Biochemistry Citric Acid Cycle II

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

In the last lesson, we talked about how the pyruvate that was formed in glycolysis is converted to acetyl-CoA in this amazing enzyme called the pyruvate dehydrogenase complex.0004

Today, we are actually going to start with the reactions of the citric acid cycle, how this acetyl-CoA enters the cycle and runs through it.0016

Let’s just jump right on in.0025

This first thing that I have here is the cycle itself but just the names of the molecules that take place.0029

In the next page, I am actually going to draw out - or it is is already drawn out - the structures themselves.0037

I wanted us to get, sort of, a big picture view of this, talk about what happens, talk about the number of reactions, what goes in, what goes out, just to get a big picture idea of what the citric acid cycle is.0044

And then, we will take a look at the structures, correlate them with the individual reactions- the 1 through 8.0055

And then, we will look at the individual reactions and their mechanisms.0061

A lot of information, so we will try to cover as much as possible.0065

And again, I encourage you to look at your books.0069

One of the things that I really, really want to stress in this particular course is that the biochemistry books are really fantastic.0073

They are an incredible resource with lots of information and lots of beautiful illustrations, and I think it is nice to be able to see it like this and then, look at the illustrations in the book and to see the correlation.0080

It, sort of, locks it in your mind; now, again, there is a difference between looking at something passively and being able to write it out actively.0090

This is something that you want to be able to write out yourself, starting with the oxaloacetate going to the citrate going to the isocitrate and so on all the way around the cycle.0099

It is very, very important to be able to produce this actively instead of just looking at it passively.0110

OK, so let’s see what is going on here.0115

Acetyl-CoA, that is the molecule that we actually formed from the pyruvate.0119

That actually enters the cycle, and what it does is it condenses with oxaloacetate.0125

In some sense, oxaloacetate, you could say, is the beginning and the end of the cycle.0131

That is where things, sort of, start; these 2 condense.0135

Water comes in; coenzyme A leaves, and you end up forming this molecule called citrate, thus, the name citric acid cycle.0139

This reaction - notice - is an arrow in one direction.0150

This particular reaction is irreversible under physiological conditions; notice, these other ones are reversible.0154

Most of these reactions are reversible.0160

Reaction 1, reaction 3 and reaction 4, under physiological conditions, are irreversible; and we will talk about that a little bit later also.0164

The first step is the conversion of oxaloacetate to citrate; that is the first reaction.0171

Now, citrate loses water, and it becomes something called cis-aconitate.0177

Now, notice, I have reaction 2 and reaction 2.0183

The conversion is actually from citrate to isocitrate.0186

That is actually reaction 2, but it takes place through an intermediate called cis-aconitate.0190

In some illustrations for the citric acid cycle, you may not see this.0195

In the individual reaction, they will show you what the intermediate passes through.0199

In this particular case, I thought it would be nice to actually include it in the citric acid cycle.0203

The conversion is citrate to isocitrate, but it passes through this thing called cis-aconitate.0208

Water leaves; water comes in again, and you get the isocitrate.0214

OK, now, here is your first oxidative step and decarboxylation step.0219

In this case, NAD⁺ oxidizes the isocitrate to alpha-ketoglutarate, and not only does it oxidize it, it also decarboxylates it.0224

It takes away one of the carbon dioxides; that is why you have a CO₂ leaving and you have an NADH leaving.0234

The things that I have put squares around, the CO₂ here, the NADH, CO₂, FADH₂, here and here, these are the important things that you want to concentrate on in the citric acid cycle.0239

These are the things that end up leaving and these are for example...well, we will talk about what they do in just a second.0250

Reaction number 3: isocitrate to alpha-ketoglutarate.0257

Reaction number 4: it is another decarboxylation, and it is another oxidation, and again, irreversible under physiological conditions.0260

We have alpha-ketoglutarate, which is converted to succinyl-CoA.0268

Now, the succinyl-CoA is transformed into succinate, and in this particular case, notice that GDP plus an inorganic phosphate is used to actually produce a GTP.0273

In this particular case, GDP produces the GTP, and the coenzyme A leaves.0286

ATP is also possible to produce here directly, depending on the particular isozyme that is used in this reaction.0293

It will either produce GTP directly or ATP directly.0300

But ultimately, the GTP is converted to ATP in subsequent reactions in the body.0304

OK, so now, the next reaction, the no. 6 reaction, again, we have an oxidation this time by FAD, is reduced to FADH 2, and that ends up leaving.0309

It is succinate to fumarate; water comes in, fumarate to malate, and then, the final oxidation, malate to oxaloacetate, NAD⁺ releasing NADH.0320

