Week 6 Lecture 560B on Line

8/3/2019 Week 6 Lecture 560B on Line

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Week 6 Lecture

Biochemistry of Nutrition

Dr. Charles Saladino

Further Thoughts on Enzymes

Although the topic of enzymes was covered in the previous lecture, lets make certain

additional and review points before going on to our current topic of energytransformation. A critical issue regarding nutrition is the regulation of metabolic

pathways. In order to sustain growth and maintain a homeostatic environment

compatible with life, modulations of the activity of enzymes controlling various

metabolic sequences must accomplish an approximate balance between catabolism andanabolism. Regulation of enzyme activity generally occurs either through a) induction of

the enzyme where more enzyme is synthesized; b) covalent modification of the enzyme

(such as phosphorylation/dephosphorylation); and c) allosteric control of enzyme activity.

In the case of covalent regulation, protein kinases adding phosphate groups and

phosphatases removing phosphates is common (especially as noted in the glycogensythase and phosphorylase controlling the formation and breakdown of glycogen,

respectively). Allosterism involves positive and negative regulators which bind to the

enzyme other than at the active site to activate or deactivate enzymes, respectively).

Such regulation involves altering the conformation of the enzyme structure, much in thesame way as the positive cooperative effect of binding oxygen upon hemoglobin

changing from the taut to the relaxed conformational state.

As part of this weeks assignment, email me and tell me whether you think that

regulatory enzymes which control a metabolic pathway would most like be able tocontrol reversible or irreversible reactions or both.

Introduction Energy Concepts

If we really think about it, the bottom line of the metabolism in which we engage is about

the production of energy whether it be derived from a bowl of pasta or heaven forbid

a Big Mac. This energy might be needed to drive chemical reactions that require

energy, or to build structures on the molecular, cellular, or tissue level, or it might berequired for moving structures and biomolecules, as well as many other cellular

housekeeping chores. I suggest that ultimately, however, that all these energy-requiring

endeavors are designed to keep the cells alive by down-modulating the rate at which thetotal entropy of our living system and its surroundings is spontaneously increasing. Said

in simplistic terms, we produce energy to maintain the organization necessary to keep our

living system doing just that. Remember, dont be fooled. Just because entropy isobviously decreasing (increasing organization) in one place (such as in the synthesis of

DNA), the Second Law of Thermodynamics tells us that entropy must increase

(decreased organization) in another place (such as in the burning of glucose for fuel).


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The folding of a protein illustrates this. Obviously, when a protein folds, it increases its

organization and decreases its entropy. So wheres the compensation? The answer lies

in the properties of water. In brief, the water surrounding a protein can interact with thepolar amino acids of the protein. However, interactions between water and the non-polar

portions of the proteins are not as favorable, and the water will form cages within the

vicinity of these non-polar areas of the protein, where the water becomes well-ordered,compared to water molecules in free solution. Remember, we said that the interior of

proteins tends to exclude water. So when the protein starts to fold, water becomes

excluded from the interior (non-polar regions) of the protein, causing the organized water(the cages) to become less organized. This is the roughly the entropy compensation for

the more organized protein structure. Not so hard to understand, right?

Now energy can take different forms within cells, including electrical and heat energy.Lets focus on the latter form. Heat is a manifestation of the kinetic energy of random

molecular motion. In contradistinction, energy can be present as potential energy, which

means that energy released from some process was locked up in a chemical bond. Within

chemical and biochemical systems, potential energy is related to the likelihood that atomscan react with one another.

As I just said, in the living cell, energy is stored in chemical bonds, such as in the

phosphate bond of adenosine triphosphate (ATP). Thus, when the first phosphate bond is

broken from the ATP molecule, its potential energy is released in the form of 7300

calories (a measure of heat), with perhaps 40% available as useful energy (-G) (and H, as in exothermic). The free energy (-G), is the energy to dowork (such as synthesize a large molecule from smaller building blocks).

