Defenders of Mitochondria

Alright my Biochem peeps,

Here is my 2nd review article. It is based on the target therapies of cancer treatment. In particular, this article focuses on the drug known as dichloroacetate which targets a mitochondrial enzyme known as pyruvate dehydrogenase kinase. This enzyme inhibits the action of pyruvate dehydrogenase as well as increase the metabolism of cancer cells that facilitate cancer proliferation. This article is quite interesting and thought-provoking. It will aid your understanding of the concepts of glycolysis, Krebs, ETC and oxidative phosphorylation. It is also marvelous to read the mechanism of action of cancer cells and the intricacies of their metabolism that fosters survival and proliferation. Take a read and see for yourself.

Michelakis, Evangelos D. and Gopinath Sutendra. 2013. “Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology.” Frontiers in Oncology 3(38): 1-11. doi: 10.3389/fonc.2013.00038

Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology.

This article is not a study as in the previous posting, but rather delves into the strategies utilized in cancer treatment in particular targeting more efficient, effective and less destructive methods of cancer therapy. This article deals mainly with investigating the effects of inhibition of an enzyme, pyruvate dehydrogenase kinase (PDK) as well as the applications of similar techniques that promote glucose oxidation as a viable option in metabolic targeting of neoplastic cells. The authors also explore the pre-clinical and clinical evidence of the effectiveness of these inhibitors on different cancers.

Neoplastic cells have a unique metabolic property in which there is both molecular and genetic signalling that converts glucose oxidative phosphorylation in the mitochondria of cells to glycolysis in the cytosol even under normal conditions. This results in the increase in glucose consumption as well as lactate production in cancer cells. This process is referred to as the Warburg effect after Otto Warburg who published this hypothesis in 1956. This means that cancer cells depend on glycolysis as its primary energy source as well as the source of macromolecular building blocks through glycolytic intermediates.

This is initiated by the activation of hypoxia-inducible factor 1α (HIF-1α). HIF-1α is activated in hypoxic-like environments and then stimulates the expression of glycolytic enzymes, glucose transporters and mitochondrial enzymes that increase the glycolytic pathway as well as formation of lactate. One important mitochondrial enzyme that is activated is, PDK. PDK  is considered the gatekeeper of the mitochondria since it regulates the flux of pyruvate into the mitochondria to undergo oxidative phosphorylation. PDK does this by inhibition of pyruvate dehydrogenase that performs the link reaction that decarboxylates pyruvate to Acetyl-CoA to enter the Krebs cycle. This inhibition leads to pyruvate not entering the mitochondria and therefore is converted to lactate in the cytosol by lactate dehydrogenase. Oncogenes are responsible for the maintenance of hypoxic state for cancer cells to continue to activate HIF. In cancer cells, there is also a loss of p53 gene expression that was responsible for mitochondrial DNA repair and expression cytochrome c oxidase enzymes that aid in apoptosis of the cell. Loss of p53 directly induces the expression of PDK and increases the stability and transcriptional activity of HIF. Additionally, HIF also increase pro-angiogenic and metastasis-promoting factors such as VEGF and SDF-1.

With increase in glycolytic enzymes, there is an increase in glycolysis as well as these glycolytic enzymes are utilized to aid in anti-apoptotic activities, activation of transcription in neoplastic cells, inhibition of mitochondrial function. Enzymes such include, glyceraldehyde 3-phosphate dehydrogenase and hexokinase. An isoform of hexokinase, hexokinase 2 is up regulated in neoplastic cells and then translocated to the mitochondrial transition pore, a channel responsible for the release of apoptotic factors as well as the regulation of anions the facilitate oxidative phosphorylation. Thus, hexokinase 2 facilitates resistance to apoptosis as well as increase in glycolysis in tumour cells. The glycolytic intermediates may also be funnelled to alternative pathways to produce amino acids, nucleic acids and lipids.

The mechanism of action of cancer cells lies in the alteration of the mitochondria of the cell. The mitochondria are responsible for the intrinsic apoptotic mechanisms of the cells and suppression of mitochondrial function is critical to cancer cell proliferation. One method employed by cancer cells is the alteration of the mitochondrial membrane potential of the inner mitochondrial membrane. This membrane potential is crucial to oxidative phosphorylation and electron transport chain. This membrane potential is akin to the proton motive force that is generated as hydrogen ions are pumped into the inter-membrane space of the mitochondrion during electron transport chain. This membrane potential is also critical to maintenance of intracellular calcium levels since mitochondria act like calcium sinkholes that facilitate the influx of calcium.

Neoplastic cells, inhibit glycogen synthase kinase in the cytosol of the cells that lead to the translocation of hexokinase 2 to the mitochondrial membrane. Hexokinase 2 then binds to the inner membrane and blocks and inhibits the voltage-dependent anion channel found in the mitochondrial transition pore that is responsible for the efflux of anions to maintain membrane potential of the mitochondria that is crucial for oxidative phosphorylation and ETC. This results in the hyperpolarization of the membrane and prevents the opening of the channel in the mitochondrial transition pore that is responsible for release of apoptotic factors such as mitochondrial reactive oxygen species, cytochrome c oxidase  as well as apoptotic inducing factor.

The suppression of mitochondrial functioning leads to the decrease in oxidative phosphorylation as well as the release of apoptotic factors. With diminishing functioning, the mitochondria of tumour cells begin to degenerate and further increase the proliferative potential of the neoplastic cell. However, this information can be utilized in targeting specific cancers especially solid tumours.

A drug known as dichloroacetate is able to penetrate most cell membranes and tissues that are not accessible to chemotherapy treatments such as the brain. This molecule is able to activate pyruvate dehydrogenase by inhibition of pyruvate dehydrogenase kinase and its isoforms. Dichloroacetate carboxylic group forms a salt bridge with Arg154 residue of the active site of pyruvate dehydrogenase kinase. Dicholoroacetate is able to decrease the membrane potential of inner mitochondria and aid in normalization of mitochondrial function in cancer cells. However, it does not affect non-cancer cells. It functions to increase apoptotic activity in the cancer cells as well as to increase the intake of pyruvate into the mitochondria and increase more ETC./oxidative phosphorylation. This decreases the production of lactate and the alternate pathways of glycolytic intermediates.

Subsequently, there is a deactivation of HIF since HIF is highly regulated by the TCA intermediate, alpha ketogluterate. Hence, an increase in TCA cycle leads to increase production of alpha ketogluterate that facilitates the inhibition of the action of HIF. This results in decrease tumour perfusion, glucose uptake, resistance to apoptosis as well as decrease production of macromolecules essential for tumour proliferation. DCA has also shown to increase p53 activity in cancer cells leading increase activation of apoptotic action by the mitochondria.

