Biochemistry Citric Acid Cycle I

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

We just finished discussing glycolysis, gluconeogenesis and the pentose phosphate pathway.0004

Now, we are going to continue on with our metabolic pathways, and we are going to start discussing the citric acid cycle.0010

Let's just jump right on in.0020

OK, I am going to go ahead and do just a quick overview, again, of the stages of cellular respiration, so that we have an idea of where it is that we are in the big picture; and then, we will actually start to get into the citric acid cycle and the conversion of the pyruvate that was formed during glycolysis into acetyl-CoA, and then, of course, in the next lesson, we will go ahead and start the citric acid cycle.0024

OK, the stages of cellular respiration, let's see, and what we mean by that is just the conversion of glycolysis to pyruvate ultimately to acetyl-CoA , the citric acid cycle all the way down; and we produce the CO₂, and we reduce oxygen via all those electrons that we took away from all of these oxidative steps.0045

Let's see here; we started with glucose.0080

That was our first step; from glucose, the glycolysis, we went to pyruvate.0083

We have already taken care of this first step; that was the glycolysis, the pyruvate, acetyl coenzyme A.0091

Now, from here, the acetyl-CoA actually enters the citric acid cycle.0103

OK, and over here, later on, when we actually discuss fatty acid catabolism and amino acid catabolism, the breakdown of those things, they also end up funneling into acetyl-CoA and entering the citric acid cycle.0115

So, I will just go ahead and draw that this way, OK, something like that.0132

OK, now, let me go ahead and put over here NADH and FADH₂.0141

I will go ahead and put a little rectangle around that, and then, over here, I will go ahead and put the electron transport chain.0151

The final step in cellular respiration, what oxidative phosphorylation...it is going to be our final culminating step of this thing called cellular respiration- the ultimate breakdown of the things that we ingest, whether it is fatty acids, amino acids and carbohydrates, so, amino acids, electron transport chain, and let me go ahead and put a little square around that.0165

Here, in the electron transport chain, what happens is this thing called oxidative phosphorylation, different from what we talked about earlier when we talked about glycolysis...remember we talked about the formation of ATP?0193

That was substrate level phosphorylation; this is where most of the ATP that is produced in the body is produced.0207

It is called oxidative phosphorylation, so ADP + PI is converted into ATP.0214

That is one of the major products; all of this is designed to produce ATP, so that ATP can run the body because ATP is the energy currency.0220

The other thing that happens is that oxygen gas is actually reduced to water, so H₂O, right, oxygen?0230

Normally, it has an oxidation state of 0; H₂O has an oxidation state of -2.0241

All those electrons that we took away from the breakdown of all these things - the carbohydrate, the fatty acid, the amino acids - they end up coming and reducing oxygen.0246

That is what they do, and in the process, of course, during these processes, CO₂ is also released.0254

Now, let's go ahead and just follow the electron path here.0260

I am going to do the electron path in red.0264

We have got some electrons from the citric acid cycle in the form of the NADH and the FADH₂.0268

In these cycles, in the glucose to pyruvate, pyruvate to acetyl-CoA and all the citric acid cycles, reactions that we are going to talk about, NADH is produced.0277

FADH₂ is produced; all those things come here, and they actually release their electrons.0287

Once they collect all of the electrons from the oxidations that they have done, they take their electrons, and they give them over to the electron transport chain; and that is what takes care of this.0292

Let's just follow the flow of electrons; remember, we had some NADH formed here?0303

Some electrons come here, and then, from here, there are going to be some electrons that come over here.0309

From here, there are going to be some electrons that come here; from here, there are going to be electrons that come here.0315

So, that is what these NAD and FAD coenzymes do; they are electron carriers.0320

They hold all of the electrons by oxidizing all of these things, and then, they dump their electrons onto the electron transport chain; so that those high-energy electrons can do work.0324

The work that they do, is they actually produce ATP, and they reduce oxygen to water.0334

