BIOCHEMISTRY DISCUSSION 6

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Okay - so now we’re going to talk about what happens after glycolysis, and that is the citric acid cycle. And this is where we extract most of the energy from glucose. {The chapter on gluconeogenesis is now part of the course}, but I’ll just say a few words here about it. Sometimes you need more glucose - and for one reason, your brain needs the glucose - your brain doesn’t function on anything else. So at times like that, you can run the glycolytic cycle, pretty much in reverse. There’s a few tricks you need to do in order to overcome some barriers, but you can manufacture your own glucose. But now we’re moving onto the citric acid cycle, and the first step here is preparation.

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Biochemistry: A Short Course

Fourth Edition

CHAPTER 18 Preparation for the

Cycle

Tymoczko • Berg • Gatto • Stryer

So what we’re going to talk about now is the pyruvate dehydrogenase complex, and that is how pyruvate gets turned into acetyl CoA. So it actually gets turned into the acetyl part of acetyl CoA and that is regulated by two separate mechanisms that we’re going to go into.

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Chapter 18: Outline

18.1 Pyruvate Dehydrogenase Forms Acetyl Coenzyme A from Pyruvate

18.2 The Pyruvate Dehydrogenase Complex Is Regulated by Two Mechanisms

So all of this takes place in the mitochondria - and the mitochondria are the source of the most ATP in your cells. The first parts of glycolysis take place in the cytoplasm - but after it gets broken down to pyruvate and releases corresponding ATP, that pyruvate gets transported into the mitochondrion where it is further processed. The first step of this process is the creation of acetyl CoA.

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Acetyl CoA

• Under aerobic conditions, pyruvate enters the mitochondria where it is converted into acetyl CoA.

• Acetyl CoA is the fuel for the citric acid cycle, which processes the two-carbon acetyl unit to two molecules of CO2 while generating high-energy electrons that can be used to form ATP.

Figure 18.1 Coenzyme A. Coenzyme A is the activated carrier of acyl groups. Acetyl CoA, the fuel for the citric acid cycle, is formed by the pyruvate dehydrogenase complex.

Here’s the molecule of acetyl CoA. You can recognize on the right the regular adenine and ribose with phosphate attached, a couple more phosphates attached and then a small section of crazy-looking stuff, and at the end there’s a sulfur that attaches to the acetyl group. It’s the acetyl group that is really important - everything else there is just basically a handle so that your proteins can recognize this acetyl group and pass it around easily.

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Structure of Coenzyme A

THIS IS THE IMPORTANT PART – THE REST IS JUST A “HANDLE”

Figure 18.2 An overview of the citric acid cycle. The citric acid cycle oxidizes two- carbon units, producing two molecules of CO2, one molecule of ATP, and high- transfer-potential electrons.

So an acetyl unit enters the citric acid cycle, and you can see here it is a circular cycle. Now, the first thing that it does is it gets attached to a four carbon molecule, which makes a six carbon molecule. Then 2 carbon dioxides are released, high-transfer- potential electrons are released and captured and ATP and also GTP are created in this process. And by that point you’ve fully oxidized two carbons and you’re back down to the four carbon molecule, which is then ready to accept another acetyl unit.

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A Schematic Portrayal of the Citric Acid Cycle

The pyruvate dehydrogenase complex is a large complex inside the mitochondria which takes in these pyruvate molecules and puts out acetyl CoA. So once that’s been done, the pyruvate can no longer be used for anything else. You’ve gotten rid of carbon dioxide and that is an irreversible step because the carbon dioxide leaves the system and you exhale it.

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Section 18.1 Pyruvate Dehydrogenase Forms Acetyl Coenzyme A from Pyruvate

Learning objective 1: Explain why the reaction catalyzed by the pyruvate dehydrogenase complex is a crucial juncture in metabolism.

• The pyruvate dehydrogenase complex, a mitochondrial matrix enzyme, oxidatively decarboxylates pyruvate to form acetyl CoA.

• This reaction is an irreversible link between glycolysis and the citric acid cycle.

Figure 18.3 Mitochondrion. The double membrane of the mitochondrion is evident in this electron micrograph. The oxidative decarboxylation of pyruvate and the sequence of reactions in the citric acid cycle take place within the matrix. [(Left) Omikron/Photo Researchers.]

Here’s just a electron micrograph of a mitochondrion and you can see the sort of the wavy cristae in there. It's the internal part, the large spaces, where this pyruvate dehydrogenase does its job.