So a thermodynamically unfavorable reaction (like synthesizing DNA) can be made to go

to completion by coupling it to a thermodynamically favorable reaction (such asbreaking the phosphate bond of ATP). Another way of saying that is that ATP hydrolysis

shifts the equilibrium of coupled reactions from unfavorable to favorable. We will see

this throughout our discussions of metabolism. For example, when we oxidize glucoseall the way to CO

2and water, we are producing the energy required for the formation of

the high energy-storage molecule, ATP, from ADP and phosphate by coupling the

glucose oxidation reactions to the ATP formation reaction. In fact, all the processes ofliving cells are processes of energy transformation. These energy transformations are

referred to as bioenergetics

Remember, despite what politicians tell you, there is no such thing as a free lunch.

Someone pays somewhere. We saw that in the above discussion on entropy balance. Ittakes energy to build an ATP molecule. For the most part, that comes from the oxidationof fuels that we ingest, digest, and then transform into energy (besides producing

numerous intermediary biomolecules of metabolism). This is the BIG picture I want you

to appreciate, because all cells require a constant supply of ATP and a continuous supply

of fuels to generate that ATP. The details of the metabolism of nutrition are yet to come and come they will like a pack of wild horses.


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General Comments Regarding Metabolism

I would consider metabolism as an exquisitely designed, linked series of chemical

reactions that begins with a given molecule. That molecule is then converted into another

molecule(s) in a manner that is nothing less than unbelievably thoughtful. Although theintimidating number of biochemical reactions within a given cell is literally in the

thousands, what would otherwise appear as an overwhelming task to understand becomes

possible to grasp because of recurring themes, many of which are common to a variety ofspecies. These unifying themes that include common metabolites, reactions, and

regulatory mechanisms make the understanding of metabolism more manageable.

Before moving on to the specifics related carbohydrate metabolism and other fuels to bediscussed over the coming weeks, I wish to point out what you will eventually realize are

some of the above-mentioned recurring themes of metabolism. So let us talk in general

about the stages involved in the extraction of energy from foodstuffs.

1. Large molecules contained in food are broken down into smaller units. Sopolysaccharides are hydrolyzed into simple sugars, proteins are broken down into amino

acids, and fats are oxidized to fatty acids and glycerol.2. Then, the myriad of small molecules are further degraded into a few simple units that

play a key role in metabolism. We shall see eventually that most of the simple sugars,

fatty acids and amino acids are converted into what will be a critically important

molecule called acetyl coenzyme A (acetyl CoA). Although some ATP is generated here,the yield is relatively small compared with that derived from the third stage.

3. In this stage, ATP is produced from the complete oxidation of acetyl CoA via the

Krebs cycle and oxidative phosphorylation the final common pathways in theoxidation of fuel biomolecules. In the process, four pairs of electrons are transferred

(three to NAD+ and one to FAD) for each acetyl group that is oxidized. A proton

gradient is generated as electrons flow from reduced forms of carriers to oxygen. Thisgradient is used to synthesize ATP.

(These mitochondrial processes wherein oxygen is utilized are collectively referred to ascellular respiration or internal respiration. This is not to be confused with external

respiration, which we think of as breathing or taking oxygen into the lungs.) The

formation of ATP in the mitochondria, as we shall see in subsequent lecture material, is

known as oxidative phosphorylation.

To further outline metabolism in a general way (remembering that we mentioned that key

reactions are reiterated throughout metabolism), we note an economy of design inbiochemical reactions. Specifically, we can simplify the work of understanding

metabolism by boiling the thousands of metabolic reactions down to just six subdivisions.


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These six types of reactions are:

1. oxidation reduction involving electron transfer

2. ligation requiring ATP involving covalent bond formation (C-C bonds)3. isomerization involving atom rearrangements to form isomers

4. group transfer involving transfer of a functional group from one

molecule to another5. hydrolytic involving the cleavage of bonds by adding water

6. adding or removing involving the addition of functional groups to

functional groups double bonds or their removal to form double bonds

Note: Do these classes of reactions remind you of the six classes of something else we

discussed before the exam?

