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 ATP-
synthesis.

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 support
for rotational model

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).