That is what the body does; OK, this is the general scheme of what we are doing.0340

What we have done is we have taken care of glycolysis.0346

Today, what I am going to talk about in this lesson is we are going to discuss the conversion of the pyruvate that was formed during glycolysis.0350

We are going to talk about its transformation into acetyl-CoA by an absolutely extraordinary enzyme.0357

Really, it is truly fascinating what this does; it is unbelievable is what it does.0365

We are going to talk about that conversion of pyruvate to acetyl-CoA , and then, in the next lesson, we are going to start discussing the movement of acetyl-CoA into the citric acid cycle; and then, later on, in subsequent lessons, we are going to discuss the breakdown of fatty acids.0371

We are going to discuss the breakdown of the amino acids, and we will have covered this part right here.0387

And then, later, when we get to oxidative phosphorylation after that, we will discuss this part right here - the transfer of the electrons onto the electron transport chain - and then, we will have completed our basic catabolic movement.0394

Again, we did the glucose to pyruvate; today, we are going to do pyruvate to acetyl-CoA, so let's get started.0408

OK, now, from pyruvate to acetyl-CoA, let's go ahead and go to black here.0416

From pyruvate to acetyl-CoA- I will just write it as this for now, and then later, I will add the little S when we talk about the coenzyme A.0423

OK, pyruvate that is formed in glycolysis is oxidized to acetyl-CoA - and do not worry.0441

We will be drawing out all these structures in just a minute - in the mitochondrion.0455

This definitely takes place in the mitochondrion, not in the cytosol, by an amazing - and I do mean amazing - collection of enzymes - it is an aggregation of enzymes - called the pyruvate dehydrogenase complex.0462

I am telling you, when you see what this does, it is extraordinary.0492

And all this means is that it is a collection of enzymes that are aggregated together.0501

They are stuck together; they are not far away from each other to make sure that...because the product of one enzyme becomes the substrate to the next enzyme, and the product of that one becomes the substrate to the next enzyme.0506

They are all collected in one, sort of, huge enzyme complex.0516

It is called the pyruvate dehydrogenase complex, and the overall reaction that it catalyzes is the following.0520

The overall reaction goes as follows.0528

We have a molecule of pyruvate, and let's go ahead and draw the carbonyl vertically.0533

We have that, and we have CH₃; and it is converted to C, O, CH₃ - yes - S, CoA, and this S is a sulfur attached to the enzyme, the coenzyme A.0540

This is the ultimate conversion; pyruvate is converted to CoA, and here is how it happens.0566

Let's have something coming in, so what comes in is the acetyl-CoA; and I will write SH.0572

What also comes in and leaves is NAD⁺.0586

OK, there is going to be a dehydrogenation; there is going to be an oxidation - that is what this is - and it is going to release NADH + H⁺, and another thing that actually ends up leaving is CO₂.0590

Coenzyme A comes in; NAD⁺ comes in.0603

NADH leaves; CO₂ leaves.0607

The enzyme is the pyruvate; yes, I am just going to write PDH, so when we talk about...I am not going to keep writing it out.0614

We will just say PDH- pyruvate dehydrogenase.0621

We should say PDC, but it is PDH- pyruvate dehydrogenase.0626

The PDH or if you want to say PDHC, that is fine too; it does not really matter.0632

Now, in parentheses, I will write E1 + E2 + E3 because this is made up of a collection of 3 enzymes, multiple copies of each, so it is actually pretty extraordinary.0638

I will write multiple copies of each, and I will be talking about it in just a second; and we end up producing this.0651

This is our pyruvate molecule, and it is converted into acetyl-CoA, acetyl-S-CoA.0663

You are going to write acetyl-CoA, acetyl-S-CoA.0673

Some people write the S, some people do not; I am probably going to do both.0677

You will see why in just a minute; OK, the ΔG for this reaction...let's just go ahead and write the ΔG- -33.4kJ/mol.0679

