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ATP synthase is the enzyme found in all living organisms starting from bacteria to animals. The reason for the wide occurence is that it produces ATP as currency of energy in biological systems. ATP synthase is a key enzyme of two main biological reaction cycles; oxidative phosphorylation in animals and photophosphorylation in plants. Both reactions end with ATP production which is ultimate source of energy in all living organisms (Biochemistry by Zubey, 4th edition). Oxidative phosphorylation occurs in mitochondria and hence ATP synthase is found on inner membrane of mitochondria. In plants, thylakoid membrane is embedded with ATP synthase enzyme to generate energy currency ATP of the cell (Robert K. Nakamoto, 2008). In case of photophosphorylation, ATP synthase is present in membrane of chloroplast. It is also known as ATPase as it catalyses the synthesis of ATPs (Muller V et al., 1999). The main function of the enzyme complex is to catalyse ATP synthesis using proton motive force which is generated by electrochemical potential. The enzyme's unique structure and function as motor at micro level gave a new name to the enzyme complex 'nano-molecular machine' (D.K. Robinson et al., 2007). Scientists also coined terms for the enzyme like motor protein or marvellous rotary engine (Y M Yoshida, 2000; W Junge, 1999)
The history enlightens fact that our understanding of ATP synthase is based on 'chemiosmotic theory' proposed by Peter Mitchell 1961. The theory suggested that proton gradient across membrane of mitochondria/chloroplast is the source of energy for oxidation reactions. ATP synthase mechanism shows same features in several ways in both mitochondria and chloroplast. First is electron flows through membrane bound proteins (S Nath et al., 1999). Second is proton gradient generates free energy to carry out catalytic reaction of ATP synthesis. Third is proton passage through membrane protein provides free energy for ATP synthesis catalysis coupled with photophosphorylation of ADP (S. Jain et al., 2000).
The general reaction mechanism of the ATP synthesis is depicted in the following figure. Proton gradient is generated translocation of hydrogen ions results proton motive force. This helps in catalysis of ATP synthesis reaction.
Figure: ATP synthase structure and function in Mitochondria
Experiment proving role of proton gradient in ATP synthesis:
Buffer with pH-7
Buffer with pH-9
Ref: Principles of Biochemistry by Lehninger; 5th Edition
Experiment by Walker P.G.
The experiment is arranged to check the proton gradient as energy source for ATP synthesis. The experiment uses artificial electrochemical gradient in laboratory condition and check the oxidation of the substrate. Cellular mitochondria are isolated and kept into a beaker containing buffer solution. The ATP synthesis reaction takes place in intramembrane space and matrix of mitochondria (showed as enlarged portion in figure). In this experiment, catalysis results into no oxidisable substrate.
First step of experiment (Figure-2a) shows that same KCl concentration across the mitochondrial membrane (0.1M KCl) can not generate potassium concentration gradient. This stops the oxidative phophorylation and hence no oxidisable substrate present in the solution.
In second stage of the experiment pH of the buffer is changed from 7 to 9. And also valinomycin (carrier of K+) is added which carries potassium into from intramembrane space to matrix and as a result concentration gradient is generated (Chance and Williams, 1955). This electric potential carries oxidative phosphorylation and results into absence of oxydisable substrate in the solution.
MOLECULAR STRUCTURE of ATP SYNTHASE
To understand the molecular mechanism of ATP synthase, it is necessary to understand the structure of the protein complex. The detailed structure of the protein complex is revealed by X-ray crystallography techniques and biochemical study (G I Belodugrov, 1998). ATP synthase is composed of multiple subunits (generally 8; stoichiometry arrangement K3L3QNAab2) with unique structure embedded in membrane (R H Fillingame, 1990). The size/molecular weight of the protein is approximately 530kD and similar in all organisms (A E Senior, 1988).
Morphological study of ATP synthase enzyme structure shows two main damains. One is Fo domain which is membrane bound and provide channel to pass sodium or potassium ions. It is composed of three subunits Î´, Îµ and Î³. Second domain F1 of the protein is made up of six alternatives Î± and Î² subunits mainly catalyse ATP synthesis (Schäfer et al., 1999; Leslie and Walker, 2000; Forgac, 2000). Other subunits like Î´ and b2 subunit provides supports and bind whole enzyme complex known as 'stator stalk'. A subunit functions as pump and translocates H+ ions into intramembrane space. Îµ and Î³ and epsilon subunits act as shaft around which whole F1 domain rotates.
