Biochemistry Citric Acid Cycle III

Hello and welcome back to Educator.com, and welcome back to Biochemistry.0000

Today, we are going to continue our discussion of the citric acid cycle.0004

Excuse me; in the last lesson, we talked about the first 4 reactions of the citric acid cycle.0008

Today, we are going to talk about the last 4, close out the cycle, and then say some final words about...well, fill in some of the gaps about what is going on.0012

Let's just jump right on in with reaction no. 5.0021

We left it off at reaction no. 4 with the formation of the succinyl-CoA.0025

Now, we are going to go ahead and start with reaction no. 5.0030

Let me go ahead and do this one; let's go ahead and just start with blue today, why not?0034

Reaction no. 5, this is going to be the conversion of the succinyl-CoA to succinate, and the reaction looks like this.0039

We have 1, 2, 3, and we have our 4, COO^-.0057

We have a CH₂, CH₂, and I will go ahead and put the carbonyl here, S-CoA.0067

And this one in walls, this is the reaction where we actually form our guanosine triphosphate or our adenosine triphosphate.0079

I will just go ahead and write is as the GDP + PI that actually comes in, and what leaves is the GTP; and another thing that actually ends up leaving is the coenzyme A.0090

OK, and what you end up with is the following molecule.0108

You have C, C, C and C.0112

Now, we have our symmetric molecule; now, it is at this point where we can no longer tell which one of these carboxyl groups actually is the one that came from the original acetyl-CoA.0117

At this point, we cannot tell anymore.0130

OK, let's take a look.0134

Let me go ahead and write what they are; this is succinyl-CoA, and this is our succinate.0141

OK, let me go back to blue.0152

Now, the hydrolysis, if you remember from the bioenergetics chapter, a thioester, the carbonyl and this bond right here between the carbon and the sulfur, it is a very high-energy bond.0158

So, when you break that bond, there is a lot of energy that is released.0172

It is the energy released upon breaking that bond that has enough to actually form GTP, which in and of itself, is a highly endergonic reaction.0179

Again, we are attaching an endergonic reaction to a highly exergonic reaction using that energy to form the triphosphate.0187

OK, the hydrolysis of the high-energy thioester bond of succinyl-CoA drives the synthesis of GTP, and I will just put ATP in parentheses.0197

You know what, no, let me just go ahead and leave it as GTP.0236

We will talk about the ATP in just a little bit; here is the reaction sequence.0240

OK, we have our C, C, C, C.0251

This is OO^-; this is H₂.0257

This is H₂; we have our carbonyl.0261

We have our S-CoA, and this is, of course, attached to the particular enzyme.0265

OK, and this enzyme...what ends up coming in and leaving...inorganic phosphate ends up coming in, and the CoA enzyme actually ends up leaving.0278

This inorganic phosphate ends up phosphorylating this thing.0294

They just switched places, so what you end up with is the following molecule.0299

You end up with C, C, C and C.0303

This is OO^-; this is H₂.0307

This is H₂, and what you end up with is O and PO₃^2-.0310

This CoA group is replaced by this phosphoryl group.0317

Yes, that is exactly right, and it is still attached to the enzyme; and let me go ahead and put that there.0324

Now, at this point, we will rewrite that.0335

We have C, C, C, C, COO^-, H₂, H₂.0343

We have the carbonyl; we have the S-CoA, and we have its attachment to the enzyme and a histidine residue on the enzyme.0350

What ends up happening is...what you end up with is actually not the CoA.0363

Sorry about that; we just phosphorylated that.0369

We actually have an O, and we have a PO₃^2-.0375

That is what we have; what we end up getting is we actually end up transferring this phosphoryl group to the histidine residue, and we actually end up spitting out what is left over.0381

What you end up spitting out is the molecule COO^-, H₂, H₂ and COO^-.0391

Though this is our succinate molecule, this is what is produced.0404

What you end up with is an enzyme attached to a histidine residue that has a phosphoryl group attached to it.0410

Now, at this point, here is where the GDP comes in, and it actually takes this to become GTP reforming the enzyme histidine, which can, now, continue on doing what it does.0420

