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

Zulfiqar Ahmad,Alan E Senior

doi:10.1074/jbc.m503955200

Zulfiqar Ahmad, Alan E Senior

Open Access

https://doi.org/10.1074/jbc.m503955200

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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 betaE catalytic site of ATP synthase specifically. Removal of natural betaArg-246 (betaR246A mutant) abrogates P(i) binding; restoration of P(i) binding was achieved by mutagenesis of either residue betaAsn-243 or alphaPhe-291 to Arg. Both residues are located in the P(i) binding site close to betaArg-246 in x-ray structures. Insertion of one extra Arg at beta-243 or alpha-291 in presence of betaArg-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 betaArg-246 in betaR246A caused almost complete loss of transition state stabilization, but partial rescue was achieved in betaN243R/betaR246A and alphaF291R/betaR246A. BetaArg-243 or alphaArg-291 in presence of betaArg-246 was less effective; the combination of alphaF291R/betaN243R with natural betaArg-246 was just as detrimental as betaR246A. The data demonstrate that electrostatic interaction is an important component of initial P(i) binding in catalytic site betaE 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.

Highlights

Introduction of Asp orGlu at ␣-291 completely prevented Pi binding
This paper presents a study of the role of positive charge in the Pi binding site of Escherichia coli ATP synthase, the enzyme responsible for ATP-driven proton extrusion and ATP synthesis by oxidative phosphorylation
Growth Properties of New Mutants of E. coli ATP Synthase—A series of mutants was generated to modulate charge in the proximity of residue ␤Arg-246, which was shown earlier to be a key residue for binding of Pi into the catalytic sites on the F1-sector of ATP synthase [11]

Summary

Introduction

Introduction of Asp orGlu at ␣-291 completely prevented Pi binding (Z. Ahmad, unpublished work) indicating proximity of the side chain to bound Pi and ␤Arg-246.binding (Fig. 3), but the combination of ␣F291R/␤N243R (two extra charges) abrogated Pi binding. A similar pattern of effects was seen when transition state stabilization was assessed by assaying inhibition of ATPase activity by the transition state analogs MgADP-fluoroaluminate and MgADP-fluoroscandium. Ref. 11 that both inhibitors are potent against wild-type ATP synthase but inhibit ␤R246A mutant only to small extent, indicating that ␤Arg-246 is intimately involved in transition state stabilization. Raising the number of positively charged residues to two (␤N243R and ␣F291R mutants in wild-type background) had an adverse effect as reflected by lesser inhibition of ATPase; and raising the number of local positive charges to three reduced transition state stabilization right back to where it was in ␤R246A. Restoration of Pi binding in ␤E catalytic sites by charge compensation is not sufficient by itself to restore full function

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