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Electron Micrograph and Diagram of the Mitochondrion

Figure 18.4 The link between glycolysis and the citric acid cycle. Pyruvate produced by glycolysis is converted into acetyl CoA, the fuel of the citric acid cycle. Fatty acid degradation is also an important source of acetyl CoA for the citric acid cycle (Chapter 27).

So in the overall flow of glucose to ATP, we’re going to be covering this central part, which shows the pyruvate being turned into acetyl CoA.

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Diagram of the Link between Glycolysis and the Citric Acid Cycle

? Arrow should bend the other way!

Now the pyruvate dehydrogenase complex has got three subunits - well, I mean three different types of subunits. One of them is pyruvate dehydrogenase, one is dihydrolipoyl transacetylase, and the other one is dihydrolipoyl dehydrogenase. We're just going to call them E1, E2, and E3. These are large complexes and you can see that there's a number of chains of each one of these in the complex, as well as prosthetic groups - remember prosthetic groups are there to allow proteins to do things that are not normally possible with your limited set of 20 amino acids. These things, in this case, include the oxidative decarboxylation of pyruvate (in other words, just pull off a carbon dioxide), transfer of an acetyl group to CoA, and then regeneration of the system.

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Table 18.1 Pyruvate Dehydrogenase Complex of E. coli

(LEFT) Electron micrograph of the pyruvate dehydrogenase complex from E. coli. [Courtesy of Dr. Lester Reed.]

(RIGHT) Figure 18.5 Structure of the pyruvate dehydrogenase complex from B. stearothermophilus. The image of the complex, which was derived from cryo- electron microscopic data, shows an inner core consisting of the E2 enzyme. The shell surrounding the core consists of E1 and E3 enzymes, although only the E1 enzymes are shown in this structure. Two of the 60 lipoamide arms are shown (red and yellow). [Donald Bliss, National Library of Medicine.]

These are electron micrographs of these pyruvate dehydrogenase complexes. This is from E. coli. You can see they’re kind of cube shaped.

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Image of the Pyruvate Dehydrogenase Complex from B. stearothermophilus

A schematic representation of the pyruvate dehydrogenase complex. The transacetylase core (E2) is shown in red, the pyruvate dehydrogenase component (E1) in yellow, and the dihydrolipoyl dehydrogenase (E3) in green. The number and type of subunits of each enzyme is given parenthetically. Each red ball represents a trimer of three E2 subunits. Notice that each subunit consists of three domains: a lipoamide- binding domain, a small domain for interaction with E3, and a large transacetylase catalytic domain. The transacetylase domain has three subunits, with one subunit depicted in red and the other two in white in the ribbon representation.

On the upper left you’ll see a different representation that looks more or less cube shaped, and the E1 (yellow) molecules are on the outside, and the E2 are buried on the internal part, and the E3 are these green balls. And there are several of these subunits in this complex. The E2 subunit of this complex has got eight different components, and they’re all trimers, and each element of these trimers is a fairly large complex with three separate domains. The transacetylase domain you can see on the right on the bottom, and that is a large domain that is actually made up with three separate sub-domains. And then the domain interacting with the E3 component is one of the structural domains. And the lipoamide domain is one of the catalytic domains for the capture and transport of acetyl groups.

Figure 18.7 Reactions of the pyruvate dehydrogenase complex. At the top (left), the enzyme (represented by a yellow, a green, and two red spheres) is unmodified and ready for a catalytic cycle. (1) Pyruvate is decarboxylated to form hydroxyethyl-TPP. (2) The lipoamide arm of E2 moves into the active site of E1. (3) E1 catalyzes the transfer of the two-carbon group to the lipoamide group to form the acetyl– lipoamide complex. (4) E2 catalyzes the transfer of the acetyl moiety to CoA to form the product acetyl CoA. The dihydrolipoamide arm then swings to the active site of E3. E3 catalyzes (5) the oxidation of the dihydrolipoamide acid and (6) the transfer of the protons and electrons to NAD+ to complete the reaction cycle.

So this is just an overall picture of what happens in the pyruvate dehydrogenase complex. On the upper left you’ve got a diagram showing the E1, E2, and E3 subunits. The first step is the addition of pyruvate to TPP, and that is accompanied by the release of carbon dioxide. The second step is the attachment of that acetyl group to the lipoamide of the E2, and that’s that little pentagonal thing with the two sulfurs. That's forming a thioester bond, which we know from previously is a high energy bond and is a good way of conserving the energy during this transfer process. The step labeled four there is the transfer of the acetyl group to coenzyme A, and this is accompanied by the {REDUCTION - WRONG IN AUDIO} of the sulfur groups. And finally, the fifth step will regenerate the original pentagonal ring. And the sixth step then reduces the NAD molecule, harvesting a bit more energy from the system.