That is it; that is all that happens.0442

Now, the GTP that is formed, actually, the GTP that is formed, it can react as follows.0445

GTP + ADP, they can actually switch the phosphoryls to form ATP + GDP, and the enzyme that catalyzes this is called nucleoside diphosphate kinase.0452

Here is our nucleoside diphosphate kinase; we are attaching a phosphoryl group to it to produce the ATP.0481

So, even if it produces ATP, one of the isozymes actually produces ATP directly instead of GTP, well, that is fine.0487

It can do it directly, but if it produces the GTP, well, a GTP ends up reacting with the ADP to ultimately form adenosine triphosphate.0494

The ultimate effect is you are still forming adenosine triphosphate.0503

OK, now, we are almost there, so reaction no. 6.0508

Let's go ahead and go back to black here, so reaction no. 6: oxidation of succinate to fumarate.0512

We have got C, C, C, C.0536

We have that, and this time, I am going to actually write the individual Hs so that we see it, OO^-; and what you end up with is...OK.0543

Here is where FAD actually comes in, and what leaves is the FADH₂.0557

This is an oxidation by flavin adenine dinucleotide.0563

This is succinate dehydrogenase.0569

It is going to end up pulling off a couple of these hydrogens here- this one and this one or this one and this one, depending on how you look at it - and what you end up with is this very, very interesting molecule, double bond, and it is actually a trans, not a cis.0574

You remember alkene nomenclature - trans - the main groups are opposite each other.0594

This is the fumarate, and this is the succinate.0600

We pulled off a couple of these hydrogens - either this one and this one, or this one and this one, or this one and this one, it does not matter how you look at it - to produce this trans molecule, which is fumarate.0606

OK, now, reaction no. 7: fumarate to malate.0619

Now, what happens is the following; you take this one and you add water.0632

I will do it as HOH, just so you can see what actually happens; this is catalyzed by fumarase, and what you end up with is the following: C, C.0652

I will go ahead and write is this way, and I will just put H here and H here.0678

Because there are 2 Hs attached to the same carbon, there is no chirality at that molecule, so I am not going to actually represent any of it with the dashes and wedges.0683

What you end up forming actually here is the L-malate from the fumarate, and here is what it actually passes through.0691

What actually ends up happening is the following; it passes through, apparently, a carbanion intermediate.0703

So, what you have is C; you have the C, the OH, the H, the COO^-.0711

There is a minus charge there, COO^- and H.0722

Apparently, what happens is that the hydroxy actually comes in, and it attacks this carbon pushing these electrons on the carbon to form a carbanion - a carbon that is actually carrying a negative charge - and at that point, what you have is an H⁺ come in.0726

This will actually grab an H⁺ from some source, and then, it produces the L-malate.0743

Apparently, it does pass through this carbanion intermediate, but that is it- OK, nice and straightforward.0750

Now, notice something about this, this is the trans isomer.0757

Fumarase is highly stereo-specific.0760

There is another isomer, this; there is this cis isomer, right?0763

There is this one.0767

Fumarase will not take that and convert it to L-malate.0775

It is highly stereo-specific; that is what enzymes are.0780

They are very, very, very specific about what they want and what they will react with.0783

Fumarase is highly stereo-specific meaning it only will deal with a specific stereo isomer of the molecule, and only catalyzes the conversion of trans isomer, which happens to be the - we are going from...yes - fumarate, the trans isomer.0787

Yes, not the cis isomer, which is actually called maleate, in case you wanted to know.0840

OK, reaction no. 8: this is going to be the oxidation of malate to oxaloacetate.0856

This is the final reaction, the oxidation of L-malate to oxaloacetate to begin the cycle again.0865

We have got...let's see, C, C, C, C.0879

This is OO^-; this is H₂.0886

Let's go ahead and put the OH here; let's go ahead and put the H here.0891

Let's go there; OK, and again, the oxidation here is the electron carriers NAD⁺ releasing NADH, and this is catalyzed by malate dehydrogenase, and by now, we are very, very, very familiar with what dehydrogenases do.0896

