The (αF³⁵⁷C)₃(βR³⁷²C)₃γ Subcomplex of the F₁-ATPase from the Thermophilic Bacillus PS3 Has Altered ATPase Activity after Cross-Linking α and β Subunits at Noncatalytic Site Interfaces

Abstract

In crystal structures of bovine MF(1), the side chains of alpha F(357) and beta R(372) are near the adenines of nucleotides bound to noncatalytic sites. To determine if during catalysis these side chains must pass through the different arrangements in which they are present in crystal structures, the catalytic properties of the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the TF(1)-ATPase were characterized before and after cross-linking the introduced cysteines with CuCl(2). The unmodified mutant enzyme hydrolyzes MgATP at 50% the rate exhibited by wild type. Detailed comparison of the catalytic properties of the double mutant enzyme before and after cross-linking with those of the wild-type subcomplex revealed the following. Before cross-linking, the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex has less tendency than wild type to release inhibitory MgADP entrapped in a catalytic site during turnover when MgATP binds to noncatalytic sites. Following cross-linking, ATPase activity is reduced 5-fold, and inhibitory MgADP entrapped in a catalytic site during turnover does not release under conditions wherein binding of ATP to noncatalytic sites of the wild-type enzyme promotes release of MgADP from the affected catalytic site. When assayed in the presence of lauryldimethylamine oxide, which prevents turnover-dependent entrapment of inhibitory MgADP in a catalytic site, ATPase activity of the cross-linked form is 47% that of the unmodified mutant enzyme. These results suggest that, during catalysis, the side chains of alpha F(357) and beta R(372) do not pass through the extremely different relative positions in which they exist at the three noncatalytic site interfaces in crystal structures.

The Ionic Track in the F1-ATPase from the Thermophilic Bacillus PS3

“…On the basis of the catalytic characteristics of other mutant R 3 β 3 γ subcomplexes of TF 1 (23), this indicates that subcomplexes containing the βE 395 C substitution have a greater propensity to entrap inhibitory MgADP in a catalytic site during turnover. Previous studies have shown that nucleotide-depleted MF 1 (24) and the nucleotide-depleted wild-type R 3 β 3 γ subcomplex of TF 1 (14) hydrolyze 50 µM ATP in three distinct phases: an initial In an earlier study, we demonstrated that the ATPase activity of the oxidized (RF 357 C) 3 (βR 372 C) 3 γ double mutant containing R-β cross-links at two noncatalytic site interfaces was stimulated 13-fold by LDAO, whereas the ATPase activity of the non-cross-linked double mutant was stimulated 5.7-fold by the detergent (17). In crystal structures of bovine MF 1 , the 392 GMDELS 397 segment is the turn in the helixturn-helix motif in the C-terminal domain of β subunits.…”

“…Inactivation of five subunit TF 1 (R 3 β 3 γδ ) with 5′-p-fluorosulfonyl[ 3 H]adenosine is caused by derivatization of the equivalent of βY 368 in MF 1 (26). After cross-linking the introduced cysteines in the (RF 357 C) 3 (βR 372 C) 3 γ subcomplex of TF 1 , the ATPase activity was reduced to 10% of the rate catalyzed by the wild-type subcomplex (17). Therefore, it is reasonable to assume that substitution of βE 395 in the 392 GMDELS 397 segment with Cys or Ala might affect the orientation of the R subunit with respect to the β subunit at the noncatalytic site interface, thereby altering the affinity of noncatalytic sites for nucleotides or affecting the conformational equilibrium between noncatalytic and catalytic sites.…”

“…To test this possibility, the R 3 (βE 395 C) 3 γ mutant subcomplex was carboxymethylated with iodo[ 3 H]acetate. An earlier study showed that the naturally occurring cysteine in position-193 of the R subunit was not labeled after prolonged incubation of the wild-type subcomplex with iodo[ 3 H]acetate under the conditions described in the legend of Figure 6 (17). Therefore, it is probable that only the introduced cysteines were carboxymethylated when the R 3 (βE 395 C) 3 γ mutant subcomplex was treated with the reagent.…”

Aloe-emodin Attenuates Staphylococcus aureus Pathogenicity by Interfering With the Oligomerization of α-Toxin

“…A 280-nm excitation wavelength with a 5-nm bandpass and a 345-nm emission wavelength with a 10-nm bandpass were used for the measurements. Details of performing the measurements were described previously (Bandyopadhyay et al, 2002; Jurasekova et al, 2009).…”