Structural Topology of ATP synthasefrom E. coli
Genetics of the Enzyme
ATP synthase is a very complex enzyme composed of approximately 12 subunits. GenomicDNA sequence analysis in Neurospora crassa gave detailed information about genes encoding Î± and Î² subunits (Emma Jean Bowman and Tracey E. Knock, 1992). The research also focused on regulation of the gene expression as N. crassa is best model to study gene regulation (Sebald and Kruse, 1984). Genes atp-1 and atp-2 code for Î± and Î² subunits of ATP synthase enzyme. The genes are conserved through evolutionary time and also through out species (Walker et al., 1985). The study of the molecular genetics also carried out in another well known fungal species Saccharomyces cerevisiae (Rodney J. Devinish et al., 2000). Bowman and Tracey
TYPES OF ATPases
A-type of enzyme catalyses reversible ATP synthesis reaction and functions as proton or sodium pumps (Mitchell, 1961). A-type of ATPaases can synthesise ATP using proton motive force generated by Ao subunit of the protein (Schäfer et al., 1999). The structure and functional relationship in this type of enzyme is not clear. It functions using Ao subunit for proton passage to generate proton motive force and A1 for ATP hydrolysis to catalyse the reaction (Grüber et al., 2000).
The F-type of ATP synthase enzymes are found in mitochondria, bacteria and chloroplasts. This type of enzyme drives upward transmembrane proton transfer using energy from ATP hydrolysis Schäfer et al., 1999). The molecular insight suggests that Fo protein provides passage and outer F1 protein drive the reaction using energy gained from ATP hydrolysis. F-type ATPases catalyse reversible reaction and proton gradient provide energy for that (Leslie and Walker, 2000). In that sense F-type are real ATP synthases enzymes and thought to be existed first in evolution order (Forgac, 2000). As it synthesise ATPs it plays major role in mitochondria, chloroplast and bacteria and in archaebacteria (Ref: Principles of Biochemistry by Lehninger).
V-type of ATPase contains twelve different subunits and involved in ligang trafikking, cell signalling and nutrient uptakes (Wieczorek et. al., 1999). These types of ATPases are called so as they are vascuolar and generally acidify the intramolecular compartment during the reaction. V-type of ATPase is structurally similar to F-type but shows different function and hence found in higher plants and fungi with vacuoles (Boekema et. al., 1997). Secondary function of V-type of ATPase if to acidify endosomes, golgi body complexes in animal cells (Pedersen et. al., 2000). The enzyme shows similar activities of Vo and V1 proteins like F-type But in detail mechanism of V-type ATPase is unknown (Ref: Principles of Biochemistry by Lehninger).
MECHANISM of ATPases
It is well known that ATP synthase shows as 'rotary mechanism'. The whole F1 domain moves around Fo protein using energy generated from proton motive force (PMF). During this movement the enzyme catalyse phosphodiester bond formation between ADP and Pi. Functional aspect of ATP synthase is closely associated with molecular structure (Principles of Biochemistry by Lehninger, 5th edition). The enzyme shows unique mechanism and hence has been interest of detailed study. It is widely referred as molecular motor as it works in similar fashion like mechanical motor.
In earlier time, it was believed that sequential change in alternative subunit of enzyme leads to catalysis of the reaction. This changes the binding affinity of subunits with enzyme and substrate molecules (P.D. Boyer 1993). Boyer suggested that each subunit initially act as binding sit, than catalytic site and finally act as product release site. All three reactions can be carried out by different site at same time (P.D Boyer 1989). The mechanism involved three assumptions. First is, only one subunit can perform reaction at a time. Second is all the reactions are reversible in nature. And third is the energy is utilized in binding of substrate (ADP and Pi) and product release (ATP) and not in actual catalysis. But actin filament experiment and detailed study by X-ray crystallography depicted the real mechanism of catalysis process.
Rotary Mechanism Evidence by Actin Filament Experiment
The experiment was carried out by Yoshida and his co-workers in1998 to check the actual mechanism of ATP synthase. They created F1 domain mutant Bacillus strain. They used histidin tagged F domain attached with Nikle beads surface. Moreover, Actin filament was attached to Epsilon subunit and gamma subunit using steptavidin and biotin interaction. The biotin is also labelled with fluorophore to be detected using fluorescence spectroscopy (Milan Hofer, 1966). The spectroscopic analysis showed that epsilon subunit can rotate during catalytic reaction. The rotation was anticlockwise and was quicker when both epsilon and gamma subunits are tightly attached with each other. Moreover, frictional force hinders the rotational movement but strong coordination among subunits balance the force and carries out rotation of the motor (Moore and Pressman, 1964; Pressman, 1965).
