Oxidative Phosphorylation and the Electron Transport Chain - Assignment Example

Assignment on Oxidative Phosphorylation and the Electron Transport Chain Question From these first studies which biochemical pathway is likely tobe dysfunctional in patient X. From the experiment 1 it becomes clearly evident that ADP and Pi are needed for the synthesis of ATP from them with the help of the ATP synthase enzyme located in the inner mitochondrial membrane. When the ADP and Pi stores are some what present it binds to the loose site of the ATP synthase molecule and as Hydrogen ions starts entering the inner mitochondria through the ATP synthase half proton channels the ADP and Pi bound to the loose site (or L site) now becomes placed on the Tight site (or T site) where the free energy of electron transport chain translates to the synthesis of ATP. As the ATP synthase molecule rotates further by 120 degree the bound ATP occupies the open site or O site from where it is released from the Open site (Yoshida et al, 2001). As the newly formed ATP comes into the mitochondrial matrix there is another transporter located in the inner mitochondrial membrane called the ADP/ATP transporter (Leys and Scrutton, 2004). This transports the ATP formed from the mitochondrial matrix to the cytoplasm and at the same time antiports the transport of ADP into the mitochondrial matrix to become bound to the open site, then loose site and once again to the Tight site where it binds with Pi (carried into the matrix by Pi transporter) and forms ATP again. This is the way the cycle continues by ejecting out ATP into the cytoplasm and at the same time inflowing ADP for the next cycle of ATP synthesis. Since ADP is not present, hence it does not go through this antiporter (protein molecules that helps to move two substrates in different directions) to form the new cycle of ATP. Hence when ADP was not there , no antiport occurred however when the ADP was replenished the oxidative phosphorylation returned to normal as ADP entered through the antiporter in exchange of ATP to start the new cycle of ATP synthesis by oxidative phosphorylation(Yoshida et al, 2001). Question 2a: Briefly describe how pyruvate plus malate can act as substrates for cellular respiration. Mostly during the tricarboxylic acid cycle and beta oxidation of fatty acids the pyruvate and malate can become oxidised by NAD+- dependant dehydrogenases, because these enzymes are able to transfer electrons from these substrates to the NAD+ owing to their lower redox potentials or the electron transfer potentials than the NAD+ because of the prevalence of the concentration of the molar oxidant/molar reductant ratios of these substrates and of the NAD+ in the mitochondria. Oxidation- reduction comprises of the flow of the electrons between the two redox couples which differs in their affinity for electrons (Calhoun et al, 1994). Hence electrons flow from the reductant ( pyruvate or malate) to the oxidant of another redox couple which is NAD+ in this case. Thus NAD+ gets reduced to NADH2 which further transfers the electrons through ubiquinone, cytochrome C to donate it finally to the oxygen molecule which is the ultimate terminal electron acceptor in the mitochondria, this generates free energy which is utilized to synthesize ATP from ADP and Pi. The other part is pyruvate is converted via pyruvate dehydrogenase to Acetyl Co-A which reacts with oxaloacetate to form citrate and the TCA cycle starts to further generate NADH2, which again donates the electrons to molecular oxygen to liberate free energy which is utilized to synthesize ATP from ADP and Pi. In many tissues like the cardiac muscle malate first enters through the mitochondrial matrix from cytoplasm by tricarboxylate transporter or malate transporter and the it reacts with NAD+ to form oxaloacetate which reacts with acetyl Co-A to form citrate and TCA cycle starts to further generate NADH2, which again donates the electrons to molecular oxygen to liberate free energy which is utilized to synthesize ATP from ADP and Pi (Mitchell et al, 1961) (Mitchell et al, 1967). Question 2b: Describe how glucose in the presence of hexokinase can function as a phosphate acceptor. Glucose in the presence of hexokinase acts as the phosphate acceptor because the terminal phosphate oxygen atom is shielded by Magnesium ions in the hexokinase molecule, so the C6 hydroxyl of glucose can make a nucleophilic attack on the phosphate atom and hence glucose acts as an acceptor of phosphate in presence of hexokinase (Yoshida et al, 2001). Q3. Fig A : Indicates the oxygen consumed in micromoles/ gram of the muscle tissue in a normal person with or without phosphate acceptor. Fig B : Indicates the oxygen consumed in micromoles/ gram of the muscle tissue in a patient X with or without phosphate acceptor. Q4. As the terminal phosphate acceptor used in the study was glucose in presence of hexokinase it means that in its presence there will be continued generation of ADP for ATP synthesis and this fact is true in the first graph that the increased ADP produced gets into the inner mitochondrial