The NADH is here, these 3, and the FADH₂s, those are, of course, carrying the high energy electrons from the oxidation.0331

Those ends up being funneled into the electron transport chain for oxidative phosphorylation, for complete oxidation to water.0339

OK, and there you have it, oxaloacetate, and then the cycle starts all over again.0349

Acetyl-S-CoA comes in; it condenses with the oxaloacetate, and it runs through this process.0353

What you want to learn is the sequence.0359

Acetyl-CoA + oxaloacetate or you want to start...I usually start with citrate, myself: citrate, cis-aconitate, isocitrate, alpha-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, oxaloacetate to start the cycle all over again.0364

This is the big picture of what is happening; again, concentrate on the things that are leaving.0378

Reaction 1, reaction 3, reaction 4, irreversible under physiological conditions; the rest of them are reversible.0384

The formation of NADH, NADH, NADH, 3 times, the formation of FADH₂ once, and the release of 2 carbon dioxides, and the formation of 1 guanosine triphosphate or adenosine triphosphate.0391

OK, now, I am going to reproduce this picture on the next page, but now, I am going to deal just with the structures.0404

I would like you to see what the structures of these molecules look like.0410

I do not have all the other information, the H₂O, the CO₂.0414

I just wanted you to see what the structures look like just to get a global view, and then, of course, we will deal with the individual reactions themselves- not a problem.0417

OK, here we go, oxaloacetate, again, citrate, cis-aconitate, isocitrate, alpha-ketoglutarate, succinyl-CoA, succinate, fumarate, malate and back to oxaloacetate.0424

Alright, oxaloacetate is this molecule right here, and it is going to condense with the acetyl-CoA; and what I have done is, I have written the acetyl-CoA in red.0438

OK, this is the group that is actually going to attach because coenzyme A is going to leave.0449

I have written that group in blue; now, notice, blue, blue, blue, blue, blue.0456

At this point, I did not forget to put this into blue.0460

At this point, what you end up having is a symmetrical molecule.0464

Once we get to the succinate, we are not going to be able to tell anymore which acetyl group, CH₂, COO^-, whether it is this one or whether it is this one because, now, the molecule becomes symmetric.0468

And now, you cannot tell which of these acetyl groups actually comes from the original acetyl-CoA.0480

Up to here, you can; that is another important point for this.0487

Again, we go to reaction no. 1; we form citrate, and again, this is the acetyl group that comes from the acetyl-CoA.0492

We have got this; a water leaves to form a double bond, the cis-aconitate intermediate, and we end up forming the isocitrate.0499

Really, all we have done is we have taken this OH group from this carbon, and we have moved it to this carbon.0505

We have switched the OH and the H.0511

Let me actually make one little change here.0515

Let me go ahead and write the H that way because that way, the OH and the OH switched0520

That is why it is citrate and isocitrate; OK, reaction no. 3.0525

This is one of the decarboxylations; I did write the CO₂ leaving this one because these are important.0530

These are the 2 decarboxylation steps, 2 of the oxidation steps.0534

Isocitrate becomes alpha-ketoglutarate; notice, what has happened here.0538

A CO₂ is gone, right?0541

We have taken this CO₂ away, and we have taken this OH, and we have oxidized it with the dehydrogenase.0545

We have turned it into a ketone, a carbonyl, so we have oxidized that carbon.0551

OK, we lose another CO₂; we lose this one.0555

The carbonyl stays, but we oxidized that carbon with an acetyl, I am sorry, with a coenzyme A group- the S, a further oxidation.0562

OK, now, you have the succinyl-CoA, and at this point, it is converted to succinate, which is your symmetrical molecule.0572

And from here, it goes to fumarate, double bond, and then here, OH is attached, malate; and then, of course, oxidized to oxaloacetate, and then, it starts again.0579

These are the structures; again, this is citrate.0590

This is the cis-aconitate; here is the isocitrate.0594

This is the alpha-ketoglutarate; this is the succinyl-CoA.0598

This is the succinate, fumarate, malate and oxaloacetate, and here is your acetyl-CoA.0603

Just, sort of, take a look at this, whether you want to use this particular diagram.0612

Again, I encourage you to take a look at the diagrams in your book.0617

I think it is always great to see multiple diagrams, not just one, because each one brings just something a little subtly different.0621

And there might be something in there that strikes you, that speaks to you, that maybe is more comfortable for you, that makes more sense to you.0626

Maybe there is more room, and maybe it is clearer; maybe the structures, somehow, make more sense to your particular eye.0634

It is not a problem; what is important is, again, the cycle, the sequence and what is going on where.0640

