A-ATP Synthase

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Contents 1 Introduction 2 Structure 3 Transition State Stabilization 4 Conclusion

Introduction

The A1Ao ATP synthase from archaea represents a class of chimeric ATPases/synthase , whose function and general structural design share characteristics both with vacuolar V1V0 ATPases and withF1Fo ATP synthases ^[1] A1A0 ATP synthase catalyzes the formation of the energy currency ATP by a membrane-embedded electrically-driven motor. The archaeon in this study, Pyrococcus horikoshii OT3 is an anaerobic thermophile residing in oceanic deep sea vents with optimal growth at 100degrees. Anaerobic fermentation is the principle metabolic pathway. The membrane-embedded electrically-driven motor (A0) is very different in archaea with sometimes novel, exceptional subunit composition and coupling stoichiometries that may reflect the differences in energy-conserving mechanisms as well as adaptation to temperatures at or above 100 degrees C. ^[2] Because some archaea are rooted close to the origin in the tree of life, these unusual mechanisms are considered to have developed very early in the history of life and, therefore, may represent first energy-conserving mechanisms. ^[3]

Structure

The ATP synthase is composed of two domains consisting of nine subunits A3:B3:C:D:E:F:H2:a:cx. that function as a pair of rotary motors connected by central and peripheral stalk(s). The A0 domain is the hydrophobic membrane embedded ion-translocating sector that uses the H+ gradient to power ATP synthase in domain A1. A1 is water soluble and undergoes a conformational change upon binding substrate. It is a ring with three-fold symmetry of alternating A,B subunits similar to F-ATP synthase ATP synthase alpha/beta subunits. The A subunit is catalytic and the B subunit is regulatory, with a substrate-binding site on each. Within the catalytic A subunit there are four domains, the N-terminal [residues 1-79, 110-116, 189-199], non-homologous [residues 117-188], nucleotide binding alpha-beta P-loop [residues 80-99, 200-437], and the C-terminal alpha helical bundle [residues 438-588).figure 1.

The P-loop or phosphate binding loop is conserved only within the A subunits (as compared to the F-ATP synthase where it is present in both alpha and beta) and is a glycine-rich loop preceded by a beta sheet and followed by an alpha helix. It interacts with the phosphate groups of the nucleotide and with a magnesium ion, which coordinates the β- and γ-phosphates. ^[4] This P-loop has an arched conformation unique to A-ATP Synthase, indicating that the mode of nucleotide binding and the catalytic mechanism is different from that of F-ATP syntheses.

Transition State Stabilization

Five steps inside the catalytic A-subunit are critical for catalysis. Substrate entrance, phosphate and nucleotide binding, transition-state formation, ATP formation, and product release. The vanadate bound model mimics the transition state. Orthovandate is a transition state analog and because it can adapt both tetragonal and trigonal bipyramidal coordination geometry. Fig. 1. The Avi structure can be compared to the As sulfate bound structure and the Apnp AMP-PNP bound structure. "'As'" is analogous to the phosphate binding (substrate) structure, and "'Apnp"' is analogous to the ATP binding (product) structure. A reaction coordination is generated from freeze frame picture of reactants such as "'As"' "'Avi"' and "Apnp". The movement of specific residues to stabilize the transition state is demonstrated by comparing the deviations between the three structures.

There are three major positions that interact with ligands in the P-loop, S238 L417 and F236.

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Residue [S238] is polar and interacts with the nucleotides via a hydrogen bond during catalysis. The distance between residue [S238] is longest in "'As"', shortest in "'Avi"' and intermediate in "'Apnp"' . In"'As"' a water molecule bridges the gap, which is removed in '"Avi"'. Dehydration of the transition state active site is reversed when ATP forms. [20] "In "'Apnp"' the water molecule interacts with the y-phosphate of ATP [10].

In "'F-ATP Synthase"' the homolog to S238 is the non polar A158. Since A158 cannot form hydrogen bonds to interact with the substrate, the P-loop undergoes a conformational change. In A-ATP Synthase the close proximity needed between S238 and the vandate during transition state is achieved with a hydrogen bond, not a conformational change in the P-loop.

L417 Is involved in a bifurcated hydrogen bond.

K240 T241 interact with Mg+ [10]

R349-

Residues that stabilizes the arched P-loop include P235 ----F236*subunit beta in moving towards the y-phosphate of ATP during catalysis. also stabilized by weak non-polar interactions and polar. K162+ R189+ E188-

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Although not at bonding distances the residues K240 R264 E263 move closer to the vanadate with respect to the two other structures and are proposed to stabilize the transition state during catalysis.[table 4]

These increased proximities of the catalytically important residues clearly demonstrate that structural rearrangement occurs during catalysis in subunit A.

The second vandate is positioned in a region exactly opposite the nucleotide-binding site, where the ATP molecule transiently associates on its way to the final binding pocket in subunit "'B"'. [25] Similar binding behavior was observed for "'As"' [10] indicating that the substrate molecule has a similar path of entry to the active site in both the "'A"' and '"B"' subunit of the A-ATP synthase and that they have a transient binding position near the P-Loop. It is proposed that Pi binds first to the catalytic site and sterically hinders ATP binding, thereby selectively allowing binding of ADP [14] The "'Avi"' structure confirms this, since although both ADP and Vi were present in the crystallized solution, the catalytic A-subunit first permits only the binding of the phosphate analogue Vi. Hence the present "Avi"' structure represents a trapped initial transition state showing for the first time both the entering path and the final Vi-bound state in the catalytic subunit.

Conclusion

The active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme.[29] As a result, the substrate does not simply bind to a rigid active site; the amino acid side chains which make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function. Stabilization of the transition state supports the induced fit model. A-ATP synthase lowers the activation energy by creating an environment in which the transition state is stabilized (e.g. straining the shape of a substrate—by binding the transition-state conformation of the substrate/product molecules, the enzyme distorts the bound substrate(s) into their transition state form, thereby reducing the amount of energy required to complete the transition).

when the enzyme is complementary to the substrate, the E.S. complex is more stable, has less free energy in the ground state than substrate alone. You increase the activation energy.

Avi "loose state"-closes up around molecules and binds them loosely (transition state has more free energy than both S and P)

(P loop intermediate S238 closest G234 residue sidechain k240 P loop closer)

Adp(ADP bound) Apnp (AMP-PNP bound) "tight state"- forces molecules together, binding ATP with high affinity

(P loop closest S238 int K240 significant)

possible sources of error can be the fact that ADp is not bound during transition state? is synthase reversible? where is it located? absence of ADP, may not affect the formation of transition-like state because of example