Atp Synthase

ATP SYNTHASE 1997 Nobel Prize was for the enzymatic mechanism underlying the synthesis of adenosine triphosphate Its function There are several ATP synthase enzymes and they have been remarkably conserved during evolution. The bacterial enzymes are essentially the same in structure and function as those from mitochondria of animals, plants and fungi, and the chloroplasts of plants. The early ancestery of the enzyme is seen in the fact that the Archaea have an enzyme which is clearly closely related, but has significant differences from the Eubacterial branch. The H+ATPase found in vacuoles of the eucaryote cell cytoplasm is similar to the archaeal enzyme, and is thought to reflect the origin from an archaeal ancestor. In most systems, the ATP synthase sits in the membrane (the "coupling" membrane), and catalyses the synthesis of ATP from ADP and phosphate driven by a flux of protons across the membrane down the proton gradient generated by electron transfer. The flux goes from the proton chemically positive side (high proton electrochemical potential) to the proton chemically negative side. The reaction catalyzed by ATP synthase is fully reversible, so ATP hydrolysis generates a proton gradient by a reversal of this flux. In some bacteria, the main function is to operate in the ATP hydrolysis direction, using ATP generated by fermentative metabolism to provide a proton gradient to drive substrate accumulation, and maintain ionic balance. In mitochondria, the positive side is the intermembrane space, and the negative side is the mitochondrial matrix; in bacteria, the positive side is the outside the cell and the negative side the cytoplasm; in chloroplasts, the positive side is the thylakoid lumen and the negative side the stroma. Subunit composition of the ATP synthase There are minor differences between bacteria, mitochondria and chloroplasts in some of the smaller subunits, which leads to a confusing nomenclature. The simplest system is that from E. coli. The ATP synthase can be dissociated into two fractions by relatively mild salt treatments. A soluble portion, the F1ATPase, contains 5 subunits, in a stoichiometry of 3a:3b:1g:1d:1e. Three substrate binding sites are in the b-subunits. Additional adenine nucleotide binding site in the a-subunits are regulatory. The F1 portion catalyzes ATP hydrolysis, but not ATPsynthesis. Dissociation of the the F1 ATPase from the membranes of bacteria or organelles leaves behind a membrane embedded portion called FO. This consists (in E. coli) of three subunits a, b and c, with relative numbers of 1:2:9-12. The c-subunit is very hydrophobic, and forms a helix turn helix structure which spans the membrane twice, with a hydrophilic loop on the side of attachment of F1. There is a conserved acidic residue half-way across the membrane in the C-terminal helix. After dissociation, the membranes are permeable to protons. The proton leak can be stopped by addition of inhibitors, which are also inhibitors of ATP synthesis in the functional complex. Two "classical" inhibtors are known. The antibiotic, Oligomycin, binds at the interface between F 0 and F1; dicyclohexylcarbodiimide (DCCD) binds covalently to the conserved acidic residue in the c-subunit of F 0. One DCCD per ATPase is sufficient to block turn-over, suggesting a cooperative mechanism. The action of these inhibitors indicates that the proton permeability of the F0 is a part of its functional mechanism.

Structure of the F1 ATPase

The structure of the soluble (F1) portion of the ATP synthase from beef heart mitochondria has been solved by X-ray crystallography. The pictures below are from Abrahams, J.P., Leslie, A.G., Lutter, R. and Walker, J.E. (1994) Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621-628. The protein was crystallized in the presence of ADP, and an ATP analogue, AMP-PNP, in which the the two terminal phosphates of ATP were replaced by the non-hydrolysible imidodiphosphate group. The three a-subunits each contained an AMP-PNP. The three b-subunits contained either ADP (bDP), AMP-PNP (bTP), or no nucleotide (bE). Mechanism of the F1 ATP-ase The ATP synthase operates through a mechanism in which the three active sites undergo a change in binding affinity for the reactants of the ATP-ase reaction, ATP, ADP and phosphate, as originally predicted by Paul Boyer. The change in affinity accompanies a change in the position of the g-subunit relative to the a, b-ring, which involves a rotation of the one relative to the other. In the direction of ATP synthesis, the rotation is driven by a flux of H+ down the proton gradient, through a coupling between the g-subunit, and the c-subunit of F0. This rotation has now been demonstrated experimentally. Experimental for rotational model

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This rotational motion has been captured in dramatic videos from the laboratory of Masasuke Yoshida. In this work, the F1-ATPase was tethered to a glass surface by the b-subunit, using a His-tag engineered into the protein at the N-terminus, and NTA-ligand on the glass. (see illustration from Junge et al. TIBS article, below). The motion was detected by attaching an actin filament to the g-subunit, which was tagged with fluorescent groups to make it visible, and recorded using a video camera attached to a microscope. The motion was seen only under conditions of ATP-hydrolysis, and the direction of motion was always counter-clockwise when viewed from the Fo portion, giving the sign of the catalytic mechanism. Hiroyuki Noji, Ryohei Yasuda, Masasuke Yoshida & Kazuhiko Kinosita Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature, 386, 299 - 302.

