Monday, November 19, 2012

Biochemistry Tutorial Essays: Glycolysis

I have been for almost seven weeks here at Oxford, an now that the first term is rapidly approaching its end, I would like to draw some conclusions and share some experiences from here.

As we center our study on biochemical essay writing, this term on carbohydrate and lipid metabolism, I thought I could share the experience, by sharing the essays and the essential aspects outlined in them as well as the key aspects of certain topics.

Essay 1: Glycolysis & Gluconeogenesis


In the first week, we were assigned an essay with the topic "How is glucose metabolized to pyruvate? Describe how the pathway can be reversed to accommodate gluconeogenesis in the liver, indicating the importance of key enzyme steps."

I began my essay as following:

Glycolysis, an essential process for cellular respiration, represents the main metabolic pathway of converting glucose into pyruvate. In this way, glucose, one of the most used biological fuels by living systems, is converted into a compound with lower energy potential, pyruvate. Also, by the means of glycolysis, essential metabolic intermediates of the process are supplied to the cells for bio-synthetic purposes.

Chemically speaking, glycolysis is a set of ten reactions that transform glucose, one step at a time, into pyruvate. The reaction involves ten metabolic intermediates, all of which offer access to entry points in the process for products of other metabolic reactions. This plays a key role in the metabolism of sugars as the intake of sugar in living organisms also consists of saccharides other than glucose.

Glycolysis is comprised by a set of ten chemical steps, seven of which take place near their equilibrium state, the other three being far from equilibrium. This fact is useful in the process of glycolysis as the process thus becomes irreversible in 3 major points of the process. By this means, not only metabolic intermediates resulting from glucose are stopped from being transformed into other intermediates previous in the chain, but also those from other metabolic chains are prevented from being converted to glucose or other intermediates, originating from glucose. In other words, the irreversible reactions ensure the unidirectionality of the process, an important mechanism in the cells’ efforts of producing energy in the form of high-energy phosphate molecules (ATP).

The first five steps of the glycolytic pathway are preparatory steps. They are also energy consuming, in the form of ATP, their aim being the consecutive phosphorylation of the glucose molecule followed by the cleavage of the molecule. In opposition to this, the last five steps, also known as the pay-off phase, return the ATP investment and generate high-energy phosphorylated molecules simultaneous to the degradation of intermediate products, which eventually leads to pyruvate.

The first step of glycolysis is the transformation of glucose to glucose-6-phosphate, by hexokinase, an enzyme responsible of transferring phosphoryl groups from high-energy phosphorylated molecules, such as ATP, to lower-energy acceptor molecules, in this case glucose. Nonetheless, hexokinase would also catalyse the reaction between ATP any other hexoses (D-mannose or D-fructose), as it is a rather nonspecific enzyme. With this born in mind, it is also important to notice the presence of more specific kinases, in the liver, such as the glucokinase, an enzyme only responsible for the conversion of glucose to glucose-6-phosphate with a view to maintaining the blood-sugar levels within normal physiological ranges.

The hexokinase enzyme, catalysing this reaction, will form a complex with glucose and the ATP-Mg2+ complex, both essential for the on-set of the reaction. A very important factor in the outcome of the reaction is the presence of the Mg2+ ion, as it will form a complex with two oxygen atoms placed on the two outermost phosphoryl groups of the ATP molecule. This way, the negative charges of the oxygen atoms will be shielded and nucleophilic attack on the phosphorous atom will be facilitated. Even more, the binding of glucose to the active site of the protein will create a conformational change of the protein, thus placing the hydroxyl on the 6th carbon atom of glucose and the external phosphoryl group of the ATP molecule very close one to another, making the reaction possible. Also, this prevents smaller molecules, such as water, that could cause the hydrolysis of ATP, to undergo this reaction as this would be energetically unfavourable for the cell.

The next step of the reaction-chain is the isomerisation of glucose-6-phosphate in order to be transformed into a metabolisable form. In this case, the reaction will be facilitated by an enzyme called phosphoglucose isomerase and will convert glucose-6-phosphate into fructose-6-phosphate. As the reaction is pH dependent it is believed that the mechanism of the reaction involves electrically charged side-chains, localized in the active site of the protein, that are bound to facilitate ring opening of the glucose derivative so that it can be isomerised. Subsequently, the Hydrogen atom α to the aldehyde group, slightly acid due to its location, will be absorbed by a basic side-chain (such as that of Histidine). The cis-enediolate thusly formed will rapidly suffer a replacement on the C1 carbon atom, simultaneous to the tautomeric shift of the C=O double bond to C2. This step is followed by ring closure and release of the newly from intermediate product: fructose-6-phosphate.

