Prof. Pinaki Sar
Department of Biotechnology
Indian Institute of Technology-Kharagpur
Lecture - 10
Review of Cellular Metabolism - Part E
Review of cellular metabolism will be discussed in this lecture in terms of how the metabolic flux
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is controlled between the important glycolytic reactions like EMP pathway and pentose phosphate pathway. This will help us to understand the control of these important fueling reaction, which is representing the major part of the central carbon metabolism of any actively metabolizing cell. Fermentation reactions will also be discussed.
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As it is discussed earlier, there are three major glycolytic pathways, which are responsible for representing the fueling reactions within cellular system. So within these three glycolytic pathways the glucose molecules or the representative other hexose sugar molecules are converted into the hexose monophosphate pool as we already discussed.
And then, if it is utilizing the EMP pathway as the major pathway then it will be converted to the pyruvic acid through the substrate level phosphorylation producing two moles of ATPs per molecule of glyceraldehyde 3-phosphate and also one mole of NADH H+ per mole of glyceraldehyde 3-phosphate. So together it will be four moles of ATP and two moles of NADH H+ per moles of glucose metabolized through EMP pathway.
The reactions which represent the pentose phosphate pathway and ED pathway also oxidize the glucose into pyruvate or different other intermediate metabolites and thereby allowing the cells to convert the hexose monophosphates into pyrophoric acid.
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Now the flux through glycolysis is adjusted in response to conditions both inside and outside the cells. The rate of conversion of glucose into pyruvic acid is regulated to meet the two major cellular needs. So whatever amount of glucose is transported inside the cell is actively metabolized.
And this metabolism will be either through only EMP pathway or EMP pathway and pentose phosphate pathway or maybe majorly by pentose phosphate pathway or partly by ED pathway and partly by EMP and PP pathway. This all depends on what are the requirements of the cells. Now with respect to these requirements, there are two major needs of the cells.
The first one is the production of ATP, which is to be generated by the oxidation of the glucose. And the second one is the provision for building blocks for synthetic reactions. Now in actively growing cells would like to have lot of biosynthetic reactions going on to produce all the necessary macromolecules.
And those biosynthetic reactions would require a high amount of building blocks in order to facilitate those anabolic reactions or biosynthetic reactions. At the same time, the cellular activities including growth or non-growth related activities would define the ATP requirement of the cell. So cell generally implements a highly organized and structured regulation in order to cater to the needs of these ATP as well as the building blocks.
So both these needs are highly balanced and highly organized in order to control the glycolytic pathways.
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Now enzymes which are responsible for catalyzing these reactions are found to be either irreversible type or reversible type. So some of these reactions, some of these enzymes catalyzing the reactions are found to be involved in irreversible reactions. And these enzymes which are catalyzing the irreversible reactions are the potential sites of control, control of the flux within the particular path or between the two pathways.
Now the activities of these enzymes which are controlling the irreversible reactions are regulated by reversible binding of different allosteric regulators or allosteric effectors or by covalent modifications of the enzymes. Now amount of this enzyme, so the enzyme regulation could be by virtue of the allosteric mode of regulation or by covalent modifications in some cases.
However, the control is also executed in terms of the amount of enzymes which are present in any point of time within the cell. Now this amount of the important enzymes which are involved in the irreversible reactions are controlled in terms of regulating the transcription of the relevant enzyme coding genes.
So in accordance with the changed metabolic needs or the metabolic requirements, which are present within a cellular system, the cells can control the transcription or
the even the translation of those required enzyme genes and thereby controlling the amount of the enzymes which are to be present or which are actually to be present in a cellular system as well as their activities.
So both the activities and the amounts of these enzymes are controlled by different mechanisms.
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Now the major control points within the EMP pathway and PP pathways are discussed.
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Now there are two major sites or reaction sites where these the flux between these pentose phosphate pathway and flux between the EMP pathways are controlled. As
we can see over in these two pathway schemes, that glucose is converted to the glucose monophosphate pool and this monophosphate pool is able to be utilized or providing the carbon backbone for both this pentose phosphate pathway as well as the EMP pathway.
In case of EMP pathway, it will be converting to fructose 1,6 bisphosphate and then by the substrate level phosphorylation and oxidation of these glyceraldehyde 3-phosphate would lead to the production of pyruvic acid. Whereas, in case of the pentose phosphate pathway, it will be oxidized to and decarboxylated. This oxidation will facilitate the synthesis of two moles of NADPH.