DCA is therefore very effective at selectively targeting cancer metabolism by inhibition of pyruvate dehydrogenase kinase. Clinical evidence of this has been observed in non-small cell lung carcinoma, breast carcinoma, glioblastoma, colon cancer, prostate and endometrial cancer as well as leukaemia.

However, other therapies may mimic the action of DCA and acquire similar results. Some therapies include the inhibition of lactate dehydrogenase to facilitate the channelling of pyruvate into the mitochondria to undergo oxidative phosphorylation. Similarly, decrease activation of the M2 isoform of pyruvate kinase that is expressed in neoplastic cells may prove to be another selective target for cancer treatment.

In concluding this article, the authors note that the aforementioned techniques are not a permanent solution to cancer therapy since there is no absolute action of these inhibitors on the metabolism of cancer.

My initial thoughts of this paper, was that it was long-winded and boring. However, as I continued past the introduction, the content became more interesting and captivating. The explanations and the organization of the information laid a good base level to which anyone reading was able to easily follow. It was greatly surprised at the intricacies of cancer metabolism and the very dynamic alterations made for the survival and proliferation of these cells. I knew that the mitochondria of cells were integral to the optimal functioning of the cell, nevertheless, I had no knowledge of the specific actions involved in mitochondrial functioning.

The authors aptly summarized the pathophysiology of neoplastic cells and then linked this to the action of the small DCA molecules and its efficiency in cancer treatment through selective inhibition. The importance of pyruvate dehydrogenase kinase was also elucidated. I was also intrigued by the importance of glycolysis to cancer metabolism and how effective glycolytic pathway is to the rapid growth and sustainability of the tumour.

However, it must be stated that even though DCA and the other techniques mentioned in this paper would prove highly valuable to oncology, there is no absolute solution since neoplastic cells have the ability to adapt and evolve to avoid the actions of these target drugs. There is no certainty that these therapies will show significant reduction in tumour proliferation in cancer patients and is subjected to individual differences. These therapies also have the potential to produce effects on non-cancer cells with prolonged use and high doses. Therefore, I am hesitant to believe that DCA and similar inhibitory therapies are a permanent solution to reducing the prevalence of cancer.

Overall, I think that this is a groundbreaking and lifesaving research in oncology since it will prove to reduce the mortality and morbidity of cancer. It is quite exciting to note that this method is a good step forward in finding more advance and effective treatment for a serious medical condition. Clinical evidence thus far has been positive and with more research, the methods of cancer treatment may develop into a simple cure like penicillin.



Parasite and Neoplastic cells Villain

Hey My Biochem peeps,

Hereunder is a review of a new study on the discovery of a new irreversible enzyme inhibitor that may be useful in the treatment of the parasitic infection, Leishmania as well as the treatment of cancers. This inhibitor targets pyruvate kinase, the last enzyme in the glycolytic pathway. Pyruvate kinase is responsible for the production of pyruvate as well as ATP. So I hope you find it as interesting as I did. Enjoy and Happy studying…….

Morgan, Hugh P., Martin J. Walsh, Elizabeth A. Blackburn, Martin A. Wear, Matthew B. Boxer, Min Shen, Henricke Veith, Iain W. McNae, Matthew W. Nowicki, Paul A. M. Michels, Douglas S. Auld, Linda A. Fothergill-Gilmore and Malcolm D. Walkinshaw. 2012. “A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase.” Biochem. J. 448: 67-72. doi:10.1042/BJ20121014.


A new family of covalent inhibitor block nucleotide binding to the active site of pyruvate kinase.

Pyruvate kinase is the last enzyme in the glycolytic pathway and catalyses the substrate-level phosphorylation of phosphoenolpyruvate to pyruvate and converting ADP to ATP in the process.  Human Pyruvate kinase consist of four isozymes: RPYK (Erythrocytes), LPYK (Liver), M1PYK (Muscle), M2PYK (Embryonic Tissues and Neoplastic cancer cells). Most of these isozymes are allosterically regulated by fructose 1, 6-bisphosphate meaning that increase in in fructose 1, 6-bisphosphate will lead to increase activation of pyruvate kinase. M1 isoform is constitutively active.

Leishmania mexicana is a trypanosome parasite that causes Leishmaniasis. It is transmitted by the bite of infected sand flies (Tsetse Fly). This infection results in skin sores, fever, weight loss as well as splenomegaly and hepatomegaly (Medline Plus 2012). Leishmania mexicana possesses similar pyruvate kinases as human however, trypanosome pyruvate kinase (LmPYK) is regulated by fructose 2,6-bisphosphate.

According to Morgan et al., parasitic infections are usually treated by the sulphur drug, Suramin that inhibits 7 out of the 10 glycolytic enzymes of Trypanosoma brucei. This drug works by mimicking ATP/ADP and competitively binds to the active site of pyruvate kinase to inhibit the formation of ADP. It is believed that saccharine inhibitors may have the same effect on LmPYK as suramin. This paper utilizes the saccharine derived compound to inhibit LmPYK activity and hence induce cell death in this parasite. The inhibitor is known as {4-[(1,1 dioxo-1,2-benzothiazol-3-yl)sulfonyl] benzoic acid} or DBS.

Through experimentation, this study showed that DBS was able to bind to the active site of pyruvate kinase, specifically form covalent bonding with the Lys335 residue present in the rim of the active site of pyruvate kinase. This covalent binding leads to the modification of Lys335 that prevents ADP from binding and forming ATP. This process leads to cessation of glycolysis and ATP  generation as well as pyruvate production in the parasite leading to cell death.

First, utilizing purified and characterized wild type and mutant k335R forms of LmPYK, an inhibitor assay was made in order to determine the rate of inhibition of DBS on LmPYK. The inhibitor assay contained a buffer, NADH, lactate dehydrogenase, LmPYK, Phosphoenolpyruvate and inhibitor mix of DBS. A control was created in the same manner except for replacing the inhibitor mixture with buffer solution. The inhibitor assay and control were incubated at 25oC water bath throughout the experiment. ADP was then added to the reaction mixtures and the decrease in absorbance from 340 nm was measured every 20 minutes for 300 mins. This gave the rate of inhibition by DBS.

This experiment illustrated that maximal inhibition was approximately 80% after 250 mins. However, there was never complete inhibition of 100% since after 10 hours, there was heat denaturation of LmPYK due to prolonged incubation in the water bath. The small percentage of pyruvate kinase activity was attributed to the flexibility of the side chains of Lys335. Lys335 residue in the active site has a very flexible R-group side chains that facilitate conformation to allow small access of ADP to the active site of pyruvate kinase to form ATP. However, the affinity of pyruvate kinase to ADP was greatly reduced by the covalent binding of DBS. However, Morgan et al,  posited that 80% inhibition of pyruvate kinase was sufficient to induce cell death in the parasite.