It is essential for the on-going of the glycolytic process that the multiple types of sugar (lactose, galactose, sucrose, starch, etc.) inherent to the nutritive intake of the cells be transformed into fructose-6-phosphate at this point in time, since this compound is the only phosphorylated monosaccharide able to undergo further chemical transformations along the glycolytic pathway.

In the third step, fructose-6-phosphate is converted to fructose-1,6-bisphosphate. This third step of the pathway is also determinant for the on-going of glycolysis, as it is also essentially irreversible under normal, physiological conditions and in the intracellular environment. This is due to the very large negative free energy of the reaction. It also represents a major point of regulation within the process of glycolysis, being influenced by the ATP/ADP& and fatty acid concentrations in the cytosol, a very significant issue to be covered later on in the essay.

Again, the course of this reaction is ATP and Mg2+ dependent, since the enzyme catalysing the reaction is also a kinase, called phosphofructokinase-1 (PFK-1). This is the second ATP consuming reaction, a necessary energetic investment in the glycolytic process.

The following reaction in the process is that involving the cleavage of the fructose-1,6-bisphospate molecule, caused by aldolase. The reaction products of this reaction are glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). The reaction, basically the reverse of an aldol condensation, is a reversible reaction not necessarily demanding the presence of an enzyme as it can be catalysed in-vitro also by the OH- anion.

In order for the pathway to yield pyruvate, it is essential that the cleavage reaction take place between C3 and C4. This way, only products with a three carbon atom backbone will be generated in the cleavage process. Furthermore, this also explains why glucose-6-phosphate has to be converted to fructose-6-phosphate, as a carbonyl group on C2 and a hydroxyl on C4 are required for the cleavage to take place exactly between C3 and C4.

There are two classes of aldolases, able to perform this cleavage. Out of the two, Class I Aldolase is found in animals and plants, whereas Class II Aldolase is to be found in less evolved biological systems, such as fungi, algae and prokaryotes. It is thought that the reason for being more than one enzyme able to do this specific task is probably that both enzymes evolved early and separately in the evolution. Proof sustaining this theory exists, as some organisms have assimilated both types of aldolase, a physiological aberrance eliminated in the course of millions of years by natural evolutionary forces in most of the living organisms.

For Class I Aldolase, the mechanism of action includes five major steps, beginning with substrate binding. The active site of the enzyme shows one Lys and one Asp side chain. These play a very important role, as the carbonyl group of fructo-1,6-bisphosphate will condensate will the ε-amino group of lysine. A Schiff base will thus be created. Consequently, cleavage takes place simultaneously to the subtraction of a Hydrogen atom from the C4 hydroxyl group, which will become an aldehyde group for glyceraldehyde-3-phosphate, the triose produced in this cleaving step. The next step involves the tautomerization and protonation of the enzyme-Schiff base product, immediately followed by the releasing of dihydroxyacetone.
When it comes to Class II Aldolase, the enzyme does not form a Schiff base with the substrate. Instead, through stabilization of the enolate oxygen by the means of metal ions, such as Zn2+ or Fe2+.

Since the fourth step yields two products, only one of them can go to be further metabolized into pyruvate. Therefore, this fifth and final step of the preparatory phase of glycolysis is bound to convert dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. The reaction is catalysed by an enzyme called triose phosphate isomerase, which has been described in the literature as a perfect enzyme. This statement relies on the fact that the reaction occurs spontaneously and immediately on collision of the substrate with the enzyme, the reaction rate not being able to rise any further.

The sixth reaction of the ten reactions of the glycolytic pathway is rather special due to the fact that the esterification taking place shadows the real chemical process, the oxidation reaction. In this reaction, glyceraldehyde-3-phosphate is converted into 1,3-bisphosphoglycerate. The reaction happens only in the presence of NAD+ which is transformed into NADH. What happens is an initial nucleophilic attack of the SH group on the aldehyde group, followed by the formation of the thiohemiacetal. The thiohemiacetal undergoes further oxidation by the donation of a hydride ion to NAD+. By doing this, the previous thiohemiacetal becomes a thioester, which will undergo subsequent nucleophilic attack by inorganic phosphorous. In the end, the 1,3-bisphosphoglycerate thusly formed will be released by the enzyme.