And then the pentose sugars, the pentose sugars eventually will convert into a number of intermediate sugars and some of the sugars will be used in the anabolic reactions representing the precursor molecules or some of the intermediates might convert into the fructose 6-phosphate or to the glyceraldehyde 3-phosphate thereby coming back to the main oxidative scheme of the glycolysis.
Now how do we control these? Because in these two pentose phosphate pathway and EMP pathways, the basic objectives or the basic deliverables of these two pathways are different. Although these two pathways are highly interconnected, the pentose phosphate pathway is responsible for producing the reducing power in the form of NADPH whereas, in case of EMP pathway, it is the NADH.
So there are differences between the use and how these reducing equivalents are further oxidized into different reactions. The other important difference between these two pathways are in terms of the deliverables are the intermediates which are produced.
The intermediates which are produced over the pentose phosphate pathway are mostly the pentose sugar, the erythrose 4-phosphate and other sugars molecules like sedoheptulose 7-phosphate, which are responsible for supporting distinct set of biosynthetic reactions. Whereas in case of EMP pathway, the intermediates like glyceraldehyde 3-phosphate and phosphoenolpyruvate etc., are responsible for a different set of biosynthetic reactions.
Now if we look into these two pathways, these both these pathways are starting from a common intermediate molecule which is the glucose 6-phosphate. So glucose 6-phosphate is converted to the phosphogluconate through the enzyme complex, which is glucose 6-phosphate dehydrogenase.
So this enzyme represents one of the very fundamental and irreversible reaction towards the pentose phosphate pathway. And the same type the phosphofructokinase or abbreviated as PFK is responsible for converting the fructose 6-phosphate to fructose 1,6 bisphosphate.
Now this glucose 6-phosphate dehydrogenase enzyme which is responsible for taking the flux of the carbon towards the pentose phosphate pathway is strongly regulated by the NADPH to NADP ratio. Whereas, the phosphofructokinase or PFK enzyme is allosterically regulated by several effectors where it is activated by adenosine monophosphate, ammonia, phosphate or fructose to 6-bisphosphate, it is inhibited by ATP.
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Now if we look at this conversion of fructose 6-phos to fructose 1,6 bisphosphate because this PFK which is controlling the flux towards the EMP pathway, this is one of the most well studied example, where we can see the how the metabolic flux between the two pathways are controlled very tightly. Now this phosphofructokinase enzyme is a kind of a complex allosteric enzyme with multiple regulators.
So one of the regulators is the ATP. Now ATP binding to this PFK molecule is facilitated through a particular regulatory site. So ATP can bind to this PFK in a particular regulatory site not at the catalytic site and thereby it decreases the affinity towards fructose 6-phosphate.
That means, if the cellular system or the cell is having high concentration of ATP, the ATP will bind to the PFK in the regulatory site because PFK is having a different regulatory site for PFK and if ATP is in excess the PFK will be negatively affected and the enzymatic capability of the PFK towards converting the fructose 6-phosphate to fructose 1,6 bisphosphate will be declined as the affinity of those ATP bound PFK will be less than the non ATP bound PFK.
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Now the ATP elicits the negative effect as we have discussed just now by binding to the specific regulatory site, which is distinct from the catalytic site. Now interestingly, the same enzyme PFK can be controlled positively. That means, its affinity towards fructose 6-phosphate or the negative effect of ATP can be reversed with binding of adenosine monophosphate into this enzyme.
Now AMP reverses the inhibitory effect of ATP thus increasing the activity particularly and this is increased when the ATP/AMP ratio is lowered. So cell they represent the energy status in terms of the level of ATP or level of AMP, if there is a
high concentration of AMP that represents that this cellular energy charge is low. So under this condition PFK will be activated.
And this activated PFK will be having a very high affinity towards fructose 6-phosphate thereby able to convert the fructose 6-phosphate to fructose 1,6 bisphosphate.
However, if the concentration of AMP is low, that means if the concentration of the ATP is high in the cellular system, then the PFK will be negatively controlled by the high ATP concentration in the way that the affinity of the PFK towards fructose 6-phosphate will be lowered and less amount of fructose 1,6 bisphosphate will be produced.