Carbohydrate Background

The term carbohydrate is derived from the fact that a great many molecules from thismajor class of biomolecules have the empirical formula, CH2O. Now I am sure you are

well aware that you can classify carbohydrates into three main categories, the first being

the simplest sugars, called monosaccharides. On pages 1124 and 1126 of your text, are

examples of these simple sugars (note: they are biochemically-usefull D-form isomers),

glucose, fructose, and galactose. Also notice that some of these (example, glucose,fructose, and galactose) are all isomers of one another, having the same chemical formula

but showing different structural, three-dimensional arrangements. Note the different

ways of drawing the same structures such as Fisher projections on page 1125, thecyclized Fisher projection shown at the middle right ofpage 1126, and the Haworth

projection right below those on page 1126 where the sugar is shown in the cyclized form,

which is the way the sugars are really configured in the living cell. Each representationhas a useful aspect for understanding the respective structures. For example, the Haworth

structures give us a better idea of how an internal hemi-acetal bond (you can look thischemistry up for yourself butwhere basically one part of the molecule reacts withitself (forming a cyclic ring within the sugar). The Fisher projection makes it easier to

understand why glucose and galactose are considered reducing sugars, because they have

a free aldehyde group on the end carbon, whereas fructose does not (it contains a ketone

group on carbon number two). Thus, sugars with ketone groups are referred to asketoses, and those with aldehyde groups are called aldoses. The total number of carbons

is the basis for naming monosaccharides trioses, tetraoses, pentoses, hexoses, and

heptoses (3, 4, 5, 6, and 7 carbon sugars, respectively). Specifically, by way of example,

glucose is an aldohexose (6 carbons and an aldehyde group), fructose a ketohexose (sixcarbons with a keto group). Finally, under physiological conditions, the sugars are

generally in the cyclized form.

Disaccharides are formed by the bonding of two monosaccharides together(see page1028 of your text). This bond is called a glycosidic bond, formed by a dehydration

synthesis reaction meaning removal of water by taking an OH group from one sugarand an H from the other sugar to which the first sugar bonds. Common disaccharides are


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unique needs? Even though you are not necessarily familiar with all the biomolecules we

are about to mention, they will become evident and understandable as the weeks pass.

Some of the enzymes I will mention might be meaningless to you at the moment.However, if you keep this part of the lecture handy for referral over the coming weeks,

these terms will fall into place eventually. In other words, I would rather do this now

than later. Let us start with some definitions:1. Glycolysis the oxidation of glucose to pyruvate anaerobically. This pathway can

lead into the Krebs cycle.

2. Glycogenesis the formation of the storage form of glucose molecules called glycogen3. Glycogenolysis the breakdown of glycogen to provide readily available glucose

4. Gluconeogenesis the formation of glucose from either fatty acids, or pyruvate, or

lactate, or amino acids, or glycerol.

5. Krebs cycle = Citric Acid cycle = Tricarboxylic acid cycle an aerobicmitochondrial pathway that begins with the utilization of acetyl CoA and which generates

a series of intermediates that lead to a continuous cycle, with a constant input of acetyl

CoA. This cycle is coupled to the generation of major quantities of ATP, and it generates

intermediates which can also become part of several other important metabolic pathways.6. Ketones ketone-group-containing molecules often generated from a heavy degree of

fatty acid oxidation (called beta oxidation). They can cause a condition of plasmaacidosis called ketoacidosis.

Now let us start with the overall view of metabolism:

Muscle. Glucose, fatty acids, and ketones are the major fuels. However, unlike thebrain, muscle has large glycogen stores (about 1200 kcal), about 75% of the bodys total

glycogen. This glycogen is easily converted into glucose-6-phosphate (glucose-6-P)

(keep that on the back burner of your mind until next week). Like the brain, muscle lacksthe enzyme glucose-6-phosphtase, meaning that the conversion of glucose-6-P back to

glucose does not occur in either organ. Why? What is the result? Both organs retain

glucose, rather than exporting it, as glucose is the preferred fuel for activity bursts. OK?So the glucose of muscle and brain is not fro export. Thats the job primarily of the liver.

In active muscle skeletal, much of the pyuvate from glycolysis is converted into lactate.Some of that lactate gets back to the liver to generate more glucose (gluconeogenesis, as

above). This shifts part of the metabolic burden back to the liver. The muscle can

transaminate some amino acids, but it can not form urea. So muscle nitrogen is released

in the blood in the form of the amino acid alanine. The alanine goes to the liver forglucose and/or urea formation. Again, the metabolic burden shifts to the liver. The

nitrogen is also carried back to the liver in the form of glutamine (where glutamate picks

an amine group in its R-group). (Remember what happens when glutamine reaches theliver (hint: glutaminase).?