Very important from an energetic point of view, step 7 is the first step to produce ATP instead of consuming it. This is achieved through a reaction catalysed by phosphoglycerate kinase. Essentially, the action of the enzyme here is opposite to the one of hexokinase in the first step of glycolysis: it removes a phosphoryl group that gets transferred to a new molecule of ATP, leaving only 3-phosphoglycerate as a product. Since the reactions can go both ways, this reaction is generating ATP. Moreover, since the number of moles of chemical substance has doubled ever since the cleavage of the bi-phosphorylated fructose, it is intuitive that for every glucose molecule that underwent glycolysis, two ATP molecules are generated in this step. It can be easily observed that the initial investment of two ATP molecules was fully returned.

In the following step, phosphoglycerate mutase plays a very important role in moving the phosphoryl group from the oxygen atom attached to C2 to the one on C3. The mechanism of the reaction involves phosphorylation of the C2 hydroxyl group, followed by decomposition with a view to form only the 2-phoshoglycerate (2PG) compound. The next reaction is transforming 2PG intro phopshoenolpyruvate (PEP). The reaction occurs in a magnesium rich environment. It is a basic elimination reaction that dehydrates the compound in order to yield PEP. This reaction is significant as there is a rate-limiting step to it: the OH elimination.

As an energy producing metabolic pathway, the last step also produces ATP, in order not only to return the investment of ATP, but to also gain energy from the molecules of glucose undergoing this pathway. For this to become true, phopshoenolpyruvate is converted into pyruvate with the aid of the enzyme pyruvate kinase. This is also a very important step as it is essentially irreversible and thus a regulatory step in the metabolism of glucose.


Thermodynamics
By analysing the free energy change of the 10 steps of glycolysis we can see two categories of reaction.

First of all, most reactions have varying ∆G’s, but close to 0, a fact that indicates that most reactions, take place close to equilibrium, a fact that is indeed true for seven of the ten reactions. Even though the standard free energy change ( ∆G°) of the reactions may not be close to 0, most of the reactions also depend highly on the concentrations of products versus reactants in cytosol. Therefore the free energy change can be calculated by means of the Gibbs free energy formula: ∆G=∆G°+RTlnKeq . In these circumstances, even some reactions that don’t have their free energy change close to null, given the concentration ratio of products and reactants (the Keq), they still are close to their equilibrium because their ∆G is null or close to zero.

Secondly, there are the reactions whose ∆G and ∆G° are far from 0 and fairly negative. This means that the products have lower energy than the reactants and the forward reaction is favoured if not compulsory as the reverse reaction is not physically possible in the normal, physiological activity of cells.

Finally, due to the overall gain in high-energy molecules such as ATP, glycolysis is an essentially exergonic process (more than -63 kJ/mol) as the initial reactant, glucose, loses its energy when converted into pyruvate, a molecule with much lower energy.

Gluconeogenesis

Gluconeogenesis is the process that takes place with a view to creating new glucose from organic chemicals like pyruvate, the intermediate metabolites of the glycolytic pathway, or even from gluconeogenetic amino acids (alanine). Generally, gluconeogenesis is an energetically expensive process run by the cells, especially when they are in need of glucose.

The gluconeogenetic pathway is opposite and similar to the glycolytic pathway, but still, not identical. This is due to the impossibility of cells to reverse the unidirectionality of glycolysis in key regulatory steps. So, gluconeogenesis has also developed means of literally by-passing the glycolytic pathway in those key points, that will be described further on in this essay.

The first by-passing point with the gluconeogenetic pathway is the conversion of pyruvate to PEP, a process that happens in two separate steps, catalysed by two different enzymes. The first step takes place in the mitochondria of cells. This step uses pyruvate obtained either from glycolysis, or from a transamination process if it comes from alanine.

Pyruvate is transformed intro oxaloacetate, by the reaction of pyruvate and acid carbonate, in the presence of pyruvate carboxylase. Other elements are necessary for the reaction to take place, such as the biotin co-factor, or the Acetyl-CoA positive effector. This step is activated when Acetyl-CoA is present in cytosol in concentrations high-enough, as Acetyl-CoA is the direct result of fatty acid degradation. This further means that fatty acids have been metabolized, making the generation of glucose a priority for the cell, so as not to run of chemical fuel.
After oxaloacetate is synthesized in the mitochondria, as there are no protein carriers for it, it is converted through a fairly reversible reaction into malate, driven out of the cell, and reconverted back to malate. Now, the second step of pyruvate to PEP conversion may proceed. In this step, oxaloacetate reacts with GDP, another high-energy molecule, and it gets phosphorylated while giving of carbon dioxide and GDP.

