F1-ATPase, Roles of Three Catalytic Site Residues

182

209

The structure of bovine F 1 -ATPase, crystallized in the presence of AMP-PNP and ADP, but in the absence of azide, has been determined at 1.9 Å resolution. This structure has been compared with the previously described structure of bovine F 1 -ATPase determined at 1.95 Å resolution with crystals grown under the same conditions but in the presence of azide. The two structures are extremely similar, but they differ in the nucleotides that are bound to the catalytic site in the ␤ DPsubunit. In the present structure, the nucleotide binding sites in the ␤ DP -and ␤ TP -subunits are both occupied by AMP-PNP, whereas in the earlier structure, the ␤ TP site was occupied by AMP-PNP and the ␤ DP site by ADP, where its binding is enhanced by a bound azide ion. Also, the conformation of the side chain of the catalytically important residue, ␣Arg-373 differs in the ␤ DP -and ␤ TP -subunits. Thus, the structure with bound azide represents the ADP inhibited state of the enzyme, and the new structure represents a ground state intermediate in the active catalytic cycle of ATP hydrolysis.Our current understanding of the molecular mechanism of F 1 -ATPase is based on the structural analysis by x-ray crystallography of the enzyme from bovine heart mitochondria. The first high resolution structure (1), now known as the "reference" structure, determined at 2.8 Å resolution with crystals grown in the presence of both ADP and the nonhydrolyzable ATP analog AMP-PNP, 3 showed that the three noncatalytic ␣-subunits and the three catalytic ␤-subunits are arranged in alternation around an asymmetric ␣-helical structure in the single ␥-subunit. The ␣-and ␤-subunits have similar folds consisting of an N-terminal domain with six ␤-strands, a central nucleotide binding domain made of both ␣-helices and ␤-strands and an ␣-helical C-terminal domain containing six ␣-helices in ␤-subunits and seven in ␣-subunits. Because of the asymmetry of the ␥-subunit, the catalytic ␤-subunits adopt different conformations with different nucleotide occupancies. Two of them have similar conformations, but one, designated as ␤ DP , contains bound ADP, and the second, ␤ TP , has bound AMP-PNP. The third has adopted a radically different conformation in which the nucleotide binding domain has been disrupted by an outward hinging movement of part of the domain and the attached C-terminal domain in response to the curvature of the central ␣-helical structure of the ␥-subunit. This ␤-subunit has no bound nucleotide, and so it is known as the "empty" or open state, designated as ␤ E . To explain the interconversion of catalytic sites through "tight," "loose," and "open" states required by a binding change mechanism of catalysis of ATP hydrolysis by F 1 -ATPase (2), it was proposed that the interconversion of sites is effected by a mechanical rotation of the ␥-subunit, each 360°r otation taking each ␤-subunit through the three states and thereby hydrolyzing three ATP molecules. It was shown subsequently that during ATP hydrolysis, either in an ␣ 3 ␤ 3 ␥ complex or in the...

Section: Resultssupporting

confidence: 72%

Ground State Structure of F1-ATPase from Bovine Heart Mitochondria at 1.9 Aå Resolution

Bowler

Montgomery

Leslie

et al. 2007

182

209

“…b This concentration ratio was used to be consistent with our previous work on MgATP binding and hydrolysis (12,16,27 ured above, we calculated the fraction that each enzyme species contributes to the total population at different nucleotide concentrations (for details, see "Experimental Procedures"). Fig.…”

Section: Conditionsmentioning

confidence: 99%

Bi-site Catalysis in F1-ATPase: Does It Exist?