In resting skeletal muscle, metabolism is very different. Eight five percent of themuscles energy needs are met by fatty acids. Cardiac muscle, however, functions

almost exclusively aerobically (as seen by the great density of the mitochondria), and it

has no glycogen reserves. Fatty acids are the main course of fuel.


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Brain. Except for prolonged starvation, glucose is the sole fuel. This organ requires a

continuous supply of glucose, having no fuel stores. The brains consumption of glucose

on a daily basis is in the range of 120 grams, corresponding to an energy input of 420kcal and 60%-75% of the bodys glucose utilization at rest. A significant portion of this

energy is used to maintain the Na+/K+ pump essential to neural impulse transmission.

Of course, it also requires energy to synthesize neurotransmitters and their receptors.

The cell membrane-localized transporter of glucose into the brain is called GLUT 3. So

if I tell you that this transporter has a low Km value for glucose (1.6 mM), can you reachback into your recollection of the enzymes lecture and note that this means that the

transporter is saturated under most conditions? (This is an example of how I want you to

think back, correlate, and extrapolate). Now follow this: Considering that the brain is

usually provided with a continuous supply of glucose, radioactive NMR studies haveshown that the brain glucose concentration is about 1 mM when the plasma glucose

levels are normal. Glycolysis starts to slow down when the glucose levels approach the

Km (about 50 M) of hexokinase, the enzyme that traps glucose in the cell by

phosphorylating it in the #6 carbon position. The point of danger arrives when theplasma glucose drops below 39.6 mg/dL, close to the Km value of GLUT3. Now the

brain is receiving insufficient glucose.

The reason that fatty acids are not fuel for the brain is that they are primarily bound to

albumin in the plasma. Thus, they can not traverse the blood-brain barrier. In addition,

with all the other critical functions of the brain, the enzymes to oxidize fatty acids are justnot present in the cells of the brain. Ketone bodies can replace glucose as fuel during

starvation conditions, otherwise death would occur more quickly. We will deal with

these separately in another lecture.

Adipose. The amount of the bodys triaglycerols stored here is enormous. Thistranslates to about 15 kg of this lipid with an energy content of 135,000 kcal! This tissue

specializes in both the esterification of fatty acids and their release from the triaglycerols.These fatty acids are transported from the liver, the major site of fatty acid synthesis. The

details of this will be covered in our lectures on lipids. One of the essential intermediates

in the biosynthesis of lipids comes from a glycolysis (oxidation of glucose) intermediate(remember the name dihydroxyacetone phosphate). Therefore, adipose cells require

glucose for the synthesis of triaglycerols. In fact, as we shall discuss in our lipid lectures,

the glucose levels inside the adipose cell is a major determinant as to whether fatty acidsare released into the blood.

Kidney. The major role of the kidney primarily is cleansing the body of undesirable

metabolic by-products put into urine for excretion (such as urea). This is also the mainvehicle for removing metabolic waste and maintaining osmotic fluid balance. The renal

tubules filter the blood plasma about 60 times a day. Most of what is filtered out of the

blood is reabsorbed, and just a couple of liters of urine are produced. Water-solublecompounds, such as glucose and water itself, are reabsorbed to prevent unnecessary loss.

This reabsorption requires significant amounts of energy, consuming a full 10% of the

bodys oxygen needs for cellular respiration, whereas the kidney is only 0.5% of the body


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mass. Most of the reabsorbed glucose is carried into the kidney by a Na/glucose

cotransporter. This system is energy-requiring.

Liver. One could easily state that the metabolic processes taking place in the liver are

critical for providing fuel to organs, such as the brain, muscle, and many others. Even

though the liver constitutes about 4% of the body weight, it is clearly the metabolicintegration center for the whole body. Indeed, most compounds absorbed from the

intestine reach the liver via the portal system. This allows the liver to regulate the level

of many plasma metabolites.