The second bypass mechanism involves the transformation of fructose-1,6-bisphosphate to fructose-1-phosphate, a hydrolysis reaction that occurs in the presence of fructose bisphosphatase-1. Being a simple hydrolysis reaction, the secondary product is inorganic phosphorous.

The same process happens with the conversion of glucose-6-phosphate into glucose, a process catalysed by the glucose-6-phosphatase enzyme, again, a hydrolytic enzyme. Still, an interesting issue about this last by-pass route is the fact that this reaction only takes place in liver and kidney cells. The glucose produced this way is only afterwards transported towards target cells such as muscle or brain tissue, unable of sustaining this process by themselves.

Gluconeogenesis is thus a very expensive process, costing the cells 6 high-energy molecules (4ATP and 2GTP molecules) for every glucose molecule produced. Due to the different reactions catalysed by different enzymes in the major nodes of these two pathways, it is clear that both glycolysis and gluconeogenesis are irreversible processes by themselves, made thermodynamically possible by enzymatic catalysis.

Regulation

Homeostasis is an essential aspect of cellular function, and therefore, metabolism. Because of the fact that thousands of substances are being repeatedly anabolised and catabolised in the cells of all living beings, a strict control in the concentration of those substances is needed. This is very important, as any fast drop or increase in a particular substance could have great impact on the cells (damage or even cellular death).

This is also applied to glycolysis, the one of the processes responsible directly of maintaining the concentration of ATP within biological ranges. This is a difficult task to achieve, as ATP is the main fuel of the cells, hundreds of processes being linked to the usage of ATP: a major drop in ATP would cause the ceasing of most metabolic pathways, case in which the cell would probably die of starvation. This is why the glycolytic pathway is very important, and regulation of the glycolytic pathway is an essential task for maintaining the homeostasis of the sugar levels in the cell.

There are several enzymes involved in the control of allosteric feedback, such as hexokinase, phosphofructo-1-kinase (PFK) or pyruvate kinase. Any defect in function of any of these enzymes can limit the recycling of ADP to ATP, resulting in a progressively bigger loss of catalytic capacity.

The mechanism of allosteric regulation involves a binding site, different from the active site, where so called allosteric effectors bind into place, thus modifying the conformation of the enzyme. By this means, enzyme activity is increased of inhibited (non-competitive enzyme inhibition).

In the case of hexokinase, the allosteric inhibitor is actually the reaction product, phosphate-6-glucose. An elevation in the concentration of phosphate-6-glucose causes a drop the activity of hexokinase and vice versa, creating a direct negative feedback loop.

Also, a peak in ATP productions inhibits the reaction rates of hexokinase, PFK and pyruvate kinase. In contrast, rises in AMP give a signal that energy is needed into the cell; therefore the glycolytic processes are stimulated. The lack of high-energy molecules is suggested by the high concentration of AMP due to the fact that when ATP reserves run low, two ADP molecules can react and form an ATP molecule and an AMP molecule. In other words, the presence of AMP generally accompanies the depletion of ATP.

Some allosteric effectors are used for the sole purpose of regulation, not playing otherwise any role in glycolysis. For example, 2,3-bishphosphoglycerate (BPG) controls by a negative feedback loop the activities of hexokinase and PFK. Others are also part of the glycolytic pathway but are part of a positive feedback loop control system. Such an example is 1,6-bisphosphate that acts as a positive effector for pyruvate kinase.


Afterwards, in the tutorial itself, great importance and attention was paid to allosteric inhibitors and effectors. As the glycolytic pathway is very important for producing both energy and prime sources for other metabolic pathways, such as the TCA cycle, it it very important that it is very strictly regulated so that normal levels of blood sugar will be maintained (around 5 mM). It has been remarked therefore that certain enzymes in this glycolytic pathways, such as PFK, function in a similar way to hemoglobin. The substrate binding to one of the active sites of the PFK tetramer is causing a conformational change that would further cause the activation of the other sub-units. Therefore, the binding of substrate to the enzyme would represent a sigmoidal curve, rather than a normal Michaelis-Menten hyperbola. This is a very important observation as it influences enzyme kinetics, and thus the fate of glycolysis.

Much attention was also paid to the reason why the regulatory steps have to be unidirectional. According to Le Chatelier's Principle, in the case of a hypothetical regulatory enhancement of the reaction, the concentration of products would increase as opposed to the concentrations of reactants, causing a shift in the equilibrium of the reaction towards the left, thus canceling of diminishing the effect of allosteric regulation. For this reason, we concluded that only an irreversible reaction would be suitable for the regulatory steps.

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