OK, let’s go ahead and say a couple of things about this - a couple of things to note - and then, we will start with the reactions themselves.0649

OK, things to note, let’s go ahead and do this in black.0658

Well, the first thing you want to note is that steps 1, 3 and 4 are irreversible under physiological conditions.0667

That is why they have those single arrows.0677

And again, when we see reactions that are irreversible, that should be a clue that these reactions tend to be points of regulation for the particular cycle.0688

It is difficult to regulate a reversible reaction because, again, it goes back and forth.0702

It is not difficult to regulate an irreversible reaction because we can control the extent to which it moves forward or does not move forward.0706

Irreversible reactions in biochemistry are points of regulation for that particular metabolic pathway.0715

OK, now, steps 3, 4 and 8 are oxidations that yield NADH, are oxidations yielding NADH, the high energy electrons that pass to the electron transport chain.0725

Now, step 6 is also an oxidation, but it yields FADH₂ - remember that flavin adenine dinucleotide - oxidation yielding FADH₂.0750

Now, step 5, can produce, as we said, ATP or GTP directly depending on the isozyme that is used.0771

You Remember isozymes, they actually catalyze the same reaction, but they do it under different circumstances, maybe a slightly different substrate, things like that; but it is the same enzyme.0792

I am sorry, it is not the same enzyme; it catalyzes the same reaction.0801

The enzymes themselves are close to being identical, but they are not quite identical.0805

They have a different primary structure, a different amino acid sequence, but they do the same thing depending on the isozyme, yes.0810

OK, and finally 2 CO₂ molecules are released.0818

We have 2 decarboxylations, and they take place in steps 3 and 4.0828

Steps 3 and 4- very, very important steps in the citric acid cycle.0834

OK, now that we have a nice over view, a couple of things that we want to keep in mind.0838

Now, we can go ahead and start talking about the reactions themselves.0844

OK, so let’s go ahead and look at reaction 1; I think I will go ahead and do this in blue.0848

Reaction number 1, this is the formation of the citrate.0854

This is going to be condensation of the oxaloacetate and the acetyl-CoA for the formation of citrate.0858

Woo, biochemistry, there is a lot of molecules floating around with lots of names.0868

OK, we will say formation of citrate.0872

This is a condensation reaction, and then again, a condensation reaction just means we have taken 2 molecules and we have put them together somehow.0879

This is a condensation reaction.0886

OK, let’s go ahead and write CH₃.0892

Now, let me do it this way; let me start with the hydrogens on the left; H₃C, C, O, S and CoA.0897

We have acetyl-CoA is our first reactant, and then, we have, let’s go ahead and do C, C, C, C.0907

Let's go ahead and do that, and let’s put an H₂ there.0917

We have a carbonyl there, and then, we have that group.0923

Let’s go ahead and number these carbons actually; I am going to go ahead and number them 1, 2, 3 and 4.0927

It is going to be important because we want to keep track of which carbon is attaching to which carbon because this is a condensation reaction.0933

It is going to be important; OK, these are the 2 reactants.0941

Let me go back to blue here, and let me go ahead and draw my little arrow, and what is going to come in is H₂O, and what is going to leave is CoA, S, H.0944

What is going to leave is the coenzyme A, and the enzyme that catalyzes this is citrate synthase; and the final product that we get is the following.0957

Let me go ahead and do that one in blue also; let me go C, C, C.0972

Well, I wonder if I should do that in a different color.0982

Should I do that in a different color or not?0990

Yes, you know what, I will go ahead and do that in a different color; that is not a problem.0992

I will go ahead and put C there, but for the rest, I probably will not.0995

C, let’s just go ahead and put these on here first, H₂, and this is going to be OH.1001

This is going to be that one, and now, let me do this in red.1011

Actually you know what, no, I am going to go ahead and keep it in blue.1019

This is going to be CH₂, and this is going to be COO^-.1023

OK, what we have here is this; oh, let me number my carbons again.1030

This is 1; this is 2.1036

This is 3, and this is 4, so 1, 2, 3, 4.1039

Now notice, this carbon right here, that is this carbon right there.1043

This acetyl group, the methyl of the acetyl group - not the carbonyl carbon - ends up attaching to the no. 2 carbon of the oxaloacetate.1050

That is what makes this really, really an interesting reaction; what we have is this one right here.1061

This acetyl group attaches to the no. 2 carbon, not the no. 1 carbon or any other.1066

You still have this CO₂ group here; you still have this CO₂ group, and then, you end up with this molecule that has 3 CO₂ groups attached.1072

OK, now, let’s go ahead and write the ΔG for this reaction, so we have it for reference- -32.21081