The rotary mechanism reaction occurs stepwise exhibited by crystalline structure of the ATPase enzyme. The stepwise consequent binding of ADP, catalysis and release of ATP on beta subunit of the protein shows the rotational reaction (P. Senter, 1983). This is supported by anticlockwise motion of gamma subunit. Energy source for the protein movement is generated by proton motive force (H. Noji, 1997). Proton pumping is carried out by a subunit using energy from ATP hydrolysis. The asymmetric conformation of crystalline ATP synthase also adds into conclusion about stepwise reaction (Y. Kato-Yamada, 1998). According to Boyer all three catalytic sites shows incompatibility for binding of substrates but shows compatible molecular conformation for catalysis. Binding of single ATP on a subunit of protein remarkably showed slow reaction rate. Consequent binding of second ATP significantly speed up the release of first ATP (Cunningham and Cross, 1988; Milgrom et al., 1998). At a time of high ATP concentration, even all three sites are bound by ATP and coupled with ATP hydrolysis and proton pump (Muneyuki and Hirata, 1988; Muneyuki et al., 1989). Thus rotary mechanism of ATP synthase represents a model functions like mechanical motor.
Transition state of the Enzyme and Catalysis
Transition state structure study of ATP synthase has been done by J.R. Knowlesand Annu in 1980. It is generally pentacovalent phosphorus. The study of Arg376 of Î± subunit showed that the amino acid residue plays major role in achieving transition state (S. Nadanaciva, 1999). In transition state, all three beta subunits are attached with their respective substrate and end of the sate results into release of one ATP molecule (G. Oster, 2000).
Overall mechanism of ATP synthase
regulation of ATP synthase function
In almost all organisms, major way of ATP synthase regulation is by inhibition; also called as 'ADP inhibition' (Fitin, A.F. et al., 1979). Magnesium plays important role in natural inhibition in biological systems. If MgADP tightly binds with ATP catalytic subunit instead of ADP and phosphate, it stops the activity of ATP synthase (Minkov, I.B. et al., 1979). In case of bacteria, ADP acts as accelerator and phosphate hinder the catalytic activity of ATP synthase (Turina, P. et al., 1992; Galkin, M.A., 1999). Apart form that, Î³ and Îµ subunits are also found to be associated with regulation activity of ATPaes (Hirono-Hara et al, 2005; Boris A. Feniouk et al., 2006).
ATP SYNTHASE and human diseases
Mitochondrial ATP synthase produce majority of the ATP in all the mammals by its catalytic activity oxidative phosphoraylation (M. Saraste., 1999). The biogenesis of the complex multi subunit mitochondrial ATP synthase depends on the both nuclear as well as mitochondrial DNA (mt DNA). Most of the genes are encoded by nuclear DNA, only 13 but essential subunits are encoded by the mtDNA. So any genetic defect or alteration in the nuclear or mitochondrial DNA results in highly deleterious disorder of the neuromuscular system mainly in children. (Houstek et al., 2006). Till to date there is no clinical out come of these diseases. (Kucharczyk et al., 2009). There are two type of defective mechanism occurs responsible for the cause of disease, first is insufficient production (qualitative defect) and second is sufficient production but functional disability (quantitative defect)(Holt et al., 1999).
If there is a defect in the production of the ATP synthase the pathogenic mechanism become more complex results in very less ATP production and generation of free reactive oxygen species (ROS) by the respiratory chain (S.S. Korshunov et al., 1997). Increased and uncontrolled production of ROS leads to the cell death and deficiency of the cellular energy results in stopping of all the cellular activities required for the cell survival (Josef HouÅ¡tÄ›k et al, 2006).
Defective ATP Synthase and Cellular defects
ATP SYNTHASE as NANO-MOLECULAR MACHINE
TERMS and DEFINITIONS
PMF (Proton Motive Force):
It is an electrochemical potential generated by proton concentration gradient across proton impermeable membrane.
Catalysis and Biocatalysis:
Catalysis is a phenomenon by which rate of chemical reaction increases using different approaches. Certain chemicals called catalysts accelerate the reaction rate of specific reactions.
It is protein subunit where actually catalysis reaction takes place. Active site is unique to each protein molecule.
Domain is a structural part of protein which carries specific function in case of enzymes.
Transition State of enzyme:
It is time being high free energy containing state that results into either enzyme substrate or enzyme product molecule after substrate binding.