Now that means, the overall process of glycolysis through EMP pathway is stimulated as the energy charge falls. That means the concentration of ATP determines or the ATP/AMP ratio determines whether the fructose 6-phosphate will be actively converted to fructose 1,6 bisphosphate through this EMP pathway or not.
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Now there is a question that why adenosine monophosphate and why not ATP is positive regulator of PFK. Now another component or intermediate of this ATP hydrolysis is adenosine diphosphate. So similarly why not ADP is the positive regulator of PFK as well. Now it is found that AMP is a stronger signal for low energy state compared to ADP. Why so?
Because when ATP is being continuously utilized or rather utilized rapidly, there is an enzyme which is called adenylate kinase and these adenylate kinase can form ATP from two moles of ADP. Like two moles of ADP can react together and form ATP and adenosine monophosphate.
So the level of ATP is not actually so critical in terms of defining the cellular energy charge rather than it is the AMP which represent the cellular energy charge. So energy depleted cell would like to have or would have a high concentration of relatively higher concentration of adenosine monophosphate than that of adenosine diphosphate.
So the concentration of AMP as it rises the AMP gives a positive signal to the PFK binding to it and thereby lowering the negative effect of ATP or maybe ATP is not there in sufficient concentration. So the increasing the affinity of PFK towards fructose 6-phosphate and thereby allowing more flux towards fructose 1,6 bisphosphate and this fructose 1,6 bisphosphate can subsequently be oxidized and converted to pyruvic acid.
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Now the second modifier of this PFK is fructose 2,6 bisphosphate, which is actually produced from fructose 6-phosphate or it is interconvertible. So fructose 2,6 bisphosphate binding of this fructose 2,6 bisphosphate to PFK increases the affinity of the enzyme to fructose 6-phosphate many times.
So that means, if a cell is having higher concentration of fructose 6-phosphate, then the fructose 6-phosphate can be converted, part of that fructose 6-phosphate can be converted to fructose 2,6 bisphosphate by another set of reactions. And those fructose 2,6 bisphosphate can bind to PFK and increase its affinity towards fructose 6-phosphate so that it can convert fructose 6-phosphate to fructose 1,6 bisphosphate.
And then fructose 1,6 bisphosphate will be metabolized through the EMP pathway successfully. Now this regulatory profile allows the PFK to increase its activity to accommodate increasing concentration of fructose 6-phosphate. Now what is the significance of this? The significance of this is as the carbon flux or the hexose flux within the cell or a particular cell increases the fructose 6-phosphate pool.
Or the concentration is also increased, because we know that this hexose sugar phosphates they are directly connected to the sugar sources which are, the sugar substrates, which are provided to the cellular system. So the higher concentration of fructose 6-phosphate is directly connected to a higher concentration of fructose 2,6 bisphosphate.
And higher concentration of fructose 2,6 bisphosphate activates the PFK positively thereby allowing more fructose 6-phosphate to be converted to fructose 1,6 bisphosphate which can cause the level of fructose 2,6 bisphosphate to increase.
So thereby this fructose 6-phosphate, fructose 2,6 bisphosphate and fructose 1,6 bisphosphate, all the three compounds are very tightly correlated in terms of activating the affinity of PFK particularly with respect to converting fructose 6-phosphate to fructose 1,6 bisphosphate.
However, fructose 6-phosphate to fructose 2,6 bisphosphate convertion is facilitated or catalyzed by entirely different set of reactions which are two enzymes reactions are responsible for that.
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The second important criteria is the cofactors. The cofactors NADH and NADPH these two cofactors serve two different purposes in the cellular metabolism. As we have seen earlier the NADH is produced in the EMP pathway, whereas NADPH is produced in the pentose phosphate pathway. Now if we want to produce more NADPH, probably the pentose phosphate pathway would be the best one.
Whereas if we want more NADH, the EMP pathway would be the most appropriate one. Now in aerobic organisms, it is best exemplified that NADH, which is the product of the EMP pathway is involved in the generation of Gibbs free energy through oxidative phosphorylation. That is by serving as a substrate in the fueling reactions and thereby providing the electrons to the electron transport system.
Whereas the NADPH, which is the product of the pentose phosphate pathway is mainly used in the biosynthetic reactions, that is the anabolic reactions of producing the different building blocks thereby serving as a substrate in the biosynthetic reactions. Now this distinction is remarkable.