This is kJ/mol- clearly, highly exergonic, irreversible under physiological conditions also, not just under the standard conditions, which is represented by that.1091

Acetyl-CoA condenses with oxaloacetate.1102

The methyl group of the carbon, I am sorry, the methyl group of the acetyl-CoA actually attaches itself to the no. 2 carbon of oxaloacetate, and we are left with this molecule right here, which is our citrate.1106

This is citrate; this molecule here is acetyl-CoA.1120

I will write acetyl-S-CoA, and this right here, is our oxaloacetate- there you go.1127

That is the entry into the citric acid cycle.1137

Now, let’s go ahead and talk about the mechanism for this reaction- very, very interesting mechanism.1141

Let’s do that on the next page, and I think I am going to go to black ink for this one.1149

This is going to be the mechanism, and again, some of your teachers will have you memorize this mechanism and know it and be able to reproduce it.1153

Some of them, perhaps, just want you to see it; some of them will not really care about the mechanism at all, but it is important to be able to present it.1161

I would like you to see it; it is very, very important to get used to this, particularly those of you who will go on to graduate school, pharmacy school, things like that.1168

You have to get comfortable with these organic mechanisms- very, very important.1176

It will put you way ahead of the crowd if you can handle, at a very least, understand and follow a mechanism without confusion.1181

OK, let’s go ahead and draw out some molecules here; let’s draw out our oxaloacetate, C.1188

I am going to draw out this way; I am going to put just so we have a clear idea of what is reacting with what, and I will go ahead and put the C, H, H, H, and then, this is a C, our carbonyl, our S and our CoA.1196

OK, now, let’s go ahead and see if we cannot...you know what, let me draw out this down a little bit.1217

Yes, let me draw it down just a little, C, O, O, S and CoA.1232

Now, let me go ahead and draw something like that.1243

OK, so let me go ahead and let me do these enzymes in blue.1253

C, O, O, this is going to be Asp 3,75, that particular amino acid .1259

This is the enzyme right here. and this is the active site of the enzyme.1271

This is the citric synthase enzyme, and over here, we are going to have...let me see; let’s go 1, 2, 3, 4, 5.1276

This is going to be our histidine 3,20.1292

We have an N, and we have an N.1297

This is an H; that is an H.1301

And, of course, we have a little positive charge there because you have Hs attached to this histidine.1303

And let’s go ahead and do the same right here.1308

Let’s go that way, that way, that way, that way, that way.1312

Let’s go ahead and put an N there, an N there.1315

We have an H, and we have an H; there is a positive charge that is distributed between these 2.1319

That is why I wrote it that way, and this is going to be histidine 2,74.1325

This histidine residue on the enzyme, this histidine residue and this ASP residue, now, we have the active site; and, of course, this is the enzyme right here.1331

Let me do this in black.1341

This is the enzyme; that is the active site.1348

OK, here is what happens; let me make this H a little clearer.1352

OK, this, let me do the mechanism in red.1357

It is nice to have all these colors available.1363

That takes that, pushes these electrons here, pushes these electrons here, pushes those electrons onto nitrogen.1367

What you end up with is the following; now, let’s go back.1375

Let’s go ahead and draw the enzyme like that.1380

Now, what we have are the following molecules; let’s go ahead and draw our oxaloacetate again.1386

This is going to be C, COO^-.1390

We have our carbonyl; we have our CH₂.1394

We have our carbonyl again, and now, over here, this has rearranged itself.1398

Now, we have a C double bonded to a C.1403

We have an H; we have an H because we lost that H.1410

We still have our S, and we have our coenzyme A, and now, of course, we have OH - right - because it ended up taking up an H.1412

Now, we are looking at this particular molecule; let me put a couple of the electrons on there.1421

That is going to be important; now, down here, let me go ahead and go to blue and take a look at my enzymes.1426

Again, we took this H; this is our Asp 3,75, and over here, we still have our 1, 2, 3, 4.1433

Let me just draw it like this; we still have our H.1446

We have our H, and we have our histidine- still positive charge there.1452

Over here, now, what we have is - let me draw it a little further this way - here, this is N.1457

This is N; now, this is there.1467

Now, we have that - OK - and we have our H there.1469

This is our His 2,74; alright, now, what happens is the following.1478

Oh, let me do this in red - right - we are doing our mechanisms in red.1488

These electrons come and take that, push the electrons back down to there to reform the carbonyl.1490

Reformation of a carbonyl is, it wants to form; the carbonyl is a very, very stable molecule.1498

Now, it pushes these electrons over here, not onto the electrons, onto that carbon; and it pushes it, electrons there and there.1503