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Diagram of the Reactions of the Pyruvate Dehydrogenase Complex

Well, here’s an overview of the reactions involved in this process. Starting with a molecule of pyruvate on the left, you have a decarboxylation, and then an oxidation where you remove two electrons, and then transfer of the remaining acetyl group to acetyl CoA.

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The Synthesis of Acetyl Coenzyme A from Pyruvate Requires Three Enzymes and

Five Coenzymes • The synthesis of acetyl CoA from pyruvate consists

of three steps: a decarboxylation, an oxidation, and the transfer to CoA.

So the prosthetic groups here are thiamine pyrophosphate and lipoic acid, and those are pictured here.

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Structure of Thiamine Pyrophosphate and Lipoic Acid

And the first step is the attachment of pyruvate to TPP, forming hydroxyethyl-TPP and releasing carbon dioxide. Now remember this TPP is a prosthetic group, so it’s attached to the proteins and this reaction is catalyzed by the proteins.

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Decarboxylation The synthesis of acetyl coenzyme A from pyruvate requires three enzymes and five coenzymes. 1. Decarboxylation:

– Pyruvate dehydrogenase (E1), a component of the complex, catalyzes the decarboxylation. Pyruvate combines with the ionized form of the coenzyme thiamine pyrophosphate (TPP).

TPP is derived from the vitamin thiamine - and it readily forms a carbanion, which is a very active site for attacking the pyruvate molecules.

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Thiamine Pyrophosphate

The synthesis of acetyl coenzyme A from pyruvate requires three enzymes and five coenzymes. • Thiamine pyrophosphate, a coenzyme, is derived from

the vitamin thiamine.

The oxidation of this two-carbon fragment is coupled with the breakage of the disulfide bond in lipoamide and the transfer of the acetyl group there to the lipoamide.

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Oxidation The synthesis of acetyl coenzyme A from pyruvate requires three enzymes and five coenzymes. 2. Oxidation:

– The hydroxyethyl group attached to TPP is oxidized to form an acetyl group while being simultaneously transferred to lipoamide, a derivative of lipoic acid. The disulfide group of lipoamide is reduced to its disulfhydryl form in this reaction. The reaction is catalyzed by E1 and yields acetyl–lipoamide.

Lipoamide comes from the vitamin lipoic acid being attached to a lysine chain on the enzyme dihydrolipoyl transacetylase, or E2 - that addition of course being catalyzed by a different enzyme.

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Lipoamide

The synthesis of acetyl coenzyme A from pyruvate requires three enzymes and five coenzymes. • Lipoamide, a coenzyme, is formed by

the attachment of the vitamin lipoic acid to a lysine residue in another enzyme in the complex, dihydrolipoyl transacetylase (E2).

So the acetyl group is transferred from the sulfur on acetyl lipoamide to the sulfur on coenzyme A, and that’s a lateral transfer which doesn’t involve any major energy change. But it leaves the dihydrolipoamide reduced with two hydrogens and a couple of extra electrons on there. That’s where the energy has gone from the oxidation of the carbon.

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Formation of Acetyl CoA The synthesis of acetyl coenzyme A from pyruvate requires three enzymes and five coenzymes. 3. Formation of acetyl CoA:

– E2 catalyzes the transfer of the acetyl group from acetyl–lipoamide to coenzyme A to form acetyl CoA.

That energy is recovered with the regeneration of lipoamide and the reduction of FAD to FADH2. FADH2 is then oxidized and reduces NAD+ to NADH, and NADH is later used to generate energy in a different part of the cycle.

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Dihydrolipoamide Dehydrogenase

The synthesis of acetyl coenzyme A from pyruvate requires three enzymes and five coenzymes. • To participate in another reaction cycle, dihydrolipoamide

must be reoxidized. This reaction is catalyzed by dihydrolipoamide dehydrogenase (E3).

So the acetyl group is attached to the lipoamide, and it’s at the end of kind of a long chain of carbons. This makes it very flexible – and, if you recall, all these subunits of the pyruvate dehydrogenase complex are contained in this one large structure. And by attaching the acetyl group to the lipoamide, it keeps it tethered to the structure but allows it to move between the different sites on the enzyme.