They take hydrogens away across bonds, so we are left with C, C, C and C.0919

That is OO^-; that is H₂.0927

This is that, and this is that.0932

This is the L-malate.0936

This is our oxaloacetate, final.0942

This is not an infinity sign; this is a carboxyl group.0948

OK, the ΔG for this particular reaction equals 29.7kJ/mol.0955

How is it that I have this highly endergonic reaction, and yet I have this reversible condition here?0963

How is it that this reaction even actually moves forward?0969

That does not make sense; well, here is how it makes sense.0972

Under physiological conditions, the product, which is the oxaloacetate, is removed by reaction 1 of the citric acid cycle, right?0977

Oxaloacetate now condenses with acetyl-CoA; that is a highly exergonic reaction.1003

Everytime the oxaloacetate is actually formed in this circumstance, the oxaloacetate is pulled forward.1008

It is used; it is pushed forward in this citric acid cycle leaving an emptiness here.1015

Le Chatelier’s principle pushes this reaction forward, even though, thermodynamically, it might be unfavorable.1021

Under cellular conditions, it is favorable- highly favorable, in fact.1027

Reaction 1 of the cycle, that is what is going on there.1030

OK, now, let's talk a little bit about all the energy from all of these oxidations.1040

We have all of this oxidation taking place.1047

We had glycolysis that ends up producing some NADH.1050

We had the conversion of the pyruvate to the acetyl-CoA.1054

We had the citric acid cycle; we produced a whole bunch of NADH.1062

We produced FADH₂; what is happening with all of these?1064

Where is all of this energy going?1069

Let's do a little bit of an energy accounting and see what we can find out.1071

OK, let me go ahead and do this one in red.1075

What happens to the energy?1085

You know, I do not think I want to do this in red.1088

I think I want to do this in black.1093

Our question is "what happens to the energy form all these oxidations?".1098

Well, here is what happens.1116

NADH gives its electrons to the electron transport chain to form 2.5 ATP molecules per 2 electrons because NAD⁺, it pulls 2 electrons.1121

It is a hydride; the NADH, when it delivers its electrons, it is delivering 2 electrons.1147

For every 2 electrons that it delivers into the transport chain, oxidative phosphorylation, the final step, actually produces 2 1/2 ATP molecules.1151

4 electrons produces 5 ATP molecules.1160

OK, now, the FADH₂, it produces 1.5 ATP per 2 electrons.1165

Now, let's start counting electrons and seeing what we have.1180

Now, we are going to count the amount of ATP that we produce in 1 turn through the entire pathway - glycolysis, citric acid cycle, electron transport chain - in other words, the complete conversion of 1 molecule of glucose to CO₂ and water.1187

Oh, where is all this energy going?1206

From glycolysis to pyruvate to acetyl - well, from glucose, not glycolysis - from glucose to pyruvate to acetyl-CoA through the citric acid cycle to the electron transport chain, here is the accounting.1210

OK, the reaction of glucose to glucose 6-phosphate, we used up 1 ATP.1247

Remember, in glycolysis, 2 molecules of ATP were used up.1261

4 were produced for a net gain of 2.1266

So, we need to account for every ATP; that is what we are doing here.1270

1 ATP, I will write 1 ATP here.1273

Now, the fructose 6-phosphate to the fructose 1,6-biphosphate also uses up 1 ATP.1277

We are going to lose another ATP; again, we are just keeping a track of ATPs.1290

Now, the glyceraldehyde-3-phosphate reaction, upon its conversion in glycolysis to 1,3-biphosphoglycerate, remember, 1 molecule of glucose produces 2 molecules of the glyceraldehyde-3-phosphate, so what we end up with is, it ends up producing 2 NADHs.1296

OK, well, we said that 1 NADH actually produces 2.5 ATP per 2 electrons.1319

In this particular case, I am going to write, it is going to be either 5 ATP or +3 ATP, and I will explain the difference between these 2 in just a minute after I have actually done the accounting.1328

Now, the conversion of the 1,3-biphosphoglycerate to the 3-phosphoglycerate, that actually ends up producing 2 ATPs.1343