matrix to liberate out the synthesized ATP via the ATP/ADP transporter. Therefore there is a renewal of ADP and ATP simultaneously. So the oxygen consumption increases to produce the ATP. When no phosphate acceptor is used generation of ADP for ATP synthesis decreases. As ADP synthesis decreases less oxygen is consumed to form ATP. In case of the patient the oxygen consumption remained high in spite of no phosphate acceptor because as there is defective transfer of ADP into mitochondrial matrix, more and more amount of ADP was generated in anticipation to produce ATP and the oxygen consumption remained high indicating that ATP was not formed and in further anticipation to produce ATP, the oxygen consumption was also high(Yoshida et al, 2001). Q5. What is the expected effect of DNP on oxidative phosphorylation? 2, 4- dinitrophenol (DNP) is a potent uncoupler of oxidative phosphorylation. This means that the DNP molecules are proton channels that are found in the inner mitochondrial membrane through which the hydrogen ions that were ejected by the electron transfer chain leaks into the mitochondrial matrix. The effect is that though the electron transfer chains operates and eject out these protons, but in presence of DNP these protons do not enter through the half proton channels of ATP synthase and hence the rotor stator catalytic function of ATP synthase for synthesis of ATP by binding ADP and Pi to the loose site (or L site) and then to the Tight site (or T site) does not take place and ATP synthesis is prevented. This is due to the fact that the proton motive force is lost and no chemiosmosis takes place and hence no ATP is synthesized. This means the ejected hydrogen ions will enter through the DNP channels and the free energy generated for proton transfer through the electron transfer chain is liberated as heat instead of ATP synthesis. Thus the role of DNP is to convert the free energy liberated through the electron transfer chain is liberated as heat (Heytler et al, 1979). Q6. From the data in Figure 1, what can you conclude concerning the cellular respiration in the Mitochondria of the patient? The concept of the role of DNP in cellular respiration is depicted in the graphs. Considering the first bar diagram comparison, absence of DNP in the normal person, inorganic phosphate is not liberated because in that person, ATP synthesis is taking place and body does not have to depend for inorganic phosphate from other sources( Heytler et al, 1979). This means that the ATPase activity is not stimulated. However in the patient there was a problem of the ATP/ADP antiporter, hence the raw materials (ADP and Pi) are not available so there is greater breakdown of Pi from other sources to liberate energy. This indicated a higher ATPase activity in the patient. In the second bar diagram it reflected that when DNP was added it acted as an uncoupler in the normal person and his ATP synthesis must have been blocked , which forced the metabolic system to utilize high inorganic phosphate from outside like the patient, through increased activity of ATPase. On the contrary adding DNP to the patient had no impact as such because the proton gradient was missing due to the defective action of ATP/ADP antiporter and ATP already existed in the mitochondria (Miranda et al, 2006). Q7. Describe the effect of Mg2+ ions on the activity of ATPase in the normal subject in the absence of DNP. What is the effect when DNP is present? Magnesium ions act as the cofactor for the enzyme ATPase. The magnesium ions first of all bind to the Beta and gamma phosphates of ATP to form the Mg2+- ATP, which then gets bound to the enzyme to form the Mg2+- ADP or the Mg2+- PPi complex with the liberation of one inorganic phosphate or Adenosine monophosphate as the case may be. Thus the action of Magnesium ion is to form the fully active ATPase holoenzyme which functions to breakdown ATP into ADP and Pi with the liberation of free energy. This acts via a nucleophilic attack on Beta and gamma phosphates of ATP thus liberating either one inorganic phosphate or Adenosine monophosphate as the case may be. In absence of DNP in the normal person, inorganic phosphate is not liberated because in that person, ATP synthesis is taking place and body does not have to depend for energy liberation from other parts of the body via the stimulation of Magnesium ion dependant activity of ATPase and the inorganic phosphate from other sources liberated is low. This means that the ATPase activity is not stimulated to breakdown the ATP molecules at other places. However when DNP was present in the normal person it acted as an uncoupler in the normal person and his ATP synthesis must have been blocked, which forced the metabolic system to utilize sources of energy from outside to breakdown ATP resulting in the formation of increased concentration of inorganic phosphate from ATP through increased magnesium ion stimulated activity of ATPase (Riley et al, 1981). Q8. How does the data for patient X in experiment 4 differ from that of the normal subject? The data in the patient differed significantly