synthase

Abstract

This paper presents a study of the role of positive charge in the P i binding site of Escherichia coli ATP synthase, the enzyme responsible for ATP-driven proton extrusion and ATP synthesis by oxidative phosphorylation. Arginine residues are known to occur with high propensity in P i binding sites of proteins generally and in the P i binding site of the ␤E catalytic site of ATP synthase specifically. Removal of natural ␤Arg-246 (␤R246A mutant) abrogates P i binding; restoration of P i binding was achieved by mutagenesis of either residue ␤Asn-243 or ␣Phe-291 to Arg. Both residues are located in the P i binding site close to ␤Arg-246 in x-ray structures. Insertion of one extra Arg at ␤-243 or ␣-291 in presence of ␤Arg-246 retained P i binding, but insertion of two extra Arg, at both positions simultaneously, abrogated it. Transition state stabilization was measured using phosphate analogs fluoroaluminate and fluoroscandium. Removal of ␤Arg-246 in ␤R246A caused almost complete loss of transition state stabilization, but partial rescue was achieved in ␤N243R/␤R246A and ␣F291R/␤R246A. ␤Arg-243 or ␣Arg-291 in presence of ␤Arg-246 was less effective; the combination of ␣F291R/ ␤N243R with natural ␤Arg-246 was just as detrimental as ␤R246A. The data demonstrate that electrostatic interaction is an important component of initial P i binding in catalytic site ␤E and later at the transition state complex. However, since none of the mutants showed significant function in growth tests, ATP-driven proton pumping, or ATPase activity assays, it is apparent that specific stereochemical interactions of catalytic site Arg residues are paramount.ATP synthase is the terminal enzyme of oxidative phosphorylation and photophosphorylation, which synthesizes ATP from ADP and phosphate (P i ). The energy for ATP synthesis comes from transmembrane movement of protons down an electrochemical gradient, generated by substrate oxidation or by light capture. Initially, as the protons move through the interface between a and c subunits in the membrane-bound F 0 -sector of the enzyme, the realized energy is transduced into mechanical rotation of a group of subunits (␥⑀c 10 -14 ), which comprise the "rotor". A helical coiled coil domain of ␥ projects into the central space of the ␣ 3 ␤ 3 hexagon, in the membraneextrinsic F 1 -sector. ␣ 3 ␤ 3 hexagon contains three catalytic sites at ␣/␤ interfaces. In a manner that is not yet understood, rotation of ␥ vis-à -vis the three ␣/␤ subunit pairs brings about ATP synthesis at the three catalytic sites using a sequential reaction scheme (1). "Stator" subunits b 2 and ␦ are present to prevent co-rotation of ␣ 3 ␤ 3 with the rotor. Detailed reviews of ATP synthase mechanism may be found in Refs. 2-5.Binding of P i is an important step of the ATP synthase mechanism that has been extensively studied by biochemical approaches and may be directly coupled to rotation of subunits (3, 6 -11). Recent studies of the rotational mechanism have begun to illuminate which steps in the enzymic pathway of ATP synthesis an...

Role of α-Subunit VISIT-DG Sequence Residues Ser-347 and Gly-351 in the Catalytic Sites of Escherichia coli ATP Synthase

“…1B). We also established that introduction of a negative or positive charge in this location resulted in drastic modulation of P i binding (25,26,30), indicating that negative charge within the triangular subdomain was an important determinant of P i binding. Here we used the same approaches to study residues ␣Ser-347 and ␣Gly-351.…”

“…Inhibition of ATPase Activity of ATP Synthase in Membranes by NBD-Cl and Reversal by Dithiothreitol-We previously established that P i binding by mutant or wild type ATP synthase can be assayed using either membrane preparations or purified F 1 , with equivalent results (24,26). In this work we used membrane preparations that are more convenient.…”

Inhibition of ATPase activity of Escherichia coli ATP synthase by polyphenols

“…Partial inhibition of ATP synthase is not uncommon. In previous studies [34][35][36][37][38][39][40], we have noted several instances where mutant ATP synthase were incompletely inhibited by potent inhibitors like fluoroaluminate, fluoroscandium, sodium azide, or NBD-Cl. For example, detailed studies on NBD-Cl inhibition showed that fully reacted purified F 1 or membranes retained residual activity.…”

“…We consistently found that the F 1 data and the membrane data were the same for these inhibitors. Previously we established that inhibition of ATPase activity can be assayed using either membrane preparations or purified F 1 with equivalent results [34,35]. Fig.…”

Two ATPases

“…Using an assay (developed by Stuart Ferguson's laboratory) that fortuitously measures binding only in the empty site to which P i normally binds for ATP synthesis, postdoctoral fellow Zulfiqar Ahmad defined a triangular P i -binding pocket at one end of the catalytic site, involving four positively charged residues plus Ser-OH (53)(54)(55). Modulation of positive charge density within this pocket by deletion or introduction of Arg residues markedly affected P i binding (56). Not all candidate residues were "hits"; for example, from the x-ray structure, ␤-Asn-243 seemed a likely P ibinding residue, but mutagenesis showed that it was not involved (57).…”