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Flexible Linkages Allow Lipoamide to Move Between Different Active Sites

• The three enzymes of the pyruvate dehydrogenase complex are structurally integrated, and the lipoamide arm allows rapid movement of substrates and products from one active site of the complex to another.

• This increases the overall reaction rate and minimizes side reactions, making the coordinated catalysis of this complex reaction possible.

Figure 18.6 (Second Edition) A schematic representation of the pyruvate dehydrogenase complex. The transacetylase core (E2) is shown in red, the pyruvate dehydrogenase component (E1) in yellow, and the dihydrolipoyl dehydrogenase (E3) in green. The number and type of subunits of each enzyme is given parenthetically. Each red ball represents a trimer of three E2 subunits. Notice that each subunit consists of three domains: a lipoamide-binding domain, a small domain for interaction with E3, and a large transacetylase catalytic domain. The transacetylase domain has three subunits, with one subunit depicted in red and the other two in white in the ribbon representation.

So this is another look at the E2 enzymes there - and on the right you can see the structure starting big at the bottom, and then there’s a smaller domain, and then finally the lipoamide domain, and the lipoamide itself sticking out there on the top. So in this way the acetyl groups are attached to the complex.

This illustration will give you a little bit better idea of what these molecules/complexes look like. This is a large structure. I don’t believe this is human. But you can see the small blue domains all around the outside are the lipoamide domains and that pink part is the lipoamide. And so they’re able to flex and reach in various active sites on other parts of the complex.

So just once again, to review, the pyruvate initially reacts with the TPP on the outside of the complex. It then becomes attached to the E2 subunit and then gets swung over to the transacetylase domain of the E2 complex in the middle, and then gets attached to the coenzyme A. And then the lipoamide swings over to the E3 unit of the complex where the disulfide bond is restored and the hydrogens and electrons are captured by FADH2. That FADH2 is then oxidized, creating NADH, and that diffuses out to the electron transport chain. And the complex is then ready to start again.

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Diagram of the Reactions of the Pyruvate Dehydrogenase Complex

So as we’ve mentioned before, once you’ve taken pyruvate and attached it to acetyl CoA, you’ve released carbon dioxide and that makes it pretty much irreversible in animal cells. And from that point on, your acetyl CoA {or, rather, the acetyl group} can do two things - it can either be stored as fat, or it can be processed to make energy by the citric acid cycle.

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Section 18.2 The Pyruvate Dehydrogenase Is Regulated by Two Mechanisms

Learning objective 2: Identify the means by which the pyruvate dehydrogenase complex is regulated.

• The formation of acetyl CoA from pyruvate is irreversible in animal cells.

• Acetyl CoA has two principal fates: metabolism by the citric acid cycle or incorporation into fatty acids.

Figure 18.8 From glucose to acetyl CoA. The synthesis of acetyl CoA by the pyruvate dehydrogenase complex is a key irreversible step in the metabolism of glucose.

So starting with glucose, you can break it down to pyruvate. And then pyruvate could be made back into glucose following the gluconeogenesis route, or it could be processed in the mitochondria by the pyruvate dehydrogenase complex - and from there it can actually be oxidized completely to carbon dioxide, or stored as lipids, in which form it can be used to regenerate acetyl CoA and oxidized at a later point.

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Diagram of the Pathway from Glucose to Acetyl CoA

So the first step is the action of enzyme E1 and therefore that is a good site of regulation. If it is phosphorylated then it becomes inactive, but if that phosphate is removed then it becomes active again. That’s kind of backwards from the normal behavior of kinases and phosphatases but there’s exceptions to every rule, of course. Different parts of the pyruvate dehydrogenase complex can also be regulated by other elements in the chain such as ATP, Acetyl CoA, NADH, ADP, and pyruvate.

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Enzyme E1 • Enzyme E1 is a key site of regulation. A kinase associated

with the complex phosphorylates and inactivates E1. • A phosphatase, also associated with the complex,

removes the phosphate and thereby activates the enzyme. • The pyruvate dehydrogenase complex is also regulated by

energy charge. • ATP, acetyl CoA, and NADH inhibit the complex. • ADP and pyruvate stimulate the complex.

Figure 18.9 The regulation of the pyruvate dehydrogenase complex. Pyruvate dehydrogenase (PDH) kinase phosphorylates and inactivates PDH, and PDH phosphatase activates the dehydrogenase by removing the phosphoryl group. The kinase and the phosphatase are highly regulated enzymes.