Weber

Senior

2001

Self Cite

The mechanism of action of F 1 F 0 -ATP synthase is controversial. Some favor a tri-site mechanism, where substrate must fill all three catalytic sites for activity, others a bi-site mechanism, where one of the three sites is always unoccupied. New approaches were applied to examine this question. First, ITP was used as hydrolysis substrate; lower binding affinities of ITP versus ATP enable more accurate assessment of sites occupancy. Second, distributions of all eight possible enzyme species (with zero, one, two or three sites filled) as fraction of total enzyme population at each ITP concentration were calculated, and compared with measured ITPase activity. Confirming data were obtained with ATP as substrate. Third, we performed a theoretical analysis of possible bi-site mechanisms. The results argue convincingly that bi-site hydrolysis activity is negligible, and may not even exist. Effectively, tri-site hydrolysis is the only mechanism. We argue that only tri-site hydrolysis drives subunit rotation. Theoretical analyses of possible bi-site mechanisms reveal serious flaws, not previously recognized. One is that, in bi-site catalysis, the predicted direction of subunit rotation is the same for both ATP synthesis and hydrolysis; a second is that infrequently occurring enzyme species are required. F 1 is the catalytic sector of ATP synthase, the enzyme that synthesizes ATP in the last step of oxidative phosphorylation (for reviews, see Refs. 1 and 2; short reviews on individual topics can be found in Refs. 3-5). F 1 has a subunit composition of ␣ 3 ␤ 3 ␥␦⑀. The three catalytic nucleotide-binding sites are located at ␣/␤ interfaces, mainly on the ␤ subunits (6). F 1 can be isolated in soluble form and is an active ATPase. It is frequently used as a model for catalysis by the holoenzyme ATP synthase (also called F 1 F 0 ).F 1 (and F 1 F 0 ) can hydrolyze MgATP in different modes, depending on the substrate concentration. Two of these modes are clearly established and well characterized. At low, substoichiometric MgATP concentrations the enzyme binds MgATP with very high affinity, just to catalytic site one. A single turnover of MgATP hydrolysis ensues on this site, called "unisite catalysis," in which the chemical hydrolysis step is slow (0.1 s Ϫ1 in Escherichia coli F 1 , Ref. 1) and products release is even slower (0.001 s Ϫ1 ). On the other hand, at cellular (millimolar) substrate concentrations, all three catalytic sites are filled and interact with each other, and the enzyme turns over rapidly (ϳ100 s Ϫ1 ). This "tri-site" or "multisite catalysis" is the physiologically relevant working mode of the enzyme (1). In multisite catalysis, MgATP hydrolysis on the ␤ subunits drives rotation of the ␥⑀c ensemble (7-9), possibly transmitted via ␣ subunits (10). In contrast, uni-site catalysis can occur without subunit rotation (11).The contribution to hydrolysis of enzyme molecules with two substrate-filled catalytic sites ("bi-site catalysis") remains enigmatic. Originally, deviations from simple Michaelis-Menten kine...

“…Table 1). Based on the affinity constants from Senior and co-workers (61,62), and our previous work monitoring pre-steady state nucleotide binding (18), this is an adequate concentration of Mg⅐ATP to fill two sites completely and the third site partially. 32 P i produced is reported in moles of P i per mol of F 1 for untreated F 1 (Ⅺ), F 1 incubated with 50 M CuCl 2 to form the ␥-␤ crosslinked sample (f), and F 1 incubated with 10 mM DTT after CuCl 2 treatment (؉).…”

Section: Characterization Of Cys Mutants and Cross-linkedmentioning

confidence: 99%

A Rotor-Stator Cross-link in the F1-ATPase Blocks the Rate-limiting Step of Rotational Catalysis

Scanlon

Al‐Shawi

Nakamoto

2008

The F 0 F 1 -ATP synthase couples the functions of H ؉ transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located ␥, ⑀, and c subunits. To understand the ␥ subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F 1 -ATPase enzyme that uniquely fits the pre-steady state and steady state data (Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797). Here we directly test the model using two single cysteine mutants, ␤D380C and ␤E381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved ␥Cys-87. In the pre-steady state, the ␥-␤ cross-linked enzyme at high Mg⅐ATP conditions retained the burst of hydrolysis but was not able to release P i . These data show that the rate-limiting rotation step, k ␥ , occurs after hydrolysis and before P i release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the ␤Glu-381 residue for proper formation of the rate-limiting transition state involving ␥ subunit rotation.