The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

226

180

In the structure of bovine F1-ATPase determined at 1.95-Å resolution with crystals grown in the presence of ADP, 5 -adenylylimidodiphosphate, and azide, the azide anion interacts with the ␤-phosphate of ADP and with residues in the ADP-binding catalytic subunit, ␤DP. It occupies a position between the catalytically essential amino acids, ␤-Lys-162 in the P loop and the ''arginine finger'' residue, ␣-Arg-373, similar to the site occupied by the ␥-phosphate in the ATP-binding subunit, ␤TP. Its presence in the ␤DP-subunit tightens the binding of the side chains to the nucleotide, enhancing its affinity and thereby stabilizing the state with bound ADP. This mechanism of inhibition appears to be common to many other ATPases, including ABC transporters, SecA, and DNA topoisomerase II␣. It also explains the stimulatory effect of azide on ATP-sensitive potassium channels by enhancing the binding of ADP.mitochondria ͉ oxidative phosphorylation ͉ inhibition ͉ mechanism

Section: Resultsmentioning

confidence: 79%

How azide inhibits ATP hydrolysis by the F-ATPases

Bowler

Montgomery

Leslie

et al. 2006

226

180

“…Reassigning the pentacoordinate species as a transition state analogue necessarily limits the inferences that can be drawn from the structure as the bond lengths and electrostatic contacts will be governed by the intrinsic ground state properties of the analogue, and thus do not necessarily reflect the bond lengths in the TS or potential INT. However, the central MgF 3 Ϫ moiety is both isoelectronic and isosteric with metaphosphate PO 3 Ϫ and so provides a closer portrayal of the expanded TS for phosphate monoester transfer in solution than aluminum and beryllium fluoride complexes which have been more widely utilized to date (13,(26)(27)(28). The intrinsic binding constant of MgF 3 Ϫ to the enzyme-substrate complex will be considerably lower than the observed K i as MgF 3 Ϫ has a very low formation constant in aqueous solution [MgF ϩ has log ␤ 1 ϭ 1.7 and MgF 2 has log ␤ 2 Յ 3.2 (29); MgF 3 Ϫ has not been observed in solution], which means that it is only observable in the enzyme active site.…”

Section: Discussionmentioning

confidence: 99%

“…The high susceptibility of 19 F NMR chemical shifts to the local electronic environment provides a sensitive probe of subtle changes within the enzyme that would be invisible to other structural biology methods. These methods can also be applied to the study of other complexes that contain metal-fluoride species as analogues for phosphates, such as aluminum (27,33) and beryllium (26,34) fluorides, and will enable the accurate assignment of their identity in future. The potential utility of using fluoride as a probe of enzyme structure is especially powerful when used in combination with an electronically and geometrically accurate transition state mimic for phosphoryl transfer, MgF 3 Ϫ , that only exists within the confines of the active site and places the spectroscopically active atoms right into the catalytic cavity.…”

Section: Discussionmentioning

confidence: 99%

A Trojan horse transition state analogue generated by MgF ₃ ⁻ formation in an enzyme active site

Baxter

Olguín

Goličnik

et al. 2006

113

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. ␤-Phosphoglucomutase catalyses the isomerization of ␤-glucose-1-phosphate to ␤-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using 19 F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF 3 ؊ mimics the transferring PO 3 ؊ moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of ␤-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.enzyme mechanism ͉ fluoride inhibition ͉ NMR structure ͉ phosphoryl transfer ͉ isosteric isoelectronic ͉ transition state analogue P hosphate transfer reactions play a central role in metabolism, regulation, energy housekeeping and signaling (1). As phosphate esters are kinetically extremely stable, efficient catalysis is crucial for the control of these cellular processes. Although model studies have taught us much about the intrinsic chemical mechanisms (2), our understanding of the origins of the enormous enzymatic rate accelerations involved, up to a factor of 10 21 (3), is far from complete (4). A snapshot of an enzyme in a high-energy state would be immensely useful, as it would allow the very interactions that bring about catalysis to be observed (5). However, is this realistic given how elusive high-energy intermediates and transition states (TSs) inevitably are? The direct observation of TSs for simple organic reactions has required ultrafast lasers with femtosecond resolution (6) and no physical or spectroscopic method is available to observe the structure of TSs of enzymatic reactions directly. Thus transition state analogues that bind tightly in an enzyme active site have been of paramount importance in defining the structural and energetic framework for catalysis (7,8).An observation that appears to challenge this paradigm arises from structural studies with ␤-phosphoglucomutase (␤-PGM, EC 5.4.2.6): namely, that a high-energy phosphorane on the reaction pathway has been observed directly by x-ray crystallography, demonstrating how the enzyme interacts with a very high-energy, metastable species (9). The latter also apparently demonstrated that the enzyme catalyzed reaction proceeds through an addition-elimination mechanism, a reaction pathway not observed in solution for phosphate monoester anions. However, the observation of an enzyme "caught in the act" is surprising: the demands of turnover mean that the enzyme would gain no apparent advant...

“…All six of the ␣-and ␤-subunits bind nucleotides, but only the three ␤-subunits are catalytically active. The crystal structures of F 1 ATPase have proven to be enormously informative regarding the mechanism of the motor because each crystallographic snapshot provides views of three distinct states of the catalytic ␤-subunits (51,52,57). The centrally located and asymmetric ␥-subunit forms a shaft, and its orientation determines the conformation of the ␤-subunits.…”

Section: Molecular Dynamics Analyses Of F 1 Atpasementioning

confidence: 99%

“…The simulations described so far do not address the energetics of ATP binding to the various states. The essential difficulty here arises from the striking similarity in structure between the ␤ DP and ␤ TP states (52,57). A recently determined high-resolution structure of F 1 ATPase has the ATP analog ADP.AlF 4 bound at the ␤ DP and the ␤ TP sites, with tight coordination of the nucleotide in both cases (52).…”

Section: Conformational Transitions In F1 Atpasementioning

confidence: 99%

Molecular dynamics and protein function

Karplus

Kuriyan

2005

968

778

A fundamental appreciation for how biological macromolecules work requires knowledge of structure and dynamics. Molecular dynamics simulations provide powerful tools for the exploration of the conformational energy landscape accessible to these molecules, and the rapid increase in computational power coupled with improvements in methodology makes this an exciting time for the application of simulation to structural biology. In this Perspective we survey two areas, protein folding and enzymatic catalysis, in which simulations have contributed to a general understanding of mechanism. We also describe results for the F1 ATPase molecular motor and the Src family of signaling proteins as examples of applications of simulations to specific biological systems.