Physiological role Like other enzymes, the activity of F 1FO ATP synthase is reversible. Large enough quantities of ATP cause it to create a transmembrane proton gradient, this is used by fermenting bacteria which do not have an electron transport chain, and hydrolyze ATP to make a proton gradient, which they use for flagella and transport of nutrients into the cell. In respiring bacteria under physiological conditions, ATP synthase generally runs in the opposite direction, creating ATP while using the protonmotive force created by the electron transport chain as a source of energy. The overall process of creating energy in this fashion is termed oxidative phosphorylation. The same process takes place in mitochondria, where ATP synthase is located in the inner mitochondrial membrane (so that F1-part sticks into mitochondrial matrix, where ATP synthesis takes place). ATP - the universal energy carrier in the living cell The German chemist Karl Lohmann discovered ATP in 1929. Its structure was clarified some years later and in 1948 the Scottish Nobel laureate of 1957 Alexander Todd synthesised ATP chemically. An important role was that played by the 1953 Nobel laureate in Medicine Fritz Lipmann when he during the years 1939-41 showed that ATP is the universal carrier of chemical energy in the cell and coined the expression "energy-rich phosphate bonds". ATP functions as a carrier of energy in all living organisms from bacteria and fungi to plants and animals including humans. ATP captures the chemical energy released by the combustion of nutrients and transfers it to reactions that require energy, e.g. the building up of cell components, muscle contraction, transmission of nerve messages and many other functions. ATP has been termed the cell's energy currency. Adenosine triphosphate (ATP) consists of the nucleoside adenosine linked to three phosphate groups. On removal of the outermost phosphate group, adenosine diphosphate (ADP) is formed while at the same time the energy released can be employed for other reactions. Conversely, with the help of energy, an inorganic phosphate group can be bound to ADP and form ATP. Considerable quantities of ATP are formed and consumed. At rest, an adult converts daily a quantity of ATP corresponding to about one half body-weight, and during hard work the quantity can rise to almost a ton. Most of the ATP synthesis is carried out by the enzyme ATP synthase. At rest Na + , K + -ATPase uses up a third of all ATP formed. ATP synthase: an exceptional molecular machine During the 1940s and 1950s it was clarified that the bulk of ATP is formed in cell respiration in the mitochondria and photosynthesis in the chloroplasts of plants. In 1960 the American scientist Efraim Racker and co-workers isolated, from mitochondria, the enzyme "F o F 1 ATPase" which we now call ATP synthase. The enzyme can be divided into an F 1 part containing the catalytic center and the F o part coupling the F 1 part to the membrane. The same enzyme exists in chloroplasts and bacteria. In 1961 Peter Mitchell presented what is termed the chemiosmotic hypothesis for which he received the Nobel Prize in 1978. He showed that cell respiration leads to a difference in hydrogen ion concentration (pH) inside and outside the mitochondrial membrane, and that a stream of hydrogen ions drives the formation of ATP. The same applies to the chloroplast membrane. The coupling of ATP synthase to hydrogen ion transport takes place via the F o part. Paul D. Boyer began his studies of ATP formation in the early 1950s and is still highly active as a scientist. His chief interest has been to find out by isotope techniques how ATP synthase functions and particularly how it uses energy to create new ATP. His work has been crowned with unusual success in the past few years. ATP synthase has a mode of function that is unusual for enzymes, and this required much time and extensive studies to establish. John E. Walker made his first studies of ATP synthase at the beginning of the 1980s. His starting point was that a detailed chemical and structural knowledge of an enzyme is required to understand in detail how it functions. He therefore determined the amino acid sequences in the constituent protein units. During the 1990s he has collaborated with crystallographers to clarify the three-dimensional structure of ATP synthase. So far, the structure of the enzyme's F 1 part has been established. Walker's work complements Boyer's in a remarkable manner and further studies based on this structure demonstrate the correctness of the mechanism proposed by Boyer. The Royal Swedish Academy of Sciences has decided to award the 1997 Nobel Prize in Chemistry with one half to Professor Paul D. Boyer, University of California, Los Angeles, USA, and Dr. John E. Walker, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP).