GLYCOLYSIS

GLYCOLYSIS – INTRO.

Describe the role of glucose in the cell!

Glucose is a good fuel, but also a precursor for various compounds!

In what way is glucose stored in the cell, and why?

Glucose is stored as large polymers such as starch or glycogen. When the energy demand rises glucose can be released from these large polymers.

This type of storage ensures storage of a lot of hexose, whilst there is no big increase in cytosolic osmolarity.

Glucose has four major metabolic fates. Describe them all briefly!

1. Glucose may be synthesized to complex polymers, destined for extracellular matrix and the cell wall.

2. Glucose may be stored in the cell for later use as glycogen or starch.

3. Glucose may be oxidized (lose electrons) to pyruvate through a process called glycolysis in order to provide ATP and metabolic intermediates!!!

4. Glucose may be oxidized via pentose phosphate pathway to yield ribose for nucleic acid synthesis and NADPH for reductive biosynthetic processes!

Organisms that don't have access to glucose must make it. How?

Photosynthetic organisms reduces CO2 from the air to trioses, which are in turn converted to glucose.

Non photosynthetic organisms, such as our selves, make glucose from simple 3/4C-compounds through a process called gluconeogenesis!

Repetition : what does glycolysis mean?

Oxidation of glucose through ten enzymatic steps to yield two molecules of pyruvate. The point of it all is to generate energy – which is conserved in ATP and NADH. The net gain of these reactions is two ATP:s and two NADH:s. (2 ATP:s are invested in glycolysis and four are produced; hence the net gain is two ATP molecules / glucose molecules).

Glycolysis takes place where?

In the cytosol!

Citric acid cycle takes place where?

In mitochondria!

Is glucose universal or specific for mammals?

It is universal = the same in all spices. Glycolysis do differ between spices, but not in enzymes used etc, but in the regulation and in the different fates of pyruvate formed!

The oxidation of glucose is the sole source of energy for some tissues and cell types – which ones?

Most importantly : the brain!

The testis. The erythrocytes and renal medulla.

What does “fermentation” mean?

It means degradation of glucose (or other organic nutrients) in the absence of oxygen, that is under anaerobic or hypoxic conditions.

Glycolysis is separated in two different phases - describe them briefly! PIC

The first five step = preparatory phase, during which energy is invested!

The last five step = the payoff phase; because these steps generate ATP and NADH.

There are three fates of pyruvate – describe briefly! PIC

1. Oxidation through the citric acid cycle - under aerobic conditions, glycolysis is merely the first step of total glucose degradation. This means that pyruvate is further oxidized through the citric acid cycle in order to yield render energy that is used for ATP synthesis in mitochondria. These processes will be addressed thoroughly later on.

2. Lactic acid fermentation – when the muscles are extra strained they must function under hypoxic conditions. This means that NADH cannot be reoxidized (lose electrons) by giving its electrons to oxygen in order to render NAD+. But NAD+ is necessary as an electron acceptor in the further oxidation of pyruvate. Simply : there is no oxygen present to accept the electrons from NADH. Thus NAD+ cannot be regenerated. This problem has to be solved, because NAD+ is a necessary electron acceptor in the payoff phase of glycolysis. Thus electrons are transferred to pyruvate instead of oxygen, reducing pyruvate to lactate! The lactate can be recycled to pyruvate in the liver during the rest phase following intense work out.

   Some tissues in mammals reduce pryruvate to lactate even under aerobic conditions?

   Yes, retina cells and erythrocytes.

2. Ethanol fermentation -= In some microorganisms, such as yiest and protists, under anaerobic /hypoxic conditions.

Write the two equations for glycolysis!

Glucose + 2NAD+ → 2 pyruvate + 2NADH +2H+

the reaction above is exergonic : ∆G´°1 = -146 kJ/mol

2ADP + 2Pi → 2ATP + 2 H2O

This reaction is endergonic. ∆G´°2 = 2(30,5kJ/mol) = 61 kJ/mol

The sum of the equations : ∆G´°s = ∆G´°1 + ∆G´°2 = -146 kJ/mol + 61 kJ/mol = -85 kJ/mol!

This means that the overall reaction of glycolysis is accompanied by a large decrease in free energy and thus irreversible in the cell.

Which is the first “committed” step of glycolysis? (The first irreversible step, that is)

step 3 : The phosphorylation of Fructose-6-phosphate to Fructose1,6-bisphosphate. Fructose1,6-bisphosphate has no other possible fate that glycolysis.

The enzyme catalyzing the phosphorylation of Fructose-6-phosphate to Fructose1,6-bisphosphate is subject to allosteric regulation – how?

The enzyme is called phosphofructosekinase-1 and is allosterically activated by ADP and AMP – molecules that are the result of ATP consumption. High levels of ADP and AMP signals low ATP levels in the system. Accordingly; PFK-1 is allosterically inhibited by ATP!

Ribose-5-phosphate, an intermediate in the pentose phoshate cycle also activates PFK-1 allosterically – something that will be addressed later.

What is the difference between substrate level phosphorylation generation ATP and respiration-linked phosphorylation generating ATP?

Substrate level phosphorylation means that a soluble intermediate, such as 1,3bisphosphoglycerate, is phosphorylated during glycolysis.

In the latter case, membrane-bound enzymes are involved as well as transmembrane gradients of protons. I'll return to both cases further on.

Give the complete equation for glycolysis under aerobic conditions!

Glucose + 2NAD⁺ + 2ADP + 2Pi → 2 pyruvate + 2NADH + 2H⁺ + 2 ATP + H₂O

The two NADPH molecules are, under aerobic conditions, reoxidized to NAD⁺ - how?

The electrons from NADPH are passed to the electron transfer chain, in eukaryotic cells located in the mitochondria! The ultimate acceptor of the electrons are O₂. The electron transfer from NADPH to O₂ gives the energy for ATP synthesis by respiration-linked phosphorylation.

The reaction is :

2 NADH + 2H⁺ + O₂ → 2NAD⁺ + 2H₂O

Which enzymes control glycolysis? What other regulation methods contribute?

Insulin, epinephrine and glucagon. Glycolysis is also regulated through allosteric regulations of certain enzymes, such as PFK-1, and through changes in the expression of genes coding for the enzymes involved!

How is the metabolism of glucose limited in mammals?

It is limited through by uptake of glucose in to the cells and its phosphorylation by hezokinase.

How is glucose uptake into the cells from the blood mediated?

It is mediated by the GLUT family.

Describe the different members of the GLUT family and their different roles!

GLUT 1 and 2 are present in the liver tissue.

GLUT 3 is present in the brain.

The common feature of GLUT 1, 2 and 3 is that they are always present in the plasma membranes!

GLUT 4 are present in all other cells, such as muscle cells, heart cells, adipose tissue cells etc.

what separates GLUT 4 from GLUT 1, 2 and 3?

GLUT 4 are NOT present in plasma membranes, but has to “called” to go there by the hormone insulin! GLUT 4 are stored in intracellular vesicles and only comes out when insulin demands it.

How and when is insulin released?

Release by β cells in the pancreas as a response to elevated glucose levels!

Describe the reasons behind and consequences of diabetes type-1 -mellitus!

This type of diabetes is also called “insulin-dependent diabetes”. It is a condition caused by to few

β cells. Hence : no insulin or not enough insulin is released in response to elevated glucose levels. As a result of this muscle, heart and adipose tissue cells are unable to take up glucose from the blood. Glucose levels rises in the blood and eventually accumulates to extreme levels; a condition called hyperglycemia. Furthermore : when the muscle, heart and adipose tissue cells don't get access to glucose they have to get nutrition from other sources. Muscle and fat tissue thus start to use up the stored fatty acids as “new” nutrients. When these fatty acids are degraded in the liver, acetyl-CoA is the resulting compound. Acetyl-CoA in turn is transformed to “ketone bodies”. The ketone bodies can be used as alternative fuel and is critical for the brain in absence of glucose – since fatty acids CANNOT pass the blood-brain barrier.

The consequences of ketone bodies used as fuel is that they accumulate in the blood, forcing the pH to drop. The lowering of pH in blood caused by this is called : ketoacidosis – which is a life threatening condition. Diabetes type 1 is kept under control of insulin injections, causing the GLUT 4 to collect glucose from the blood.

Control questions :

What is the cause of diabetes type 1?

Not enough β cells to release insulin, a hormone which causes GLUT 4 to migrate from their intracellular vesicles to the plasma membrane in order to collect glucose into the cell.

As a result, the cell is unable to collect glucose and must rely on other nutrients. Fatty acids are degraded instead, and the ketone bodies resulting from this is used by the brain. Fatty acids them selves cannot be used by the brain, since they are unable to pass the blood-bran barrier. The result of ketone bodies being used as the primary nutrient is ketoacidosis – lowering of the pH. This is life-threating. Hence insulin must be injected on a regular bases to prevent this.

Feeder pathways...

Not only glucose is oxidized through glycolysis but also other carbohydrates, after initial transformation into what?

Glycolysis intermediates!

Name the most common disaccharides converted to intermediates in order to enter glycolysis!

lactose, maltose, trehalose, sucrose.

Name the most common monosaccharides?

Fructose, mannose and galactose.

What are the sources of alternative carbohydrates?

Intracellular stored polysaccharides such as glycogen and starch.

Or disaccharides and monosaccharides obtained through the diet!

How does degradation of starch through the diet begin?

α-amylase in the saliva hydrolyzes the internal glycosidic links.

In the stomach, the pH is to low for the α-amylase to function, but another form of the enzyme is released by the pancreas into the small intestine and the process proceeds.

Dietary glycogen has the same overall structure as starch and is hence metabolized in the same way.

What is the difference in degradation of endogenous glycogen and starch compared to the dietary dito?

Endogenous glycogen and starch can be mobilized for use within the same cell by phosphorylysis – the glycosidic bond is thus broken not by water but by Pi – a reaction catalyzed by glycogen phosphorylase. (starch phosphorylase in plants). The enzyme acts repeatably until it reaches a branch point.

The result of a glycogen unit plus the attached Pi is glucose-1-phosphate which can then enter glycolysis or the pentose phosphate pathway.

What is most beneficial energy-wise; hydrolysis or phosphorylysis?

Phosphorylation is more beneficial, because it converts the substrate directly to glucose-1-phosphate that can be converted to glucose-6-phosphate without the expense of 1 ATP that is required to convert free glucose to glucose-6-phosphate – an intermediate that can readily enter glycolysis or penthose phosphate pathway. The net gain will thus be 3 ATP:s rather than 2 ATP:s, in this case.

Hydrolysis on the other hand starts with free glucose, and is hence more expensive!

We have now concluded that phosphorylation is cheaper than hydrolysis energy-wise. But hydrolysis is still the only “method” used by the body to degrade dietary polysaccharides. Why would breakdown of dietary polysaccharides such as glycogen or starch be useless if phosphorylysis instead of hydrolysis was used?

Because a phosphoryl group on sugars in the intestines would prevent them from entering the typical epithelial cells there. The sugars most be dephosphorylated first! And disaccharides must be degraded to monosaccharides! These monosaccharides must in turn be transported to other tissues – where they are phosphorylated and enter glycolysis there!

Which “method” is used to degrade dietary polysaccharides? Which method is used to degrade endogenous polysaccharides?

Dietary = hydrolysis.

Endogenous = phosphorylysis.

What is lactose intolerance caused by?

Deficiency of the enzyme lactase. Because lactose cannot be completely degraded to monsaccharides, and cannot be absorbed into the cells of the mall intestines. Instead it passes on to the large intestine where it causes cramp and other unpleasant effects. Furthermore, osmolarity is increased with retention of water in the intestine as a result.

This is a common condition in many parts of the world, where lactose is only consumed during childhood. There are, however, more complicated conditions – all the disaccharidases are missing, which means an extremely restricted diet must be held.

What is the condition cataracts in childhood a result of?

A defect in one or all three of the enzymes – öh, böff böff.

Fourteen.three.

What happened to pyruvate after glycolysis under aerobic conditions?

It is oxidized to Acetyl-CoA (also called : acetate) and funneled into citric acid cycle – where it is oxidized even further to CO2 and H2O!

How is NADH synthesized during glycolysis and what happens to the NADH molecules under aerobic conditions?

Glyceraldehyde 3-phosphate is dehydrogenated to NADH in the step 6 of glycolysis. NADH gives electrons to O2 in the mitochondrial respiration, rendering NAD+.

What happens to NADH under aerobic conditions? What is the cause of anaerobic conditions in humans?

Very active skeletal muscles or solid tumors both produces anaerobic /hypoxic conditions. This means that no O2 molecules are available to accept electrons from NADH. But NAD+ must be regenerated somehow – otherwise no glyceraldehyde 3-phosphate would be dehydrogenated, and glycolysis would stop. The problem is solved by reduction of pyruvate to lactate.

What happens to the lactate?

It is usually recycled – it is carried by the bloodstream to the LIVER where it is converted back to glucose.

What is the chemical reason why you can't work out vigorously at the top of your capacity for much long?

The lactate that builds up in the blood because of the fermentation causes a drop in pH. Not even top athletes can keep top speed for more than a minute because of this.

Why do you have to breath heavily after a long run? Why do alligators have to take rest for hours after a hunt?

Because no oxygen was present during the straining activities, forcing the body to reduce pyruvate to lactate instead. Afterwards, lactate has to be converted back to glucose in the liver through gluconeogenesis! This requires oxygen – time to pay the oxygen dept through heavy breathing. The amount oxygen needed and payed for by heavy breathing is the amount of oxygen needed top produce the ATP needed for gluconeogenesis. Gluconeogenesis is needed as the body used up all its stored glycogen during the sprint/hunt.

What is the cycle “pyruvate to lactate, lactate to glucose” called?

The Cori cycle! Named after the Cori couple who deduced the cycle in the 1940s.

How come migratory birds can fly such long distances without resting much?

Small animals have small systems, capable of carrying oxygen fast to the muscle cells in need. Bigger animals, such as humans, have systems to big and complicated to supply oxygen for sudden, intense muscle work. Such animals are usually slow-moving under normal conditions. We don't like to engage in intense muscle work and we don't unless we really have to run from a bear or loose the belly. Then lactate fermentation is needed to provide ATP in the muscles.

What is the result of fermentation preformed by yiest and other microorganisms?

Not lactate, but ethanol. Glucose is first converted to pyruvate – then pyruvate is converted to ethanol through this reaction : rita.

What is the enzyme present in all microorganisms that produce ethanol fermentation called?

Pyruvate decarboxylase. The CO2 resulting from ethanol fermentation is responsible for the bubbles in champagne as well as the holes in swiss cheese and causes the dough to rise when mixed with fermentable sugar during baking.

Thiamine pyrophosphate (TPP) is a co-enzyme facilitating the decarboxylation reactions catalyzed by both pyruvate dehydrogenase and pyruvate decarboxylate. Which property of the TPP facilitates this reaction?

The overall reaction is : pyruvate to acetaldehyde, catalyzed by pyruvate decarboxylate. This reaction requires TPP and Mg2+. TPP is a co-enzyme of pyruvate decarboxylate, and it is derived from vitamine B1. The functional part of TPP, that enables the reaction, is the thiazolium ring. See picture below :

The second carbon of the ring has an acidic H bound to it. Loss of this proton renders a carbanion which is the active agent in the following reactions. Normally, reactions that form carbanions are highly unfavorable, but in this case the positive charge on the tetravalent nitrogen next to the carbanion stabilizes the negative charge. The thiazolium ring acts as an “electron sink”, which is a very important feature in decarboxylation reactions. Electric sinks are, by definition, such groups that pulls electrons from a reactive center (in this case a carbanion) and thus stabilize an electron-deficient intermediate or transition state.

The carbanion of the TPP can perform a nucleophilic attack on the carbonyl group on the substrate. (This forms a single bond between the TPP and the substrate.)

The detailed description below is nicked from wikipedia :

1. The target bond on the substrate is broken, and its electrons are pushed towards the TPP. This creates a double bond between the substrate carbon and the TPP carbon and pushes the electrons in the N-C double bond in TPP entirely onto the nitrogen atom, reducing it from a positive to neutral form. 2. In what is essentially the reverse of step two, the electrons push back in the opposite direction forming a new bond between the substrate carbon and another atom. (In the case of the decarboxylases, this creates a new carbon-hydrogen bond. 3. In the case of transketolase, this attacks a new substrate molecule to form a new carbon-carbon bond.)In what is essentially the reverse of step one, the TPP-substrate bond is broken, reforming the TPP ylid and the substrate carbonyl.

Is the ATP yield from glycolysis under anaerobic conditions greater or smaller than the corresponding yield under aerobic conditions?

The yield of ATP by complete oxidation of a glucose molecule to CO₂ is 30 32 ATP molecules – under aerobic conditions.

The ATP yeild from glycolysis during anaerobic conditions is only 2 ATP molecules!

This means that 15 times as much glucose must be consumed to yield sufficient ATP supply under anaerobic conditions. This is evident when studying cancer cells that initially don't have access to blood vessels – these tumors therefor consume glucose at a very high rate – a fact that is used in cancer diagnostics.

How can fermentation be used in food and industries?

Yoghurt's is produced when the bacterium lactobacillus ferments carbohydrates in milk to lactic acid. This causes the proteins to precipitate and gives rise to the characteristic texture and sour taste.

This drop in pH due to fermentation is an advantage over microorganisms usually living in higher pH – hence fermentation is a way to preserve food.

Industrial fermentation is used to different acids, such as formic, acetic and propionic acid. These fermentations are subjects to rigorous control of temperature and restricted air, of course. The factories; the microorganisms, reproduce them selves and don't give rise to many side products – a dream scenario for engineers.

How do yiest and other microorganisms regenerate NAD+?

Through ethanol fermentation

How is NAD+ regenerated in eykaryotic cells during hypoxic conditions?

Through reduction of pyruvate to lactate.

Some tissues are completely dependent on glucose as fuel – which ones?

Human brain. Testes. Renal medulla. Embryotic tissue.