from the normal person. In case of the patient, even in absence of DNP, there was high rate of breakdown of ATP took place through the activation of magnesium ion stimulated action of ATPase. This was because in the patient there was a problem of the ATP/ADP antiporter, hence the raw materials (ADP and Pi) were not available so there was greater breakdown of Pi from other sources of ATP in the body to liberate energy. This indicated a higher ATPase activity in the patient which translated in the higher concentration of inorganic phosphate liberation than the normal person even in absence of DNP. This indicated the patient had to depend on ATP from other tissues for the liberation of energy resulting in the formation of increased amount of inorganic phosphate by magnesium ion stimulated activity of ATPase (Riley et al, 1981). On the contrary adding DNP to the patient had no impact as such because even if the proton gradient was missing or present the raw materials (ADP and Pi) due to the defective action of ATP/ADP antiporter, was absent and the body had to rely on ATP from other tissues as like when DNP was absent for the liberation of energy resulting in the formation of increased amount of inorganic phosphate by magnesium ion stimulated activity of ATPase on ATP to meet the basic energy needs of the body (Heytler et al, 1979). Q9. Suggest what might be the underlying cause for the symptoms of hyper metabolism seen in patient X. The hyper metabolism or increase in the basal metabolic rate is quite evident from the case study. Hyper metabolism means breakdown of ATP from various sources in the body as there is retention of synthesized ATP by the action of ATP synthase in the mitochondria or as the raw materials like ADP and Pi are absent itself the ATP synthesis is inadequate (Yoshida et al, 2001). Therefore under these conditions the ATP cannot travel through the ATP/ADP antiporter system to the cytoplasm to produce or meet the energy demands of the body as per calorie requirement. Since ADP cannot enter so it is not present in the mitochondria to form the ATP, hence it does not go through this antiporter (protein molecules that helps to move two substrates in different directions) to form the new cycle of ATP. Hence when ADP was not there, no antiport of ADP inside and liberation of ATP outside of the mitochondria happened (Yoshida et al, 2001). This was because in the patient there was a problem of the ATP/ADP antiporter, hence the raw materials (ADP and Pi) were not available so there was greater breakdown of Pi from other sources of ATP in the body to liberate energy in the form of hyper metabolism. This indicated a higher ATPase activity in the patient for the hyper metabolic state in the patient. Hence it can be concluded that the patient had to depend on ATP from other tissues for the liberation of energy resulting in the formation of increased amount of inorganic phosphate by magnesium ion stimulated activity of ATPase and resulting in the hyper metabolic condition (Yoshida et al, 2001). Q10. Under what circumstances might the dysfunctional cellular respiration of the patient be an evolutionary advantage? The circumstances under which the dysfunctional cellular respiration of the patient can become an evolutionary advantage will be the case of obesity or overweight. The logic behind this statement is that, in obese individuals, having a normal electron transfer chain function and a normal oxidative phosphorylation potential, ATP is continuously synthesized in the inner mitochondrial matrix. After its synthesis the ATP is translocated via the ATP/ADP antiporter to the cytoplasm, where the formed ATP is broken down for the liberation of energy and the calorie is burnt for that ATP. However in case of dysfunctional cellular respiration if occurs in the obese patient, then due to inadequate uptake of ADP , ATP synthesis will not take place in the inner mitochondrial matrix and no ATP will be translocated via the ATP/ADP antiporter to the cytoplasm, where the formed ATP could be broken down broken down(Schultz and Chan, 2001). Hence the obese patient has to the patient had to depend on ATP from other tissues for the liberation of energy resulting in the formation of increased amount of inorganic phosphate by magnesium ion stimulated activity of ATPase and resulting in the hyper metabolic condition (Martin et al, 1980). This hyper metabolism will eventually lead to breakdown of more food particles in the cytoplasm for the liberation of energy and the calories which an overweight or obese person intakes will be burnt out through various biochemical steps like glycolysis or increased beta oxidation of fatty acids which will reduce in the weight of the person and will also prevent accumulation of subcutaneous fat, a hallmark symptom with patients of obesity. References Calhoun M, Thomas J, Gennis R (1994). "The cytochrome oxidase superfamily of redox-driven proton pumps". Trends Biochem Sci 19 (8): 325–30 Heytler PG (1979). "Uncouplers of oxidative phosphorylation".Meth. Enzymol. 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