This is irreversible; this is highly exergonic.0693

OK, this is the overall reaction that this pyruvate dehydrogenase complex actually catalyzes- the conversion of pyruvate to acetyl-CoA, so that it can enter the citric acid cycle.0697

Coenzyme A is used, NAD⁺.0711

Now, let's actually get into it; OK, this reaction is called oxidative decarboxylation, and is a decarboxylation - no, better learn how to spell here, wow, they really get into here - because CO₂ is removed.0715

This CO₂ group right here is lost; this CO₂ group is lost.0758

Decarboxylation, I am taking the C because CO₂ is removed or lost.0764

OK, oxidative because carbons' oxidation state actually changes, because carbons' oxidation state goes up.0771

You are normally decarboxylation and taking that something away from this carbon.0788

You are actually oxidating it some more, and here is how that looks.0792

I will describe this oxidation part; decarboxylation part is pretty clear.0798

It is the oxidation part that throws some kids off, so here is what is going on.0802

Let's take a look at pyruvate, so C, C, C.0806

That is that, and we have that; and we have CH₃.0811

The oxidation state of this carbon right here - OK - is...well, you have one carbon attached to it, another carbon attached to it, so that is 0 + 0, and you have a double bond to oxygen, so it is +1, +1, so it carries an oxidation state of +2.0816

Now, when you form the acetyl-CoA, you have that, and you have the S.0833

You have the CoA, and you have the CH₃.0838

Well, now, you have 1 carbon, which is 0; you have 2 oxygens - +1, +1 - to double bond, and then, you have sulfur.0842

Well, sulfur is more electronegative than carbon, so it is also considered...it is oxidizing the carbon.0849

It is taken away because sulfur and oxygen, they are in the same group.0854

They do the same thing, and they oxidize.0858

Now, it is carrying an oxidation state of +3.0861

That is what we mean when we say oxidative decarboxylation.0864

You are going to see that a lot in biochemistry; oxidative decarboxylation is exactly what it sounds like.0867

CO₂ has been removed, and some carbon has been oxidized.0874

Its oxidation state is changed; it does not necessarily mean that oxygen has been attached to it- it could be.0878

It could be just another electronegative element; in this case, it is sulfur.0883

Maybe it is nitrogen; we will see some of that later too.0889

Now, let's go back.0893

The NADH formed, as we said - is ultimately, well, not "is ultimately" - it ultimately gives up its electrons to the electron transport chain like all of the other NADH and NADPH and the FADH and FADH₂.0897

OK, now, let's talk about PDH; let's do this in red.0930

OK, the pyruvate dehydrogenase is a conglomeration of multiple copies of 3 enzymes.0938

The first enzyme we will call E1; it is called pyruvate dehydrogenase.0966

The second enzyme is called dihydrolipoyl transacetylase, wow.0979

OK, and E3 is dihydrolipoyl dehydrogenase.0996

How do we ever keep any of these things straight?1010

OK, now, what is amazing about the pyruvate dehydrogenase complex - well, one of things that is amazing, I mean what it does is amazing - is it actually uses 5 cofactors- 5 coenzymes.1015

The next part, I will go ahead and...yes, that is OK.1029

I will just make sure...OK, I will write PDH requires, yes, it requires 5 coenzymes, and those 5 coenzymes - I will go back to red - TPP, thiamine pyrophosphate.1034

You have already seen it.1059

Two: it requires lipoate, which we will talk about in just a minute.1063

Three: it requires coenzyme A, which we will talk about in just a moment.1068

Four: it requires FAD; remember the flavin adenine dinucleotide that we talked about when we talked about bioenergetics?1075

And five: NAD⁺, nicotinamide adenine dinucleotide- the version that is going to oxidize is going to reduce.1084

This NAD is going to be doing the oxidizing; OK, we have talked about the FAD.1096

We have talked about the NAD⁺; we have seen TPP in previous lessons when we talked about glycolysis and gluconeogenesis.1101