Like NADH is mainly responsible for the fueling reaction or generating the maximum amount of Gibbs free energy or sometimes the proton motive force through oxidative phosphorylation and the transferring the electrons to the electron transport system, whereas the NADPH is responsible for supporting the biosynthetic reactions.
So it is well demarcated within the cellular system that which one is for what function. Now depending upon the cellular requirement like if the cell is actively dividing, that means during the growth metabolism, the biosynthetic reactions will be prevalent and hence, the NADPH requirement will be higher.
Whereas, during the time of normal non-growth related metabolism, where the energy requirement would be high, maintenance related energy requirement will be high, then only the fueling reactions will be sufficient and relatively less flux may be expected through the pentose phosphate pathway.
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Now this ratio between the NADH and NAD+ and NADPH and NADP+ are maintained at different levels, but nearly constant level in case of bacteria, both in bacteria and in case of yeast. And as you can see it is NADH to NAD+ ratio is around 0.03 to 0.08 whereas NADPH to NADP+ ratio is around 0.7 to 1. In yeast also it is nearly equal except the case that in NADPH NADP ratio is slightly different, it is 0.58 to 0.75.
Now interestingly, these two coenzymes like NADH and NADPH are interconvertible. That means, in case of any exigency or any essential requirement, the cell would like to convert part of its NADH to NADPH through the enzyme nicotinamide nucleotide transhydrogenase. And this enzyme is present in many bacteria as well as in mammalian cells, but interestingly not in yeast.
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Next, we are going to discuss about the fermentative pathways. Now as the pyruvate which is produced as the end product of the three glycolytic pathways like EMP, PP and ED pathway, which will be further converted or further reacted or oxidized partly or sometimes reduced by several routes depending on the redox and energetic state of the cells.
Because pyruvate is only an intermediate of the glycolytic metabolism. So once the pyruvate is produced out of the glycolytic pathways, including either the EMP or the PPP or EMP and PPP together both as well as partly ED pathway, the pyruvate, which is produced by these pathways will be converted to different other products based on again the cellular condition or cellular requirement.
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For example, during the aerobic growth of the organism or under aerobic condition, we can expect that the pyruvic acid or pyruvate will be converted to acetyl-CoA and this acetyl-CoA will be oxidized completely through the tricarboxylic acid cycle and this complete oxidation would lead to production of carbon dioxide and water and several molecules of NADH H+.
And these NADH H+ will be NADH will be donating the electrons to the electron transport system thereby facilitating the oxidative phosphorylation. This oxidative phosphorylation will lead to production of higher concentration of Gibbs free energy. So in essence under the aerobic condition, the pyruvic acid is going to convert into acetyl-CoA and this acetyl-CoA would like to or be will be oxidized completely leading to the formation of CO2, water and high amount of Gibbs free energy.
On the contrary, under oxygen limiting conditions generally under oxygen limiting conditions, because we will be seeing some exception in this regard. So under oxygen limiting conditions, however, the acetyl-CoA production will be reduced because the enzyme responsible for pyruvate to acetyl-CoA will not be activated in oxygen depleted condition.
Rather the other enzymes which will be responsible for taking the carbons of the pyruvic acid into other pathways like the fermentative pathways will be most prevalent. So during this fermentative pathway, the pyruvic acid is going to be reduced to multiple products like lactate, acetate, ethanol etc.
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Fundamental fermentation logic and unifying themes. Now fermentation as we understand, it is going to be operated or it is operative within a cellular system under oxygen limiting condition. So fermentation uses basically the substrate level phosphorylation to synthesize the energy. How? Because it is the formation of the pyruvic acid from glucose which is included within the substrate level phosphorylation.
Now this flow of protons, if they are allowed to flow, would be exergonic, would release some amount of energy and that energy can be utilized to phosphorylate
adenosine diphosphate to ATP that is making the phosphotriester bond. Transport molecules into the cell directly because that is exergonic. So the flowing, when the protons want to flow inside back this is energy yielding reaction.
So some amount of energy can be harvested by coupling a transport of some kind of molecules which is otherwise difficult to transport because they might be against the concentration gradient. Or doing some useful work for particularly for cells, which are having prokaryotic cell in particular who are having flagella because flagellar motor requires large amount of this proton motive force or energy.
