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

Wenzong Li,Laura E Brudecki,Alan E Senior,Zulfiqar Ahmad

doi:10.1074/jbc.m809209200

Abstract

This paper describes the role of alpha-subunit VISIT-DG sequence residues alphaSer-347 and alphaGly-351 in catalytic sites of Escherichia coli F(1)F(o) ATP synthase. X-ray structures show the very highly conserved alpha-subunit VISIT-DG sequence in close proximity to the conserved phosphate-binding residues alphaArg-376, betaArg-182, betaLys-155, and betaArg-246 in the phosphate-binding subdomain. Mutations alphaS347Q and alphaG351Q caused loss of oxidative phosphorylation and reduced ATPase activity of F(1)F(o) in membranes by 100- and 150-fold, respectively, whereas alphaS347A mutation showed only a 13-fold loss of activity and also retained some oxidative phosphorylation activity. The ATPase of alphaS347Q mutant was not inhibited, and the alphaS347A mutant was slightly inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium, in contrast to wild type and alphaG351Q mutant. Whereas 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) inhibited wild type and alphaG351Q mutant ATPase essentially completely, ATPase in alphaS347A or alphaS347Q mutant was inhibited maximally by approximately 80-90%, although reaction still occurred at residue betaTyr-297, proximal to the alpha-subunit VISIT-DG sequence, near the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts inbetaE (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type and alphaG351Q mutant but not in alphaS347Q or alphaS347A mutant. The results demonstrate that alphaSer-347 is an additional residue involved in phosphate-binding and transition state stabilization in ATP synthase catalytic sites. In contrast, alphaGly-351, although strongly conserved and clearly important for function, appears not to play a direct role.

Highlights

F1Fo-ATP synthase is the enzyme responsible for ATP synthesis by oxidative or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts
This paper describes the role of ␣-subunit VISIT-DG sequence residues ␣Ser-347 and ␣Gly-351 in catalytic sites of Escherichia coli F1Fo ATP synthase
To better understand the reaction mechanism of ATP synthesis and hydrolysis and their relationship to mechanical rotation in this biological nanomotor, we have focused our efforts on determining the role of conserved residues in and around catalytic site Pi-binding subdomain

Summary

Introduction

F1Fo-ATP synthase is the enzyme responsible for ATP synthesis by oxidative or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts. It is the fundamental means of cell energy production in animals, plants, and almost all microorganisms. To better understand the reaction mechanism of ATP synthesis and hydrolysis and their relationship to mechanical rotation in this biological nanomotor, we have focused our efforts on determining the role of conserved residues in and around catalytic site Pi-binding subdomain. Knowledge of Pi-binding residues and residues surrounding the Pi-binding subdomain is imperative for accomplishing (i) the molecular modulation of the catalytic site for the improved catalytic and motor function of this enzyme, (ii) an explanation of how ATP synthase binds ADP and Pi within its catalytic sites in the face of a relatively high ATP/ADP concentration ratio, and (iii) understanding the relationship between Pi binding and subunit rotation (14 –16). This renders the potentially more convenient centrifuge column assay unsuitable with the E. coli enzyme

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Conclusion