Relevant to our upcoming discussions on carbohydrate metabolism, the liver removes

over 65% of the plasma glucose from the blood, as well as all the remaining

monosaccharides. The rest of the glucose left in the blood is utilized by other tissues. Aswe shall see in glycolysis, the glucose that enters the liver is converted to glucose-6-P by

glucokinase and by hexokinase in the other tissues (especially muscle,) taking up that

critical monosaccharide from the blood. The glucose-6-P can be stored in the liver as

glycogen (as much as 400 kcal worth at a time). Excess glucose is metabolized topyruvate, which after it enters the mitochondria is converted to that all-important acetyl

CoA. The acetyl CoA could be used to form biles salts, fatty acids, and cholesterol, or itcan commonly enter the Krebs cycle. The glycolytic pathway we shall describe this

week has an important pathway branching from it known as the pentose phosphate

pathway, to be described next week. The liver is also able to produce glucose routinely at

lower levels, and under certain conditions more vigorously by the process referred toabove as gluconeogenesis. This latter situation is especially the case when plasma

glucose levels are low, and thus plasma insulin levels are low and glucagon levels are

elevated.

As we shall learn, the liver is critical in regulating lipid metabolism. This includes,

among many other pathways, the formation of very low density lipoproteins (VLDL).Depending upon the degree of fatty acid catabolism, the liver can also form ketones.

The liver also plays a pivotal role in dietary amino acid metabolism, absorbing themajority of amino acids the rest going to the other tissues. Here the amino acids can be

directed into protein synthesis, as opposed to just being utilized as fuels. We will discuss

this distinction. Also, the transamination we discussed in an earlier lecture takes place in

the liver (with the subsequent formation of urea) and to some extent in other tissues likemuscle, but without urea formation.

The liver meets its own energy needs in several ways, especially by forming-ketoglutarate (a Krebs cycle intermediate) from the degradation ofamino acids. Further, and ultimately, a main role of glycolysis is to form the building

blocks for many biosynthetic pathways.

Thus we have an overview of some (not all) of the metabolism taking place in key

organs. A major point to remember is that all this metabolism occurring in the respective

organs is controlled overall by a variety of substances, including neurotransmitters,


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hormones, and many metabolites and second messengers. We will try to sort out some of

the more salient ones in his course.

Glucose metabolism Glycolysis

The anaerobic breakdown of glucose to carbon dioxide and ethanol (fermentation) in

yeast has been known for centuries. However, scientific investigation of this pathway

began with Louis Pasteur and others in the middle nineteenth century. Yet it was notuntil 1940 that the complete breakdown of glucose was described. This pathway is now

called glycolysis. Glycolysis is probably the most completely understood biochemical

pathway of all, and it consists of a series of ten enzymatic reactions, wherein a single

molecule of glucose is converted to two molecules of pyruvate (each consisting of threecarbons). In this process, there is a net generation of only 2 ATP molecules. This

pathway plays a critical role in energy metabolism by providing a significant amount of

the free energy required by cells of most organisms and by preparing glucose and other

compounds for further energy-yielding oxidative degradation.

So let us begin glycolysis with glucose being transported into the cells, such as in theliver. However, as fast as glucose comes into the cells, it would leave almost as fast.

This is why the first step in glycolysis is so critical. Catalyzed by glucokinase in the liver

(and hexokinase in other tissues like muscle), glucose is phosphorylated in the number 6

carbon position , forming a phosphoester linkage. Importantly, the glucose-6-P can notleave the cell, and thus the equilibrium shifts toward further oxidation, because of the

oxidative state of this sugar.

Major physiologically-relevant point: I ask you to recall our discussion of the Km of

enzymes. The liver glucokinase (when you see the word kinase, it means an enyzyme

that adds a phosphate group) has a large Km, compared to the smaller Km of thehexokinases of other tissues like the muscle. These enzymes are isoenzymes of one

another, in that they perform the same function in different tissues, varying slightly in

their respective amino acid sequences. These different isoenzymes of the liver vs. musclereflect their different roles in carbohydrate metabolism. Specifically, this difference is

that muscle consumes glucose for energy production, whereas the liver is responsible for

maintaining blood glucose levels, as well as using glucose for its own needs. The liver

does this by either producing or removing blood glucose, depending upon the prevailingglucose concentration. The 10 mM Km (remember Km is the substrate concentration

that gives half maximal velocity) of the liver glucokinase is higher than the usual

concentration of blood glucose. Also, there is rapid glucose transport into the cells thatresults in a quick equilibration of glucose concentrations between the blood and the

hepatocytes. The high Km of glucokinase allows for the liver to directly regulate blood

glucose levels. So when the blood glucose levels are high (as in after a carb-rich meal),excess glucose is transported into the hepatocytes, where the glucokinase enzyme

converts that monosaccharide into glucose-6-P. Because glucokinase is only half

saturated at 10 mM glucose, its activity continues to increase as the glucose concentration

rise to 10 mM or more. On the other hand, the hexokinase of the muscle is more easily


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saturated and can not further adjust blood glucose levels. So it could not clear the blood

of more glucose, when its concentration rise in the blood.