Now PMF or proton motive force plays a central role in prokaryotic physiology. It is not only the flagellar movement, but there are other transport events other than the generation of ATP directly.
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Now how does the proton gradient drive the ATP synthesis?
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Now we have seen this electron transport system where a number of carrier complexes are there which are responsible for carrying the electrons from the reduced cofactors like NADH or FADH2 to the terminal electron acceptor that is the oxygen and which is responsible for building a high concentration of proton. So which is the concentration of proton as well as the concentration or the charge.
It is positively charged compared to the inner side or the cytosolic site which is negatively charged. Now the electro chemical proton gradient which is created because of this efflux of these protons across the inner membrane is used to drive ATP synthesis.
Now this proton motive force which is in place now because of the formation or establishment of these electrochemical gradient because the functioning of this efflux pump of the electron carrier would drive the ATP synthesis and that is a critical component of the oxidative phosphorylation. Now how this PMF can drive ATP synthesis?
This PMF which is basically connected to again these electrochemical gradient can drive ATP synthesis by a special very special enzyme complex which is called ATP synthase which is located in the membrane itself. So that means, the membrane contains a number of very important molecules or carrier complexes, a set of carrier complexes which are responsible for the redox coupling is the first phase of coupling
which are actually responsible for transferring the electrons from the donor to the receiver through the redox potential gradient.
And the second one is this large F0 F1 ATP synthase complex which is capable of making use of this electrochemical gradient or the proton motive force.
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So this enzyme which is located on the inner membrane or the plasma membrane in case of a microbial system and it is allowing or it creates a hydrophilic pathway across the membrane that allows the protons to flow down the electrochemical gradient. We have earlier noticed the high concentration of protons are built up over here. So these protons want to flow back or come back.
So this enzyme complex facilitates or creates a hydrophilic pathway so that the charged protons can move back into the cytosolic site very favorably. And it is a favorable process because from the higher concentration to the lower concentration, they will move. But it is not only a favorable process for them, but it is also an exergonic process. That means it leads to release of energy.
So as these ions or the protons thread their way through the ATP synthase they are used to drive energetically unfavorable reaction. Because as I mentioned that these this movement is exergonic from a high concentration to low concentration so there could be a gain in energy. So that energy which is released because of this transfer of
the high protons from a higher concentration to the lower concentration is utilized to phosphorylate ADP to produce ATP.
That is the part of the phosphorylation part. So one ADP molecule can be phosphorylated to produce the ATP molecule.
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Now if we look at the theoretical stoichiometry of this oxidative phosphorylation, it is also referred as the P by O ratio. For eukaryote the P by ratio is around three moles of ATP synthesized per mole of NADH H+ oxidized or two moles of ATP synthesized for each mole of succinic acid or FADH2 oxidized. For prokaryotes it is less. It is two mole of ATP synthesized for each mole of NADH2 oxidized.
And the protons are because protons are transported only two locations. In prokaryotic system the electron transport system is more complex and it is branched also. So maybe because of that and the point of efflux of protons as electrons flow through the electron transport system are relatively less.
So because of that the amount of the ATP generation or the theoretical yield of the ATP per mole of NADH2 oxidized is significantly less in case of prokaryotic organisms. Now it is also true that in prokaryotes the incomplete coupling of the oxidation and phosphorylation is because of the extraneous processes which are driven by a proton gradient.
Because a number of transport processes particularly and flagellar movement for example, these are all directly connected to the proton gradient or proton motive force. So it is not that all the energy which is created because of the electrochemical gradient is utilized to drive the ATP synthesis by F0 F1 ATPs.
In case of prokaryotes, a large number of, a large portion of the proton motive force is actually dedicated for other functions, which are also very important like the cellular transport or the physiological, other physiological functions like the helping in the movement of the cells through flagellar motor function.
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So in this part of the lecture, we have used mainly the metabolic engineering textbook as well as two important books, one is the molecular biology of the cell that is by Alberts and the Prescott Microbiology book.
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So in summary today, we have covered in this part of the lecture, the TCA cycle and major points of energy and reducing power generation, the control sites and the factors which could control the TCA cycle, the electron transport chain and how electron transport chain is facilitating the generation of proton motive force and how proton motive force is connected to the oxidative phosphorylation. Thank you.
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