Catalytic Activity of the α3β3γ Complex of F1-ATPase without Noncatalytic Nucleotide Binding Site

Tadashi Matsui,Eiro Muneyuki,Masahiro Honda,William S Allison,Chao Dou,Masasuke Yoshida

doi:10.1074/jbc.272.13.8215

Tadashi Matsui, Eiro Muneyuki + Show 4 more

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https://doi.org/10.1074/jbc.272.13.8215

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Abstract

A mutant alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 microM; Km(2), 135 microM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below approximately 300 microM.

Highlights

A mutant ␣3␤3␥ complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides
Addition of ATP promoted single-site hydrolysis of 2؅,3؅-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below ϳ300 ␮M
Noncatalytic Sites Are Essential for Continuous Catalytic Turnover—This work provides solid support for the view that entrapping inhibitory MgADP at a catalytic site, either during incubation with MgADP or during turnover under assay conditions, causes inactivation of F1-ATPase

Summary

Introduction

A mutant ␣3␤3␥ complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. The ␣3␤3␥ complex of TF1 containing ␤ subunits with a mutation in the Walker motif A, ␤-T165S, efficiently dissociates inhibitory MgADP and exhibits a severalfold higher final rate of ATP hydrolysis than that of the wild-type complex [19]. These results suggest that F1-ATPase in the inactivated state with inhibitory MgADP in a catalytic site is reactivated by ATP binding to noncatalytic sites. The effect of noncatalytic sites on cooperative kinetics of F1-ATPase remains unsettled

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Conclusion