A-ATP Synthase

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. The catalytic action of ADP+Pi-->ATP occurs in the A1 domain. 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) 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

induced fit model 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.

The deviations between the three structures supports the hypothesis that the transition state undergoes conformational changes to induce catalysis of ATP.

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 reaction coordination=freeze frame picture of reactants*RELATE TO LECTURE substrates=higher free energy than products 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.

P 746 ####Increased proximities of catalytically important residues

Residue S238 is located within the P-loop and is involved with hydrogen bond formation between nucleotides and phosphate analog, sulfate. has an -OH group and is polar O atoms of the y-phosphate in

L417 Is involved in a bifurcated hydrogen bond Residue F236 in P-loop third position stabilizes arched P-loop (also P235 S238) *subunit beta in moving towards the y-phosphate of ATP during catalysis.

also stabilized by weak non-polar interactions and polar. K162+ R189+ E188-

Not at bonding distances-K240 R264 E263-(closer to vanadate)