Now, let's go ahead and talk a little bit about the lipoate and the coenzyme A.1109

OK, now, let's see what we can say about this.1116

Now, let's go ahead and take a look at their images just to see what they look like, and then, we will go ahead and start the actual mechanism, the enzyme, what the enzyme does; so let me move forward.1121

OK, here, I have a molecule of coenzyme A.1132

This is where the S comes from; OK, alright, this coenzyme A is made up of 3 parts.1140

This part right here, the NCCS, this is called a beta-mercaptoethylamine.1146

Ethyl- 2 carbons; ethylamine, that means one of the carbons is attached to a nitrogen.1151

Excuse me; mercapto, beta-mercapto - this is the alpha-carbon, this is the beta-carbon.1155

Mercapto means sulfur- that is it.1161

OK, this middle part right here, from here on all the way up to and including the oxygen that is attached to the phosphorus, this is pantothenic acid- that is it.1165

The acid part is when you remove this nitrogen, you end up with an OH group.1175

When you hydrolyze this, you end up with a COOH, which is actually a carboxylic acid on this end.1180

In this case, it is an amide linkage.1185

And then, of course, up here, the 2 phosphates, you have the adenine.1189

You have the ribose; the only difference is you have this extra phosphate here.1194

This is called 3-prime phosphoadenosine diphosphate- diphosphate, not triphosphate.1198

Again, that is not altogether that important.1204

I mean, it is nice if you can recognize that pantothenic acid is a part of coenzyme A, but it is the coenzyme A part that is important here.1207

Now, coenzyme A looks like this.1215

This, all of this right here, we usually abbreviate as S-CoA.1220

Acetyl-CoA actually looks like this.1230

It is CH₃, C, O, O, S-CoA.1234

This is the acetyl coenzyme A, the acetyl group, 2 carbons and then this whole, big, long thing.1239

Now, you remember in our discussion of bioenergetics or hopefully, if you remember, if not, not a big deal.1246

We will discuss it here again.1251

Thioesters, this is thioester; this is a carbonyl, but it is not attached to an oxygen that is attached to a carbon.1254

It is attached to a sulfur, so it is a thioester.1260

Thioesters have high -ΔGs of hydrolysis.1265

What that means, that means this bond right here, it wants to break.1273

It is very, very easy to break; this is an activated acetyl group.1279

They transfer acyl groups.1287

They transfer these groups - these 2 carbon groups - very, very, very easily because the bond wants to break.1296

As it is, this carbon on the carbonyl is already reactive.1301

By attaching the sulfur to it, which when it leaves, is a fantastic leaving group, it activates it.1306

It really, really wants to react, and it will with any, even moderate nucleophile; it will react.1311

We talked about the transfer of this group to another group, which is going to be the nucleophile.1319

They transfer acyl groups very easily.1326

We often say the acyl group is activated.1333

It is activated; it is ready to react.1350

OK, now, let's go ahead and talk about the other thing: lipoic acid.1353

This is lipoic acid, also called lipoate when this hydrogen is in its deprotonated form, so lipoic acid, lipoate, it is the same thing.1356

OK, this comes in 2 forms; this is where the reaction is going to take place in...well, that is where the reaction takes place with use of this coenzyme.1369

OK, I am going to call of this stuff R, and I am going to draw something here.1380

This is the oxidized version: boom, boom, boom, boom, S and S, and this is R.1386

This is the oxidized form, and remember, oxidation often means we have removed some hydrogens.1393

The reduced form looks like this.1402

There is an H attached, and there is an H attached.1406

That is the reduced form right here, and you will see the importance.1410

This is where the chemistry is going to take place, on the use of this particular coenzyme, and as it turns out, this coenzyme is actually attached to a lysine residue.1419

Notice how long this is; this is 1, 2, 3, 4, 5 carbons long.1428

If you remember the lysine, it actually has 4 carbons attached to an amine group.1435