Well, this is just a diagram showing how an active pyruvate dehydrogenase can be turned into an inactive pyruvate dehydrogenase by adding a phosphate group from a hydrolysis of ATP. So a kinase will add a phosphate group, and then it becomes inactive. Phosphatase can remove that phosphate group and make it active again. And of course, the activities of these kinases and phosphatases are regulated by other parts of the cell.

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Diagram of the Regulation of the Pyruvate Dehydrogenase Complex

Figure 18.10 Response of the pyruvate dehydrogenase complex to the energy charge. The pyruvate dehydrogenase (PDH) complex is regulated to respond to the energy charge of the cell. (A) The complex is inhibited by its immediate products, NADH and acetyl CoA, as well as by the ultimate product of cellular respiration, ATP. (B) The complex is activated by pyruvate and ADP, which inhibit the kinase that phosphorylates PDH.

And very similarly to glycolysis, the products of the process will inhibit the process itself. So if you already have a lot of the product, then you don’t need to do the process so much. NADH, acetyl CoA, and ATP all say “Hey there is enough of this stuff already so you can cool it for a bit” - whereas, if you have ADP, which shows lower levels of energy, then it will enhance the process because you need the process more. Also an excess of pyruvate will enhance the process. At the bottom you see CAC, that would be the citric acid cycle, which is the process by which acetyl CoA is broken down finally to carbon dioxide and produces ATP.

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Diagram of the Response of the Pyruvate Dehydrogenase Complex to the

Energy Charge

So what are some of the advantages of organizing the enzymes that catalyze the formation of acetyl CoA into a single large complex? Think about it for a sec, you can pause... The answer is that you’ve got your acetyl group tethered to the complex, and all of the elements of the complex are close together, so the acetyl group can visit them all very rapidly. They won’t diffuse away, and the separate enzymatic reactions can take place one after another very, very easily.

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Quick Quiz

QUICK QUIZ List some of the advantages of organizing into a single large complex the enzymes that catalyze the formation of acetyl CoA from pyruvate.

Now the first step of the pyruvate dehydrogenase complex is to attach the pyruvate to the prosthetic group that is derived from thiamine. So we can’t make thiamine in our bodies, and therefore if we don’t eat enough of it (it’s a vitamin), then the pyruvate dehydrogenase complex cannot process the pyruvate - and that leads to pathology like neuromuscular disease, such as beriberi. And thiamine is found in brown but not white rice, so that’s a good reason to eat brown rice, is to get your thiamine. People who depend entirely on white rice run the risk of getting beriberi.

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Clinical Insight: The Disruption of Pyruvate Metabolism Is the Cause of Beriberi (1/3)

CLINICAL INSIGHT The Disruption of Pyruvate Metabolism Is the Cause of Beriberi

• Thiamine deficiency results in insufficient pyruvate dehydrogenase activity.

• Insufficient pyruvate dehydrogenase activity causes neuromuscular pathologies such as beriberi.

• The vitamin thiamine is found in brown rice but not in white (polished) rice.

Furthermore, the complex can be inhibited by mercury and arsenite, both of which (I think both of which) bind the two sulfurs that are on the dihydrolipoamide that are responsible for shuttling the acetyl group around the complex. And a long time ago, when the hat makers that would make hats made of felt, they would use this mercury to make the felt and it would get into their body and cause some mental problems - and that’s where they got the term “mad hatter” from. So that’s the mechanism by which mercury causes problems, is by interfering with that dihydrolipoamide.

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Clinical Insight: The Disruption of Pyruvate Metabolism Is the Cause of Beriberi (3/3)

CLINICAL INSIGHT The Disruption of Pyruvate Metabolism Is the Cause of Beriberi

• Pyruvate dehydrogenase complex activity can be inhibited by mercury and arsenite, which bind to the two sulfurs of dihydrolipoamide.

• 2,3-Dimercaptopropanol can counter the effects of arsenite poisoning by forming a complex with the arsenite that can be excreted.

• Early hat-makers (“hatters”) used mercury to make felt, which inhibited complex activity in the brain, often leading to strange behavior. This is the origin of the phrase “mad as a hatter”.

Figure 18.13 Mad Hatter. The Mad Hatter is one of the characters that Alice meets at a tea party in her journey through Wonderland. Real hatters worked with mercury, which inhibited an enzyme responsible for providing the brain with energy. The lack of energy would lead to peculiar behavior, often described as “mad.” [The Granger Collection.]

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Illustration of the Mad Hatter