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 insert 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 ATP synthase alpha/beta subunits. The alpha subunit is catalytic and the beta subunit is regulatory, and there is a substrate-binding site on each. Within the catalytic A subunit there are four domains, the N-terminal, non-homologous, nucleotide binding a-b, and the C-terminal. Residues x-x constitute the sheet-loop-helix motif of P-loop, or phosphate binding loop.

Transition State Stabilization

vanadate

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

S238 has an oh and is polar O atoms of the y-phosphate in ATP, thus diff. mechanisms Is involved with hydrogen bond formation between nucleotides and phosphate analog, sulfate. Within the P-loop interacts with nucleotides during catalysis

L417 Is involved in a bifurcated hydrogen bond F236 in P-loop third position stabilizes arched loop (also P235 S238)

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

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

Active/Alternating Catalytic Model

The active site of B subunit is cycling between 3 states. 5 STEPS- ONE IS STANSITION STATE the deviations between the three states are concentrated within three regions… As "Open state"-ADP and Pi enter active site

P loop farthers, s238 farthest

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)

Apnp "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.