5 carbons attached to another nitrogen and 4 carbons, so you have this really, really long arm; and what this arm is going to end up doing is it is going to be a tether.1442

It is actually going to take this end of the molecule, and it is going to move it to another part of the enzyme, so that it can react, and then, it is going to bring it back around, so it can react with something else, and then, it is going to bring it back around.1450

That is what is amazing about this enzyme; it actually uses a simple mechanical feature of a long arm to take it from one end of an enzyme all the way to the other end of an enzyme, which on a molecular scale is very, very, very far away from one active site to the other active site of the enzyme, and you will see that in just a minute.1462

OK, let me go ahead and actually draw that.1483

The lipoate is attached to a lysine residue on the transacetylase- on E2.1487

It looks like this; we have N, C.1513

We have C, then, of course, it goes on this way; and it goes on that way.1518

I will just go ahead and put an H attached there.1524

We have got C, C, C, C; there are 4, right?1527

Yes, there are, and then, there is N; and then, we have our carbonyl, 1, 2, 3, 4, 5, and then, we have our S and S.1531

See how long this is; this lysine residue right here is part of the E2 enzyme.1548

This lipoate is actually attached to that, so you have this really, really long arm that allows it to move.1556

The chemistry is going to take place here, so it is going to move the substrate that is attached here to different parts of the enzyme.1561

OK, now, let's go ahead and jump right into what it is that this pyruvate dehydrogenase complex does.1571

Its 5 reactions, they all take place in this complex from one point to another, and they substrate is just going to, sort of, be moving around.1581

Let's take a look at this, and see if we can follow this particular reaction.1591

OK, alright, I have gone ahead and drawn this out beforehand.1596

Let's just take a look at what is going on here in red.1600

There are 5 reactions that take place; this first enzyme, this is E1.1608

Here, we have E2, and here, we have E3.1613

There are 5 reactions that are going to take place.1617

Here is our pyruvate; we start off with the pyruvate.1622

I am just going to run through it, and then, I will actually write everything down, specifically what is happening and what reaction is taking place.1627

The pyruvate comes in here, and what it does is, in the E1 complex, it reacts with the thiamine pyrophosphate.1633

It gives up its CO₂ group; this CO₂ group goes away.1642

Now, this 2-carbon fragment - the acetyl fragment - is actually attached to the thiamine pyrophosphate because that is what TPP does.1646

It carries the acetyl group; what it does, now, it moves over the TPP, now, comes around.1654

And notice, here is E2; the lysine, here is the lipoate.1664

The lipoate reacts with the TPP; the acetyl attached to the TPP, this acetyl group gets transferred to here.1670

OK, it gets transferred to here, and now, it is moving this way.1680

Now, you have the acetyl group, right here, is actually attached to the sulfur on the lipoate.1688

When it gives it up, now, it is TPP again, and now, it will go on and do another cycle of itself.1694

These 2 reactions take place, actually, at E1; now, what happens is now that you have the acetyl group attached to the lipoate, that is attached to the lysine in E2, coenzyme A, the other substrate of the E2 enzyme comes in.1700

It reacts with this, and it actually knocks it off.1714

It breaks this off here, right?1720

We said this is an activated group, so anytime you have some sulfur - the thioesters, sulfur, carbonyl - this bond right here between the carbon and the sulfur, it wants to break.1721

Sure enough, it does break; it is replaced by coenzyme A.1732

The acetyl group is transferred to coenzyme A, so coenzyme A comes in.1737

What leaves is acetyl coenzyme A; now, what you have is this reduced version of the lipoate.1742

Now, what happens is it swings around - that was reaction 3 - and in E3, the FAD - flavin adenine dinucleotide - actually oxidizes this back to its oxidized form, so it can continue its cycle.1750

It turns into FADH₂; well, we need to regenerate the FAD, so NAD⁺ comes in and oxidizes the FADH₂ back to FAD.1768

The NAD⁺ is, now, converted to NADH, and NADH ultimately goes and gives its electrons to the electron transport chain.1779