Next, a preparation of an inhibitor-modified LmPYK was made and crystallized in order to identify the structure of the LmPYK-DBS complex. The crystals formed were anisotropic and demonstrated covalent modification of Lys335 residues at the active sites of LmPYK. This modification was located at the adenine-binding site that leads to the blocking of ADP/ATP from binding. The saccharine group of DBS was covalently bonded to the Lys335 residue at the rim of the cleft of the active site. It was also found that the binding conformation of LmPYK-DBS complex mimics that of the drug Suramin.

Finally, using the mutant K335R, the modification of the Lys335 residue on LmPYK was confirmed. This mutant form of LmPYK was similar in action and kinetics of the wild type, however, it possessed a different Lysine residue at its active site and therefore when DBS  was added, there was no inhibition of mutant K335R. This illustrated that DBS is only able to form covalent bonding and irreversible inhibition of pyruvate kinase if Lys335 is present.

The selectivity of DBS for Lys335 was also investigated using rabbit lactate dehydrogenase and firefly luciferase. These are usually coupling enzymes with pyruvate kinase in the respective animals. However, it was found that even though these enzymes share similar characteristics with pyruvate kinase active sites such as lysine residue in the same position of the active site (LDH) or binds to the hydroxyl group of ribose sugar from NAD (Luciferase), DBS was unable to inhibit the action of these enzymes. Hence, DBS is very selective towards Lys335 residues in the active site of pyruvate kinase.

In concluding, Morgan et al., linked the fact that due to the similarity between LmPYK  and certain human PYK isoforms in terms of Lys335 residues at the rim of the active site, DBS would be an effective inhibitor to both human and trypanosome pyruvate kinases. This is especially important for humans since neoplastic tumour cells as well as erythrocyte pyruvate kinases possess these similarities  and thus DBS would provide a good target drug treatment for cancer.

My thoughts on this paper. Firstly, I was elated that it was not very long and the information was presented in a very organized, easily comprehensible manner. Methods, objectives, rationale and findings were clearly outlined and the language was not too technical so that it was difficult to follow the concepts of the paper. The topic was a very stimulating and informative since I am very interested in diseases and novel therapies to stem the prevalence. Leishmaniasis is especially critical since we are susceptible to this parasitic infection given our tropical geography.

Another interesting note, is the fact that isoforms of human pyruvate kinase such as those found in our red blood cells and cancer cells, are similar to those found in this parasite. This nugget of information may prove valuable in the formulation and production of novel cancer therapies. It has been established that neoplastic cells rely on aerobic glycolysis to generate energy and macromolecular building blocks for growth and metastasis of tumours. Therefore, targeting pyruvate kinase is essential to fighting cancers.

The selectivity of DBS, is an added bonus in drug therapies containing this saccharine since it would be able to only target pyruvate kinase that possesses the Lys335residue at the rim of the active site where ADP/ATP binding usually occurs. Thus, DBS will be able to act on specific cells while leaving the normal non-cancer or non-parasitic cells unaffected.

However, this study was the first to demonstrate the effectiveness of DBS and therefore the findings have not been established. There must be more research into DBS and other analogues to determine whether this phenomenon is a solid avenue to follow in terms of therapy. Little is known about the actual effects of DBS on the human cells since this experiment was done by enzyme testing and no live tissue analysis was performed. Therefore, even though DBS seems like a promising compound that may enhance the efficiency and efficacy of target drugs, there has to be more in-depth research into the compound and the implications of its use.

Overall, this paper was very intriguing and interesting. It brought together concepts of glycolysis, irreversible enzyme inhibition and illustrated how the knowledge of biochemical processes may be applied to medical uses. The research is very poignant since parasitic diseases as well as neoplastic ailments are predominant in modern life. This fosters a greater demand for more efficient and effective methods of treatment for disease. The finding of DBS inhibition on the Lys335 residue offers hope and positive connections to increase the efficacy of future medical interventions.

Thus, I believe that this was an enjoyable and educational reading and I am excited to see and hear about the developments in medicine and zoonoses that have arisen out of this novel study. I recommend that you take a read, the reference is listed at the top of the posting. This topic offers a good outlet to link your understanding of the key concepts of glycolysis and enzyme specificity to developing useful things for society.

Until next posting……..


Glycolysis video review

Hey Biochem folks,

Glycolisis (Biochemistry) by Pitanja sa kolokvijuma Pomozimo kolegama


Here is another video that is focused on Glycolysis. This was a long video of just over 50 minutes and the content focused on a medical based knowledge. The lecturer kept focus on the essentials towards the general board exams for medical students and touched on the key aspects of the glycolytic pathway as well as physiological and pathophysiological points to link the biochemistry of glycolysis.

This video was quite informative and interesting, even though there was much content that was not applicable to our course. I felt that the lecturer’s explanation was comprehensive and easily understandable. However, there was little animation and pictures where sometimes there were periods of just looking at the same diagram as he spoke, that was a little monotonous. However, the content was excellent and the explanations were clear and concise. All of the information he mentioned in this video, I came about in my reading and preparation for the glycolysis blog posts and therefore, I found that this video was a well-summarized and integrative approach to the topic of glycolysis.

Even though, there was a lot of skipping through the stages of glycolysis, except for the irreversible stages, there was a good summary of each stage and the focus on the three regulatory enzymes as well as how these enzymes were regulated and the implications of their regulation, was very helpful. The lecturer clearly outlined the process, reasoning behind the regulation of the enzyme and the unique characteristics of the liver that facilitates a more dynamic glycolytic pathway.

The video, however, did not go into any detail on the fates of pyruvate but rather brushed through the two main pathways of producing lactic acid or acetyl-CoA. There was a general summary on lactic acid production and the implications on the human body. However, the applications of this knowledge to athletes and their increased conditioning that may overcome the lactic acid production with extraneous, vigorous exercise, was the highlight of that section. This application created a deeper understanding into the reasons for the physiological changes that occur due to exercise. A fascinating point was the importance of 2, 3-BPG to oxygen dissociation and it importance towards oxygen transport to foetuses in pregnant women.

In summary, this video was informative, interesting and easy to understand. Even though, the content was less applicable to our syllabus, it still provided accurate and well-integrated real life information on glycolysis. The lecturer was well articulated and seemed very knowledgeable in this topic. Overall, the video was good and I recommend that you look at it in your spare time. The major points of this video are summarized below, but I would suggest that you look for yourself.


The video begins with the lecturer talking about how glucose enters the cell. Glucose enters the cell through the GLUT transporters, as he mentioned and these transporters are tissue specific and have different Km values. This means that the affinity for glucose to enter different cells is dependent on different circumstances. As he continues, the lecturer mentions that GLUT 1 and 3 transporters are found in most cells and have a low Km of 1 mmol value, which means that glucose is able to diffuse steadily into cells. However, GLUT 2 are found in the liver and pancreas and have a high Km value and follows 1st order kinetics in that the increase in glucose concentration leads to increase diffusion through these transporters in to the cell. He also mentioned about GLUT 4 transporters that are found in skeletal muscle and adipose tissues that are insulin-dependent and so will facilitate glucose uptake in response to insulin release. Interestingly enough, these GLUT 4 transporters also are increased in muscle cells in response to increased exercise. So one may allude to the fact that increased GLUT 4 transporters in muscles means an increased demand for glucose to generate energy is critical to maintain functioning.