So, we get to add 2 ATPs.1357

It produces the ATP directly from substrate level phosphorylation.1364

OK, now, the reaction of phosphoenolpyruvate to pyruvate in glycolysis also produces 2 ATPs - each reaction, right - because we have 2 molecules of the glyceraldehyde-3-phosphate, 2 molecules of the 1,3-biphosphoglycerate, 2 molecules of the PEP.1370

Each molecule produces an ATP, so PEP to pyruvate - the overall reaction - were producing 2 ATPs, so let's add 2 ATPs to our list.1391

Now, the pyruvate to the acetyl-CoA, this one produces 2 NADHs.1400

Well, 2 NADHs produces 5 ATPs.1412

Now, we had our isocitrate reaction going to alpha-ketoglutarate.1418

That one also produced 2 NADHs.1430

That is going to produce another 5 ATPs down the line during oxidative phosphorylation.1434

OK, now, we had our alpha-ketoglutarate reaction that goes to succinyl-CoA.1444

That produces also 2 NADHs, so that is another 5 ATP molecules produced in the electron transport chain.1454

OK, I hope I have enough room here; I should have probably written a little smaller.1466

Also, let's see; we have our succinyl-CoA reaction going to succinate reaction.1470

That one actually ended up producing 2 ATPs directly- 2 GDPs, 2 ATPs.1477

That is another 2 adenosine triphosphates, right?1490

Because again, the pyruvate...1 glucose produces 2 pyruvate.1493

1 pyruvate produces 1 acetyl-CoA, so 2 pyruvates produce 2 acetyl-CoAs.1498

That is why you get the 2 ATP; that is why have all these 2, 2, 2s here.1502

I just wanted to make sure that that was clear, now, succinate to fumarate.1507

That produces 2 of the FADH₂.1515

Well, 1 FADH₂ or 2 electrons produces 1.5 ATP, so we have a net gain of 3 ATP for that one.1519

Now, our last one, malate to oxaloacetate that also produces 2 molecules of NADH, which accounts for 5 ATPs.1527

When I add all of the positive ATPs and negative ATPs that we used up in glycolysis, when I add all of these, my grand total is going to be - I will put it over here - 30 or 32 ATP total.1544

One pass through the entire oxidation from glycolysis, through the citric acid cycle, through the electron transport chain, actually produces a total of 30 or 32 ATP.1563

The 30 or 32 comes from that +3 or +5 that I am going to talk about in just a second.1578

That is extraordinary; 1 sugar molecule, 1 glucose molecule, 1 monomer, ends up having enough energy to produce 30 - let's just say 30, let's take the lower number - molecules of adenosine triphosphate.1584

That is a hell of a lot of energy; that should give you an idea of the amount of energy it actually takes to run the human body.1598

It is extraordinary; it is inordinate.1605

OK, now, let's go ahead and talk about this 3,5.1608

How is it that all of a sudden the 2 NADHs...why is it that we said that 1 NADH produces 2.5 adenosine triphosphates?1613

So, that is 2 NADHs produces 5 adenosine triphosphates.1621

Where is the 3 coming from; where is the little ambivalence there?1625

Here is where it comes from; we saw that the glyceraldehyde-3-phosphate to the 1,3-biphosphoglycerate reaction, that produces NADH.1629

OK, well, the NADH takes its electrons, and it gives it over to the electron transport chain.1654

The problem is, this particular reaction of glycolysis - excuse me - it takes place in the cytosol, the electron - excuse me - transport chain that takes place in the mitochondria.1660

Somehow, the cell has to find a way to get this NADH, which is outside of the mitochondria inside the mitochondria, so that it can actually deliver its electrons.1673

Well, there are 2 ways that it can do that.1682

Let's write this out, but this NADH is produced in the cytosol because the glycolysis reaction takes place in the cytosol.1688

How can we transfer this NADH into the mitochondrion, so that it can actually deliver its electrons?1712

OK, well, there are actually 2 possible ways that the cell does this.1736

There are 2 shuttle systems, a way of taking this NADH from outside the mitochondrion to inside the mitochondrion, so it can deliver its electrons.1741