So what have we really said here? Its relatively simple. The kinetic properties of the

respective isoenzymes (of the liver vs. the muscle) determine that the liver and not the

muscle (as big as its mass is) controls blood glucose levels. To add to this knowledge isthe fact that glucokinase is not inhibited by glucose-6-P (its end product i.e., no end-

product feedback inhibition), the way it is in muscle. Therefore, it operates when the

accumulation of glucose-6-P would completely inhibit the hexokinases of the muscle,with the latter enzyme subject to end-product inhibition. Again, the properties of the

enzyme contribute to the regulation of plasma glucose levels. Thus, we have now

related the kinetics of enzymes and their inhibitory mechanisms to the critically important

need for maintaining proper glucose levels; and now you have an understanding of howblood glucose levels are maintained - not just the fact that there is some mechanism.

Now turn to Figure 15.6 in your text to view glycolysis in total. After glucose is

phosphorylated (at the cost of one ATP), the resultant glucose-6-P is isomerized intofructose-6-P. At the cost of a second ATP, the fructose-6-P is then phosphorylated at the

number one position. This is a highly regulated step, with the phosphofructokinaseenzyme being modulated negatively by ATP (and citrate-thats for later) and positively

modulated by AMP (monophosphate) and ADP (diphosphate) (is this allosteric

regulation?).

The next step is very important, because an aldolase enzyme splits the six carbon

fructose-1,6-bisphosphate into two, three carbon sugars dihydroxyacetone phosphate

and glyceraldehyde-3-P (G-3-P). Because there is an equilibrium shift away from DHAPtoward G-3-P, the result is that for every glucose oxidized, two, three carbon

glyceraldhyde-3-P molecules are formed. Thus, the rest of the reactions shown

(culminate with pyruvate formation) really occur twice.

There are some points to note in glycolysis, as shown in the Figure 15.7. In step number

6, the enzyme glyceraldehydes-3-phosphate dehydrogenase utilizes the coenzyme NAD+and puts an inorganic phosphate on the G-3-P at no ATP cost. This phosphate is

recovered in the formation of ATP in step number 7, along with another ATP formed in

the final step of glycolysis the conversion of phosphoenolpyruvate (PEP) into pyruvate.

So what is the net ATP balance? Close your eyes and think about it before reading the

next sentence. The answer is that there is a net gain of two ATP for the glycolytic

pathway. In step 1 and 3, two ATP are consumed. Because the three carbon reactionseach occur twice for every glucose that is oxidized, two ATP are recovered at step

number 7 and another two again at number 10 (the final step). Thus, glycolysis begins

with glucose phosphorylation, and it ends with pyruvate formation.

You will also note that pyruvate can be converted to lactate. This occurs when there is

insufficient oxygen in the tissue and when there is a high NADH/NAD+ ratio, both

occurring in heavily-exercising muscle, for example.


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To close this lecture, there is a very important issue for you to consider. That is, the

entire glycolysis pathway is anaerobic. No oxygen is required for this series of reactionsthat takes place in the free cytosol of the various cells. Further, and obviously, in most

cell types (excluding erythrocytes, for example, where there are no mitochondria), the

main purpose of glycolysis is not just the scant production of two ATP. Rather,important intermediates are generated, which can lead to a diverse set of metabolic

pathways, some carbohydrate metabolism, some not. One of the most prominent is when

pyruvate enters the mitochondria to form acetyl CoA as an entrant into the Krebs cycle,which is coupled to reactions that produce a much larger quantity of ATP.

Finally, I am going to ask you to play close attention to how one metabolic pathway

inter-relates to another pathway. So it is important that you are familiar with a

given pathway, so that you can integrate it with another over the coming weeks.

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