As the lecturer proceeds, he goes into depth of the process of glycolysis with the focus on the three irreversible, regulated steps of glycolysis. He mentions the importance of phosphorylation of glucose and the fact that that hexokinase required Mg ions as a co-factor. He also explains that there is another enzyme, glucokinase in the liver that performs this phosphorylation reaction to convert glucose to glucose 6-phosphate. Glucokinase is hormone-dependant and is able to trap all excess glucose in the blood into hepatocytes in response to insulin release. It was determined that hexokinase is inhibited by glucose 6-phosphate and therefore, when there is an accumulation of glucose 6-phosphate, it prevents hexokinase from converting anymore glucose. Therefore, the excess glucose is funnelled to the liver.

Next, the lecturer explains the importance of PFK-1 and how it is regulated. AMP is able to activate this enzyme and therefore increase its action to produce fructose 1, 6-bisphosphate. This is because, increase levels of AMP may occur when there is increase in exercise and hence there is more utilization of energy and so need to increase the rate of glycolysis to generate energy. ATP and citrate inhibit PFK-1 since these are both indicators of high energy states. When PFK-1 is inhibited, there is decrease in glycolysis and hence will not be able to go on to make fatty acids, which is essential to lipid production. Therefore in the liver, PFK-2 is present and produces fructose 2, 6-bisphosphate. This product over-rides ATP inhibition and forces PFK-1 to continue glycolysis in the liver. It is a very potent activator of PFK-1 to ensure that glycolysis ensues in the liver. This is essential to continue to utilize the high glucose levels in the blood. PFK-2 is driven by insulin.

Glucagon is also essential to the liver since there is inhibition of PFK-1 in the liver and decreases glycolysis and increases gluconeogenesis. This is critical for fasting states and resting states. The lecturer then explains the implications of these processes on persons with hyperglycaemia such as diabetics.

The lecture continues to explain some alternative pathways for dihydroxyacteone phosphate. He then goes on to proceed through the different stages in the energy generation phase of glycolysis. There is little focus on most of the stages in this phase by the lecturer except to note that glyceraldehyde 3-phosphate dehydrogenase generates NADH from the only oxidation reaction in the pathway and performs phosphorylation via an inorganic phosphate. There is a very brief explanation about substrate level phosphorylation. He also notes that in rbc, there is also the conversion of 1, 3-BPG to 2, 3-BPG.

The lecturer then focuses on the last stage of glycolysis catalysed by pyruvate kinase. It is noteworthy that phosphoenolpyruvate is the highest energy compound of the cell. PEP has twice the amount of energy of ATP. There was also discussion on the pyruvate kinase deficiency and the pathophysiology of the haemolytic anaemia that occurs from this deficiency. During this discussion, he explains the importance of glycolysis, NADH and NADPH to erythrocytes and why the blood glucose levels must be maintained at 5mmol.

The video continues to explain the production of lactate and the effects on the body as well as the case with athletes being able to intake high percentage of oxygen and therefore will have increased rates of glycolysis and increased rates of energy production through TCA and ETC. The lecturer then ties this concept into the oxygen transportation of rbc and the release of oxygen from haemoglobin and the importance of 2, 3-BPG. There is an explanation of oxygen saturation curve and the role of 2, 3-BPG. The video ends with the description of the physiology of oxygen transport from mother to foetus in pregnancy.

Hope you will enjoy it as much as I did. Until next posting….


Fates of Pyruvate

In determining the fate of pyruvate after glycolysis, two questions need to be asked;

  1. 1.   Is there oxygen present i.e. aerobic conditions?
  2. 2.   Are there any mitochondria present to facilitate TCA cycle and ETC?


In aerobic conditions, with mitochondria present, the two molecules of pyruvate produced from one molecule of glucose, is then transported to the mitochondria of the cell where it enters the TCA cycle. Via the TCA and ETC, energy is then generated and NAD+ is regenerated in the ETC.

Pyruvate cannot readily enter the TCA cycle and therefore, must be converted to alternative compound. Pyruvate is converted to Acetyl-CoA in the matrix of the mitochondria by the enzyme PYRUVATE DEHYDROGENASE. Pyruvate dehydrogenase is an enzyme complex that consists of three subunits. It also requires five co-factors in order to effectively catalyse the conversion of pyruvate to Acetyl-CoA. These 5 co-factors are Co-A, TPP, lipoate, FAD and NAD+. During this process, there is loss of a carbon atom from pyruvate to form carbon dioxide. Additionally, NAD+ is reduced to NADH to facilitate the oxidation of pyruvate to acetyl-CoA. Hence, this reaction is an OXIDIZING DECARBOXYLATION.

At the end of the reaction, 2 molecules of pyruvate form 2 molecules of Acetyl Co-A + 2 molecules of carbon dioxide + NADH. This reaction is also IRREVERSIBLE. Once Acetyl CoA is formed, it is combined to oxaloacetate in the matrix of the mitochondria to form citrate in the TCA cycle. This occurs in plant, animal and some microbial cells with the essential requirement that aerobic conditions exist and mitochondria are present.

Pyruvate may also be converted to oxaloacetate, which also enters the TCA cycle to generate energy. This reaction is catalysed by the enzyme, PYRUVATE CARBOXYLASE that is dependent on biotin. Pyruvate undergoes a carboxylation reaction to form oxaloacetate. This is an essential process to replenish the intermediates of the TCA cycle and provide substrates for gluconeogenesis


In anaerobic conditions and in the absence of mitochondria, Fermentation reactions occur. Fermentation may occur by two methods, which result in either the formation of ethanol or lactic acid. The main purpose of fermentation is to regenerate the limited supply of NAD+ in the cell to continue reactions including glycolysis

In yeast cells, fermentation occurs in two steps. 2 molecules of pyruvate are converted to two molecules of ethanol and 2 molecules of carbon dioxide. Firstly, pyruvate is converted to acetaldehyde by the enzyme, PYRUVATE DECARBOXYLASE.  This enzyme utilizes similar co factors to pyruvate dehydrogenase such as TPP, but may also utilize magnesium ions. In this decarboxylation, there is the release of carbon dioxide. This is an IRREVERSIBLE REACTION. Acetaldehyde is then converted to ethanol via ALCOHOL DEHYDROGENASE. During this process, NADH is oxidized to NAD+ and thus, performs a REDOX reaction. This reaction is reversible, however.