The answer is: there are 2 shuttle systems exist to accomplish this.1750

Now, I am not going to go ahead and go through the shuttle systems now.1771

Later, when we talk about oxidative phosphorylation directly, we will actually draw out the shuttle system, but I just want to name them here, so that you understand, so that they are available to you, so that you know what is happening here.1777

The first one is called the malate aspartate shuttle.1790

This particular one actually produces...when the malate aspartate shuttle brings the NADH from the cytosol to the mitochondrion, this particular process actually produces the 2.5 ATP per 2 electrons, which ultimately yields 5 adenosine triphosphates.1801

The 5 comes from the transfer via the malate aspartate shuttle.1828

OK, the liver, the kidney and the heart muscles, the heart cell, the liver, kidney and the heart- that uses primarily the malate aspartate shuttle.1833

The other one is called the glycerol 3-phosphate shuttle, and this particular one, when the body uses this one, it produces the 1.5 ATP per 2 electrons; and this is where the 3 ATP comes from.1850

Now, you know where those 3 and 5 come from.1879

In liver, heart and kidney, it is going to end up producing the 32 ATP per glucose molecule.1883

In other parts of the body, where it uses the glycerol 3-phosphate shuttle to actually bring it in from the cytosol to the mitochondrion, it is going to end up producing 30 ATP per glucose molecule.1889

OK, brain uses the glycerol 3-phosphate shuttle and skeletal muscle.1901

OK, now, let's talk a little bit more...final words on the citric acid cycle up here, let's see what we can come up with.1912

Let me go back to black here; for aerobic organisms, in other words, organisms that require O₂, the citric cycle is something called amphibolic, and basically, that just means it works in both directions.1928

It works in the anabolic pathways, and it works in the catabolic pathways.1956

So, it is a very, very central hub.1961

Basically, it just means it works in both anabolic and catabolic pathways.1966

What we have seen is a catabolic pathway, the breakdown of glucose.1979

OK, now, the intermediates of the citric acid cycle - in other words, all of these molecules, the citrate, the isocitrate, the alpha-ketoglutarate, the succinyl-CoA - are used as precursors for many biosynthetic processes.1988

What happens is that as this citric acid cycle is proceeding, let's say somewhere along the way, it is going to produce the alpha-ketoglutarate, well, the body will take that alpha-ketoglutarate.2032

It will siphon it off, so instead of it actually continuing on the cycle, the body will take some of it to go do what it does for some biosynthetic processes.2045

So, many of the intermediates of the cycle are actually pulled away from the cycle for anabolic pathways.2053

The citric acid cycle is very, very central.2059

It participates in catabolic pathways, as well as anabolic pathways - OK - for many biosynthetic processes.2064

Let's list some of these processes.2071

The alpha-ketoglutarate is one of the intermediates that is actually pulled away, and it can be used to produce glutamate; and glutamate can be used to form some amino acids - glutamine, proline, arginine - and it can also be used to form some of the purines.2075

OK, oxaloacetate is a very, very, very important biosynthetic precursor, and it is used to actually synthesize aspartate, which can go on to form the amino acids asparagine and the pyrimidines.2117

OK, oxaloacetate again, it can actually go on to form PEP, which can go on to form glucose if necessary; or the PEP can be used to form things like serine, glycine, cysteine and a host of other amino acids.2142

Succinyl-CoA is another one of the intermediates that can actually be siphoned off from the citric acid cycle for some biosynthetic processes- very, very important.2170

It is used to actually form the phorphorines and heme for hemoglobin and myoglobin- very, very important.2183

OK, now, here is the issue.2192

As these intermediates are siphoned off, are pulled out of the cycle to be used for these particular biosynthetic processes, whatever they happen to be, they have to be replenished somehow.2197

As these intermediates are pulled out of the cycle, they must somehow be replenished in order for the cycle to continue, otherwise, if it comes to a stop, bad things happen.2220

They must be replenished, and the body has ways of replenishing.2232

They must be replenished, so the cycle can continue.2237

Well, these reactions that actually replenish things that are taken away from a particular biosynthetic pathway, they are called anaplerotic reactions.2247