5 reactions, 3 enzymes, multiple copies of these enzymes, 5 coenzymes, pyruvate comes in.1789

What comes out the overall reaction; we lose a CO₂.1799

We produce acetyl-CoA, and we produce NADH.1803

We say that again; pyruvate comes in.1808

We lose CO₂; it gives up a CO₂.1810

The acetyl group is converted to acetyl-CoA, which can go on and enter the citric acid cycle; and NAD is ultimately produced, which goes on and gives its electrons to the electron transport chain.1814

This conglomeration of enzymes is absolutely amazing.1825

I mean, look at that, 5 different coenzymes, 3 enzymes locked together in this amazing dance- absolutely fantastic.1830

OK, now, let's talk about some of the reactions and what it is that is going on here.1837

Let me go ahead and use black; at E1, which is the pyruvate dehydrogenase, we have reaction 1.1841

The reaction 1 is the decarboxylation- taking the CO₂ off.1855

OK, that is reaction 1; now, also at E1, the enzyme 1, reaction 2 takes place.1863

Reaction 2 is the dehydrogenation and the transfer.1872

It is the dehydrogenation and acyl transfer- acetyl because it is 2-carbon, acyl, normal.1883

This OH is converted to a carbonyl; OK, the OH is con...there is an H group here.1897

This H and this H, it is dehydrogenated; that is what this enzyme does.1905

It converts it to a carbonyl; there is decarboxylation, and there is dehydrogenation, and there is acyl transfer.1909

It is transferred over to the lipoate.1915

At E2, this is the dihydrolipoyl transacetylase.1920

We have...reaction 3 takes place.1926

That is this reaction right here; that is the transesterification.1930

Here, the acetyl group is involved in an ester bond with the lipoic acid, with the lipoate.1940

Here, the acetyl group is involved in an ester bond and a thioester bond with CoA, with coenzyme A, so it is a transesterification- from 1 ester to another ester.1948

The acetyl group just changed the ester group.1959

OK, now, E3, this reaction, this is the dihydrolipoyl dehydrogenase.1964

Now, we have reaction 4; this is oxidation of the dithiol of the lipoate.1971

This is the reduced version; we are converting it back to its oxidized version, so it can continue its cycle.1992

Reaction 4 oxidizes that.1997

And, of course, our final reaction, which is reaction no. 5, it is the oxidation of FADH₂ back to FAD - we are taking hydrogens back away from it- by NAD⁺.2002

In this case, NAD⁺ is doing the oxidizing; FADH₂ gets oxidized.2033

There we go; OK, once again, the products of this reaction: CO₂, acetyl-CoA, NADH.2040

This is the pyruvate dehydrogenase complex- absolutely extraordinary, extraordinary enzyme.2050

OK, I think I will just say 1 final word, and then, we will leave it alone; and next lesson, we will go ahead and actually start discussing the citric acid cycle.2058

This is an example - we actually give it a name, this whole, well, we will see in a second - of substrate channelling.2069

OK, substrate channelling is where the intermediates of a reaction sequence - like we have here, we had a 5-reaction sequence - they never leave the complex.2087

They just move from 1 point to another; everything is just channelled.2112

It is channelled until it gets to where it wants to be, and then, it releases it.2116

Again, biochemistry, science in general, we like to give names to everything.2126

I personally do not like that fact; I think it is better just to always see in terms of a big picture because a lot of times you get lost in all these names.2130

What does this mean; what does this mean?2138

What does this mean?2139

Well, we call it substrate channelling; I mean, you know what it is doing.2142

I mean, your intuition tells you that basically, your substrates are staying near the complex until all the reaction sequence is completely done, but we decide to go ahead and give it a name.2145

OK, that is the PDH complex; that is the conversion of pyruvate to acetyl-CoA, and next lesson, we will start discussing the citric acid cycle.2156

Until then, take good care, and thank you for joining us here at Educator.com, bye-bye.2167