Funny enough, alcohol dehydrogenase is found in hepatocytes and aid in the metabolism of ethanol/alcohol after consumption. This enzyme also exists as isoforms, which is mutated in Asians and people of Asian ethnicity. This means that there is defective functioning of alcohol dehydrogenase and thus these people cannot metabolize alcohol quickly and suffer the toxic effects of alcohol quickly.


Fermentation may also occur in human cells under certain conditions. During vigorous exercising, skeletal muscles are unable to obtain an adequate supply of oxygen and therefore there are states of anoxia where the muscles have to undergo anaerobic respiration. In these circumstances, skeletal muscle undergoes fermentation to convert pyruvate to lactate. This process is essential to replenish the supply of NAD+ in order to ensure that glycolysis continues. However, lactic acid is toxic to cells since it causes a change in the pH and leads to acidosis.

In this fermentation reaction, pyruvate is converted to lactate by the enzyme, LACTATE DEHYDROGENASE. This is a slightly reversible reaction. During this process, NADH is oxidized to NAD+, which facilitates the reduction of pyruvate to lactate. Fermentation to form lactic acid also occurs in erythrocytes. Red blood cells lack mitochondria and therefore is unable to carry out TCA cycle and ETC, thus it relies on glycolysis for energy generation. This means that lactate is produced to ensure the quick regeneration of NAD+ to continue the glycolytic pathway.

Classes of the Glycolytic enzymes

Tying in enzymes into glycolysis. I just want to give you an idea of the class of enzymes that the ten enzymes in glycolysis fall into:

–      Hexokinase: transfers a phosphate group and therefore belong to class II TRANSFERASES

–      Phosphoglucose isomerase: converts a compound to an isomer, belongs to class V ISOMERASES

–      PFK-1: transfers a phosphate group, belongs to class II TRANSFERASES

–      Aldolase: splits a compound , belongs to class IV LYASES

–      Triose phosphate isomerase: belongs to class V ISOMERASES

–      Glyceraldehyde 3-phosphate dehydrogenase: performs redox reaction, belongs to class I OXIDOREDUCTASES

–      Phosphoglycerate kinase: belong to class II TRANSFERASES

–      Phosphoglycerate mutase: belongs to class V ISOMERASES

–      Enolase: cleaves by removal of a water molecule, belongs to class IV LYASES

–      Pyruvate kinase: belongs to class II TRANSFERASES



Glycolysis: The most ancient Energizer


Alrighty then folks let us get into the nitty, gritty of this glycolytic pathway.

As you may know, glycolysis is the process of converting glucose (a simple sugar) into pyruvate and producing energy in the form of ATP.

However, you might be asking, why is this important and why do I even need to know about this conversion?

Well the obvious answers are, because you have to know it for exam and the more important one….some cells in your body rely heavily on this pathway for energy generation. Therefore, it is prudent to understand and appreciate the dynamics of this process. I am going to take you through the various stages of glycolysis, mention the fates of pyruvate, and give some inclination about other methods of conducting the glycolytic pathway and then some extra info. That may interest you about the pathway.

Now I touched on the basics of glycolysis in a prior posting, which I have copied and pasted below to jog your memory.

Glycolysis is the first stage of cellular respiration and is involved in the conversion of glucose into a three-carbon compound known as pyruvate. This process is essential for energy generation for the cell and facilitates the control of blood glucose levels by ensuring that there is a steady intake and utilization of glucose from the blood.

As you would know, I went into why high blood glucose levels are hazardous to our tissues and why it must be metabolized quickly and efficiently. (Check out the Highs and Lows of glucose on my blog).

Glycolysis is the most primitive energy producing reaction that occurs in bacteria, fungi and even us humans. It occurs in the absence or presence of oxygen. Glycolysis occurs in the cytosol of the cell and therefore can occur in both prokaryotes as well as eukaryotes since it does not require an organelle to occur.

 Of importance, is the fact that erythrocytes or rbcs’ as I call them, depend solely on energy generated from glycolysis to carry out its functions. Now, I hope that you know that red blood cells do not possess organelles; hence, this process is ideal for them. You see rbcs’ transport oxygen via haemoglobin, which if you are still unsure, is the most important job in the body, so in order to become more efficient and effective in their duties, rbcs’ shed most of their organelles to make room for the haemoglobin. Hence, you might derive the fact that inability for glycolysis to occur, will lead to degeneration of function of rbcs’ leading to a little known ailment as anaemia.

Glycolysis involves 10 enzymes that catalyse three irreversible reactions and seven reversible reactions to yield two molecules of pyruvate (3C), four molecules of ATP (net ATP equals two), two molecules of NADH and two molecules of water. All of this results from one glucose molecule.

Now, ATP is a nucleotide that comprises of adenosine and three phosphate groups. These phosphate bonds have high chemical energy stored. This energy is released by the hydrolysis of the phosphate bonds to yield ADP. Therefore, production of one molecule of ATP stores high levels of energy, enough to adequately power the functions of the cell. NADH is also a high-energy compound. It is the reduced form of NAD, which is a co-enzyme that is found in tissue. NADH is able to transport electrons to the electron transport chain in order to yield ATP or energy.

Pyruvate may move on to the Krebs cycle or TCA cycle as the hip people call it, in the mitochondria or to produce lactic acid if there is no oxygen present as seen in muscle cells that undergo the Cori cycle. TCA further metabolizes the pyruvate to yield more energy and produce electron transporters. NADH produced is then transported to the inner mitochondria through the electron transport chain to yield energy/ATP. The ATP produced may be utilized to facilitate the different functions of the cell that require energy such as contraction of actin and myosin fibres.

The initial process of glycolysis involves the conversion of glucose to glucose 6-phosphate which occurs in the cytosol of all cells. But you must be wondering how does glucose enter the cell? Well here is a brief idea of how this occurs.

Glucose from the diet enters the bloodstream from the digestive system, where it is first transported to the liver and then to the rest of the body. Unfortunately, glucose cannot easily diffuse into cells and so require two mechanisms to enter cells. One system involves a sodium-monosaccharide co-transporter system and the other a facilitated diffusion transport system, the latter of which is performed by the use of transporters in cell membranes of cells.

The facilitated diffusion mechanism involves the use of special glucose transporters known as GLUT transporters. There are more than 14 types of these transporters but they exhibit tissue-specificity and hence, one particular type is found on certain cells. These transporters found in the cell membrane are existent in two conformational states. When glucose binds to these transporters, it induces a conformational change that allows for movement of the glucose molecule across the cell membrane. Now, as I mentioned before, GLUT transporters show tissue-specificity and therefore certain isoforms are found on particular cells. Hereunder is a list of the most common isoforms:







Okay, so you get the idea, but if you want to check out the full list and get some more information, visit this site below where there is an entire paper devoted to reviewing these transporters.

These GLUT transporters facilitate the diffusion of glucose and other hexoses by following a concentration gradient i.e. diffusion of glucose from a state of high concentration to a state of low concentration. However, these isoforms operate a little different in their uptake. GLUT 1, 3, 4 usually uptake glucose from the blood and does this periodically. However, GLUT 2 transporters uptake glucose primarily in states of hyperglycaemia or release glucose in fasting states of hypoglycaemia. GLUT 4 transporters have also been known to increase in activation in the presence of high insulin levels and therefore are stimulated by increased blood glucose levels.

GLUT 5 is a special case since it usually does not transport glucose but rather another hexose sugar, FRUCTOSE. But can you guess why it does this? Let me put your mind to rest, as I mentioned before, these transporters are mainly found in the testes. And what is the testes responsible for? You guessed it, producing sperm which main source of energy is from fructose. So when it needs to make that long voyage up the vaginal canal and uterus to the fundus of the fallopian tubes for fertilization it possesses fructose around the head of the sperm stored to generate energy. GLUT 7 is also a peculiar case since it actually mediates glucose flux across the endoplasmic reticulum membrane in gluconeogenic cells rather than uptake of glucose from the blood.

The mechanism of action of GLUT transporters

Glucose/hexose sugar present in the extracellular space encounters the transporters in the cell membrane and bind to the transporters. This binding causes the transporter molecule to change in conformation and close off at the extracellular space end and open into the cytosol. This opening into the cytosol allows glucose to move across the cell membrane and into the cell.


The other method mentioned was sodium monosaccharide co-transporter system. As you might figure from the title, it is a process of transportation against the concentration gradient and therefore will require energy in the form of ATP, in order to occur. This is a carrier-mediated process in which glucose transport is coupled with sodium influx from cells. This process is known as SGLT or sodium dependent glucose transport in which one molecule of glucose is pumped into the cell while sodium ions are pumped into cells. This may be jogging your memory of Biology in high school and therefore it is no surprise that this type of transport occurs in the epithelial cells of the intestines, renal tubules and the choroid plexus of the ventricles in the brain.

However, that is enough of that. Here is where the fun begins, with the aid of the mnemonics I created prior; we will dive into this exciting process. Again for a recap, hereunder is the mnemonics for glycolysis:



Phosphoglucose isomerase



Triose phosphate isomerase

Glyceraldehyde 3-phosphate dehydrogenase

Phosphoglycerate kinase

Phosphoglycerate mutase


Pyruvate kinase


  • H = Helen
  • P = Paints
  • P = Pictures
  • A = Along
  • T = The Training
  • G = Grounds
  • P = Praying
  • P = People
  • E = Enjoy
  • P = Paintings

The substrates



Fructose 6-P

Fructose-1, 6-Bisphosphate

Glyceraldehyde 3-P + Dihydroxyacetone-P

1, 3-bisphosphoglycerate






  • G = Gross
  • G = Guys
  • F = Favour
  • B = Big Boobs but
  • G = Gorgeous + D = Dreamy
  • B = Boys
  • P = Prefer
  • P = Pretty
  • P = Petite
  • P = Photogenic girls


Well, we have glucose in our cytosol, so what now?

Here we come to the first phase of glycolysis, THE ENERGY INVESTMENT PHASE. This involves five reactions, two of which are irreversible reactions and there is the consumption of two ATP molecules. Therefore, we are using up some energy to get some energy.




Glucose is converted into glucose 6-phosphate by the enzyme HEXOKINASE. This is the phosphorylation of glucose in which hexokinase facilitates the transfer of a terminal phosphate group from ATP to glucose.


This is performed in order to trap the glucose in the cells. Glucose can readily diffuse out of the cells by use of the aforementioned transporters and thus we have to ensure that they remain in the cell to carry out the reactions. Phosphorylated sugars cannot readily leave cells since they cannot penetrate the cell membrane. This is due to two main reasons. The first is the fact that it cannot diffuse across the cell membrane and there are no transporter molecules that may transport glucose 6-phosphate across the cell membrane. The GLUT transporters cannot transport a phosphorylated compound. Another reason is that the phosphate group is highly negative and therefore will be repelled by the negative polar bi-lipid layer of the cell membrane. This effectively traps the glucose 6-phosphate in the cytosol and hence committing it to undergo reactions.


This phosphorylation of glucose is referred to as the first priming reaction of the cell. This means that essentially, the addition of the phosphate group to glucose causes an increase in the reactivity of glucose and fosters the forward movement of the pathway. This reaction is conveniently enough, the first IRREVERSIBLE REACTION. This reaction is irreversible since it has a very negative ΔG value that means that it is a highly energetically feasible reaction and will occur spontaneously. However, the conversion of glucose 6-phosphate to glucose will require a very high positive activation energy and hence will not be energetically feasible. Therefore, conversion of glucose 6-phosphate to glucose will not occur spontaneously. Thus, the conversion of glucose to glucose 6-phosphate is IRREVERSIBLE.


A bit about hexokinase.

 Hexokinase is an isozyme, meaning that it belongs to a family of enzymes that catalyse the same reaction but possess different enzymatic kinetics such as km and Vmax. Hexokinase is not absolutely specific to glucose and may act on other hexose sugar such as fructose but does possess a high affinity for glucose. Hexokinase is one of the three regulatory enzymes found in the glycolytic pathway and found in most cells of the human body. However, in certain cells such as hepatocytes and beta islet cells of the pancreas, another isoform of hexokinase is present, GLUCOKINASE. Glucokinase aka hexokinase D, is responsible for the phosphorylation of glucose in the liver and islet cells of the pancreas. Glucokinase in the pancreas acts as a glucose sensor that aids in glucose homeostasis by aiding in the regulation of release of insulin from the beta cells of the pancreas. Glucokinase has a higher km than other hexokinases.


Whether hexokinase or glucokinase, the mechanism of action involves transfer of a high-energy terminal phosphate group from ATP and adding it to the glucose molecule. Hexokinase requires magnesium ions as a co-factor to ensure its efficacy and efficiency. When ATP molecule binds to hexokinase, it causes slight conformation to induce a complimentary fit that facilitates better binding of glucose to the active site of hexokinase. However, ATP is highly unstable since it possesses a large negative charge from its three phosphate groups. Therefore, the positively charged magnesium ions must be present in order to stabilize the ATP molecule and hence allow for the phosphorylation to occur. In this reaction, when the high-energy phosphate group is removed from ATP, there is the formation of ADP.


Hexokinase is inhibited allosterically by glucose 6-phosphate and therefore build up in glucose 6-phosphate prevents the phosphorylation of glucose. Glucokinase is however, not inhibited by glucose 6-phosphate but may be inhibited indirectly by fructose 6-phosphate which is produced in the next step.


Summary of stage: glucose + ATP + Mg2+ –> glucose 6-phosphate + ADP by hexokinase/glucokinase.




Glucose 6-phosphate is then converted to fructose 6-phosphate via an isomerization reaction. This is a reversible reaction and is catalysed by the enzyme PHOSPHOHEXOSE ISOMERASE. This reaction involves the conversion of an aldose sugar to a ketose sugar.


Something to note however, this reaction is not the only reaction that occurs with glucose 6-phosphate. Glucose 6-phosphate may be utilized in other reactions and its fate is not only in the glycolytic pathway. Glucose 6-phosphate may be utilized as a precursor molecule in many pathways that involve glucose such as the pentose phosphate pathway and glycogenesis. Glucose 6-phosphate may also be fed into the glycolytic pathway through other pathways such as glycogenolysis, gluconeogenesis from non-carbohydrate sources as well as from the pentose phosphate pathway.


Summary of step: glucose 6-phosphate –> fructose 6-phosphate by phosphohexose isomerase



This reaction is another IRREVERSIBLE REACTION. It is also known as the 2nd priming reaction in glycolysis. Fructose 6-phosphate is converted to fructose 1, 6 bisphosphate by the enzyme PHOSPHOFRUCTOKINASE-1 (PFK-1). PFK-1 performs a phosphorylation of fructose 6 phosphate by transferring a high-energy terminal phosphate group from ATP to fructose 6-phosphate to form fructose 1, 6-bisphosphate. This is a similar reaction to hexokinase. PFK-1 is the MOST REGULATED enzyme in the glycolytic pathway. It is considered the rate-limiting reaction and is the committed step in the pathway.


PFK-1 is inhibited allosterically by elevated levels of ATP meaning that in high-energy states there is a decrease in this reaction to conserve cell resources. This inhibition may also occur with high levels of citrate, which is formed in the TCA cycle by the combination of acetyl-CoA and oxaloacetate. This negative feedback is an indicator that there is a high-energy state in the cell and therefore, there is a decrease demand for energy production and pyruvate by extension.


However, PFK-1 may also be activated in low energy states in the cell by AMP, which signals that there is low energy available in cells. The most potent activator of PFK-1 is fructose 2, 6-bisphosphate. This substance is formed by phosphofructokinase-2 which converts fructose 6-phosphate to fructose 2, 6-bisphosphate.


This phosphorylation involves the addition of a high-energy phosphate group to the C-1 atom on fructose 6-phosphate, thus there is a phosphate group on two different C atoms and hence is a bisphosphate compound and not a diphosphate compound which possesses two phosphate groups on the same atom.


Summary of the step: fructose 6-phosphate + ATP –> fructose 1, 6-bisphosphate + ADP by phosphofructokinase-1



This is referred to as the splitting reaction. In all the other steps prior, the products were 6C sugar molecules but in this step, there is the splitting of the 6C sugar to form two 3C sugar molecules. Fructose 1, 6-bisphosphate is converted into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate by the enzyme ALDOLASE. This is not a regulated step and is reversible.


Thing to note is that there is an isoform of aldolase enzyme known as Aldolase B which is responsible for cleaving fructose 1-phosphate in the liver and facilitating the feeding of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate into the glycolytic pathway (will touch on this later).


Summary of step: fructose 1, 6-bisphosphate –> glyceraldehyde 3-phosphate + dihydroxyacetone phosphate by aldolase.



This is the final step in the energy investment phase and involves the conversion of dihydroxyacetone phosphate to glyceraldehyde 3-phosphate by the enzyme, TRIOSE PHOSPHATE ISOMERASE.  This is also a reversible reaction and is not regulated involving isomerization of dihydroxyacetone phosphate to glyceraldehyde 3-phosphate.


A thing to note with triose phosphate isomerase is that this enzyme is kinetically perfect. It gains this characteristic since as the substrate binds to the active site, there is an immediate reaction. This occurs since the intermediates formed in this reaction are quite labile and unstable and therefore to avoid the formation of these intermediates, there is the quick formation of the product.


Summary of the step: dihydroxyacetone phosphate –> glyceraldehyde 3-phosphate by triose phosphate isomerase


This is the end of the energy investment phase and thus far, one molecule of glucose is converted into two molecules of glyceraldehyde 3-phosphate. Two ATP molecules were consumed during this phase and there were two irreversible reactions catalysed by hexokinase and PFK-1 respectively. These enzymes catalysed a phosphorylation reaction in which a high-energy terminal phosphate group from ATP is transferred to a hexose sugar molecule. There were also three reversible reactions within this phase, two of which were isomerization reactions and one was a splitting reaction.


Let us now proceed to the next phase in the glycolytic pathway, the ENERGY GENERATION PHASE. This phase also involves five reactions, with one of them being irreversible. This phase involves the generation of four ATP molecules, two NADH molecules and two molecules of pyruvate. This stage involves the conversion of two molecules of glyceraldehyde 3-phosphate to two molecules of pyruvate.




This reaction is the only OXIDATION REACTION in the glycolytic pathway. 2 molecules glyceraldehyde 3-phosphate are converted to 2 molecules of 1, 3 bisphosphoglycerate by the enzyme, GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE.  In this reaction, glyceraldehyde 3-phosphate dehydrogenase, first utilizes NAD+ to perform the oxidation of glyceraldehyde 3-phosphate. NAD+ is reduced and gains an H+ ion from glyceraldehyde 3-phosphate to form NADH + H+. This redox reaction causes a release of energy. This release of energy is then utilized by glyceraldehyde 3-phosphate dehydrogenase to drive a PHOSPHORYLATION REACTION using an inorganic phosphate group. As you might realize, this phosphorylation does not involve ATP but rather an inorganic phosphate group. Thus, the conversion of glyceraldehyde 3-phosphate to 1, 3 bisphosphoglycerate involves an oxidation as well as a phosphorylation reaction. This reaction is a reversible reaction however and does not regulate the pathway.


To note, NAD+ is a scarce commodity in the cell and therefore found in small quantities. Thus, this must be replenished in order to ensure that glycolysis continues as well as other reactions in the cells. Also, arsenate (pentavalent arsenic) may prevent production of ATP and NADH by glycolysis by competing with inorganic phosphate to bind to the active site of glyceraldehyde 3-phosphate dehydrogenase. In effect, it acts as a competitive inhibitor of glyceraldehyde 3-phosphate dehydrogenase, causing the formation of a complex that is spontaneously hydrolysed to 3-phosphoglycerate. This skipping or bypassing of the 1, 3-bisphosphoglycerate causes the prevention of NADH formation as well as the generation of ATP.


Additionally, 1, 3-bisphosphoglycerate does not only proceed through glycolysis but may be converted to 2, 3-bisphosphoglycerate via the bisphosphoglycerate shunt utilizing the enzyme bisphosphoglycerate mutase. This occurs in red blood cells in order to regulate oxygen binding to heme. 2, 3-bisphosphoglycerate serves as an inhibitor of allosteric binding of Oxygen to heme on red blood cells. 2, 3 bisphosphoglycerate may be dephosphorylated by a phosphatase to 3-phosphoglycerate that enables feeding into the glycolytic pathway again.


Summary of the step: 2 glyceraldehyde 3-phosphate + NAD+ + Inorganic phosphate –> 2 NADH + H+ + 2 1, 3-bisphosphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase




This reaction is the first SUBSTRATE-LEVEL PHOSPHORYLATION. 1, 3-bisphosphoglycerate is converted to 3-phosphoglycerate by the enzyme, PHOSPHOGLYCERATE KINASE. Phosphoglycerate kinase transfers a high energy phosphate group from the C-1 atom of 1, 3-BPG to ADP and forms ATP.  The energy released from the removal of the phosphate group is utilized to drive the formation of ATP. This reaction is also a reversible reaction.


Summary of the step: 2 1, 3-BPG + 2 ADP –> 2 3-phosphoglycerate + 2 ATP by phosphoglycerate kinase.



2 molecules of 3 phosphoglycerate are converted to 2 molecules of 2 phosphoglycerate by the enzyme PHOSPHOGLYCERATE MUTASE. This involves the shift of the high energy phosphate group from the C-3 atom to the C-2 atom. This reaction is freely reversible as well and enables an isomerization reaction.


Summary of the step: 2 3-phosphoglycerate –> 2 2-phosphoglycerate by phosphoglycerate mutase



The conversion of 2 phosphoglycerate to phosphoenolpyruvate is catalysed by ENOLASE. This is the hydrolysis of 2-phosphoglycerate to form a very high energy enol phosphate compound by removal of a water molecule. This reaction is however reversible even though phosphoenolpyruvate is a high energy compound.


Summary of the step: 2 2-phosphoglycerate –> 2 phosphoenolpyruvate + 2 molecules water by enolase



This is the final step in the glycolytic pathway and also a regulated step. It is the final IRREVERSIBLE REACTION in the pathway and involves the 2nd SUBSTRATE-LEVEL PHOSPHORYLATION. 2 molecules of phosphoenolpyruvate are converted to 2 molecules of pyruvate by the enzyme, PYRUVATE KINASE. As in the other substrate-level phosphorylation, there is the transfer of a high energy phosphate group from phosphoenolpyruvate to ADP to produce ATP.  This step allows for a net gain in ATP in the glycolytic pathway.


A bit about pyruvate kinase. Pyruvate kinase may be activated by fructose 1, 6-bisphosphate in a feed forward regulation. Therefore, activation of PFK-1 leads to the increase in fructose 1, 6-bisphosphate and this then leads to increased activation of pyruvate kinase leading to increased production of pyruvate and ATP. Hence, regulation of glycolysis occurs at this point. This feed forward regulation is observed in hepatocytes of the liver.


Another key aspect of pyruvate kinase involves RBC. In erythrocytes, there is the absence of mitochondria and therefore cannot undergo TCA Cycle or ETC like other cells of the human body. Therefore, erythrocytes depend heavily on glycolysis for energy generation in the form of ATP. ATP is utilized to fulfil all the metabolic requirements of rbcs including maintenance of its biconcave shape and flexibility in blood vessels. However, mutations to the structure of pyruvate kinase enzyme may occur leading to altered kinetics and function of the enzyme progressing to a deficiency of pyruvate kinase. This deficiency leads to decreased gain in ATP from glycolysis and hence will result in alterations to the metabolism of erythrocytes. This alteration to the metabolic activities of the red blood cells also causes alterations to the cell membrane and morphological features of the RBC. This results in sickling or loss of biconcavity and prone to be destroyed by phagocytes in the blood. The increased phagocytosis of RBC is referred to as haemolytic anaemia. Decreased RBC count means that there is decrease oxygen transportation in the blood and therefore affects the metabolism of other aerobic tissues of the body.


Pyruvate kinase deficiency is the 2nd most common cause of enzyme-related haemolytic anaemia and usually requires blood transfusions to rectify the loss of RBC. When RBC are lost, there is a compensation by the remaining RBC to increase the production of 2, 3-BPG. 2, 3-BPG binds to deoxyhaemoglobin molecules on RBC and decreases the affinity of haemoglobin for oxygen. This facilitates the increased release of oxygen from Haemoglobin into tissues.


Summary of the step: 2 phosphoenolpyruvate + 2 ADP –> 2 pyruvate + 2 ATP


At the end of the Energy Generation phase, 2 molecules of glyceraldehyde 3-phosphate was converted to two molecules of pyruvate. During this phase, 4 molecules of ATP were generated as well as 2 molecules of NADH.  This involved 4 reversible reactions and one irreversible reaction. The irreversible reaction was catalysed by pyruvate kinase, which converted phosphoenolpyruvate to pyruvate via a substrate-level phosphorylation. This was the last reaction in the glycolytic pathway and was the last of the three regulated reactions of glycolysis. Another phosphorylation reaction was catalysed by phosphoglycerate kinase in the conversion of 1, 3-BPG to 3-phosphoglycerate. However, this reaction was reversible. Glyceraldehyde 3-phosphate dehydrogenase also performed the only oxidation reaction in glycolysis. As well as enolase catalysed the hydrolysis of 2-phosphoglycerate to phosphoenolpyruvate which resulted in the release of water molecule.


Glycolytic pathway resulted in the conversion of one glucose molecule into two pyruvate molecules. In this process, 2 molecules of NADH and 4 molecules of ATP are produced. However, two molecules of ATP were also consumed in the energy investment phase. Therefore, there was a net gain of 2 molecules of ATP.


Okay, then folks…. That was an obscene amount of information that I just threw at you. But this is just the surface and I hope that the explanation of each stage of the pathway, will aid in better conceptualizing and understanding of the functioning of this important pathway. I suggest that you take a read through and apply the mnemonics I created in the previous postings and then summarize the key points to note. Look out for subsequent postings on the feeder pathways as well as the fates of pyruvate.

Peace out peeps and happy studying.




Kumar, Vinay, Abul K. Abbas, Nelson Fausto and Richard N. Mitchell. 2007. Robbins Basic Pathology. 8th ed. Pennsylvania, PA: Saunders Elsevier.

Rhoades, Rodney A. and David R. Bell. 2009. Medical Physiology: Principles for Clinical Medicine. 3rd ed. Baltimore, MD: Wolters Kluwer.

Champe, Pamela C., 4th ed. 2008. Lippincott’s Illustrated Reviews: Biochemistry. Baltimore: Wolters Kluwer.

Marks, Allan D., Michael Lieberman and Colleen Smith. 2007. Marks’ Essentials of Medical Biochemistry: A Clinical Approach. 3rd ed. Baltimore, MD: Wolters Kluwer.