Let's go ahead and write this in blue.2259

The anaplerotic reactions, that is what they do.2264

The anaplerotic reactions, they actually replace what has been taken away.2272

OK, let me go ahead and draw a little diagram here - just like before - of the basic anaplerotic reactions.2276

We are going to start off with pyruvate to acetyl-CoA.2284

OK, what I am drawing here is the citric acid cycle version, so one of the reactions and one of the reactions.2291

One of the things that we form is oxaloacetate, of course, and another one of the intermediates is malate.2301

Now, let me go ahead and go oxaloacetate and PEP.2311

One of the reactions that actually takes place, one of the anaplerotic reactions, as oxaloacetate is pulled out of the cycle, is this one, reaction no. 1, which actually takes acetyl-CoA, and it converts it to oxaloacetate.2317

As the oxaloacetate concentration drops or is pulled away, acetyl-CoA, instead of going directly into the citric acid cycle, it directly forms the oxaloacetate.2336

OK, let me write it over here.2347

This is catalyzed by a pyruvate carboxylase reaction, pyruvate carboxylase enzyme.2352

That is the one that actually catalyzes reaction no. 1.2362

OK, again, we do not have to replace these other intermediates like the succinyl-CoA or the alpha-ketoglutarate directly.2366

We just need to make sure that the cycle does not stop.2374

So, by producing oxaloacetate, the cycle will continue and produce the alpha-ketoglutarate, the succinyl-CoA, the malate, the fumarate- whatever is necessary.2378

These are very, very important reactions, in fact, the first one is probably the most important that you should know that acetyl-CoA converted to the oxaloacetate via pyruvate carboxylase.2387

OK, this one is reaction no. 2; that is another one of the anaplerotic reactions, and this is phosphoenolpyruvate carboxykinase.2399

I need to write PEP carboxykinase.2415

Another reaction is no. 3.2425

It takes phosphoenolpyruvate, another enzyme.2429

It uses PEP under another set of reactions, again, to produce oxaloacetate.2432

It has more than one way of producing the oxaloacetate.2438

This is the PEP carboxylase reaction, and no. 4: free pyruvate.2443

This is reaction 4: malic enzyme.2458

This thing called malic enzyme will actually take free pyruvate and convert it directly to malate, so that it will actually go on and keep the citric acid cycle going.2463

OK, now, let's see; pyruvate...no wait, I am sorry.2473

A little bit of a mistake here, sorry; I have my arrows mixed up.2485

All these names are making me a little...it is not acetyl-CoA to oxaloacetate.2489

It is pyruvate to oxaloacetate because it is the pyruvate carboxylase.2492

Yes, that is one of our first bypass reactions from gluconeogenesis, if you remember right.2498

OK, reaction 1 - OK - is an important one to know.2502

I think if there is any reaction that your teacher may actually ask you to know, as far as the anaplerotics, it is going to be this one- the conversion of pyruvate directly to oxaloacetate.2521

Reaction 1 is an important one to know and to recall.2530

Recall, it is the first bypass reaction of gluconeogenesis that we talked about a couple of lessons ago.2538

We said that pyruvate + bicarbonate + adenosine triphosphate, under the action of the coenzyme biotin, which is a coenzyme for this pyruvate carboxylase, actually produces our oxaloacetate.2556

Remember, in the second bypass reaction was the oxaloacetate to phosphoenolpyruvate.2588

So, this anaplerotic reaction actually takes the free pyruvate, converts it to oxaloacetate, to replenish all of the intermediates that have been pulled away from the citric acid cycle.2593

This is the reaction: pyruvate + bicarbonate + ATP under the action of pyruvate carboxylase via the biotin - remember that biotin, that arm that swings over - to oxaloacetate.2604

If you want to take a look at the mechanism of this reaction, we talked about it back when we talked about it in gluconeogenesis.2617

OK, now, there you have it.2625

These 4 reactions, the primary ones that you probably want to concern yourself with.2631

More than any other, this is probably the one that you want to know because it happens to be the first bypass reaction of gluconeogenesis.2636

With that, we will go ahead and close out our discussion of the citric acid cycle.2644

Thank you so much for joining us here at Educator.com; we will see you next time.2647

Take care.2651