This N from the histidine takes this H, reforms the carbonyl.1520

OK, the electrons come back down to reform the carbonyl here, pushes these electrons out to here, so that this carbon is, now, going to attach to this carbon.1525

It is going to push these electrons out to here to grab an H that is available, and these electrons in the NH bond are going to jump onto the nitrogen to stabilize that.1536

What you end up with is the following.1547

Now, you end up with this thing attached to this thing at that carbon.1552

These 2 carbons, that carbon and that carbon, are going to be attached now because that is what this is.1559

These electrons are moving there, and they are bringing this carbon with it to attach it to this.1566

It is a condensation reaction; OK, so what you end up with is this following molecule.1571

Let me go ahead and just leave it in red; actually no, let me go back to black because we are doing this in black.1578

What you end up with is this C; now, you have the OH. right?1584

COO^-, that is the oxaloacetate; now, you have the CH₂, COO-S-CoA, so that carbon attached to that carbon.1592

Now, we have CH₂ and COO^-.1605

At this point, now, what takes place is a simple hydrolysis.1610

Water comes in; CoA-SH actually leaves.1616

Water replaces that group; what you end up with is our citrate molecule.1621

Let me draw it as C, C, C.1628

This is going to be COO^-.1633

This is going to be COO^-.1639

Let me go ahead and put H₂ there; this has an OH.1643

This is CH₂, and it is also COO^-, carboxylic acid, carboxylic acid, carboxylic acid and hydroxide.1648

This is citric acid; the deprotonated, so it is citrate.1657

That is all that is, citrate, citric acid, benzoate, benzoic acid.1661

It just means that the hydrogens have been taken off.1665

OK, this oxygen right here, this oxygen actually comes from the water, just so you know.1667

I do not think it is a big deal but it is nice to know where it comes from.1674

This O, it comes from the water because of the hydrolysis.1679

This water is going to be attacking this carbon; it is going to be breaking this bond.1687

Water is going to attach; it is going to lose one of its protons.1694

It is going to lose 2 of its protons actually, and then, this S right here, that is what leaves.1697

This is that, and this comes from here.1703

This comes from here - OK - comes from the water.1708

There you go; this is citrate.1713

OK, and at this point, you just have release by the enzyme- that is it.1717

That is the mechanism; OK, now, let’s move on to reaction no. 2.1727

Now that we have our citrate molecule, let’s go ahead and go back to black.1735

Now, let’s go ahead and write our citrate, CH₂, COO^-.1741

We have C, C.1749

Let me draw it a little bit over to the right here, so C, C, C and C.1753

I have got CH₂ - woo, these carbons, oxygens, hydrogens, they are making me crazy - COO^-.1764

I have got COO^-, and I have got my OH, - right - and I have got my...let me go ahead and draw both hydrogens individually, and, of course, I have my COO^-.1772

This is our citrate.1783

OK, now, let’s do this.1790

The citrate is going to pass through an intermediate; we are going to go from citrate to isocitrate, and we are going to pass to an intermediate called cis-aconitate.1794

OK, it is going to go like this.1802

I wonder if I should do it as 3 here and here.1807

Well, let’s see if I can get this actually all in one line.1813

The enzyme that catalyzes this is the aconitase, and H₂O is going to leave.1820

It is going to be this H₂O that is going to leave; well, let me write the reaction, and then we will see what happens.1827

So, C, C, C, C, C, and this is actually a double bond.1835

This is CH₂, COO^-, and this is going to be COO^-; and this is going to be COO^-, and this is 1 to - oh no, this is, wait - 1, 2, 3, 1, 2, 3.1842

No, this is H not C; there we go, the way you, sort of, draw it, and then, what happens is, water comes back in.1862

This is our cis-aconitate, and then, water comes back in; and you end up with C, C, C and C.1873

You know what, I am going to do it over here.1892

This is our cis-aconitate; now, it is going to end up over here, and water is actually going to be coming in here and what you end up with is the final molecule 1, 2, 3 and 4.1897

We have CH₂, COO^-, and then, we have our COO^- there.1915

Now, we have our H and our OH here, and we have our OO^- here.1922

Basically, all we have done is we have taken this OH and H, and we have switched places- that is it.1928

We first removed the water to form the double bond and then, we added the water back to the double bond except in the other order- that is it.1936

Aconitase, that enzyme is what accomplishes this particular reaction- that is it.1945

OK, now, let’s go to reaction no. 3.1954

OK, this is another one of the irreversible reactions, and this is the isocitrate to alpha-ketoglutarate.1963

Reaction 3 is isocitrate to alpha-ketoglutarate, and this is an oxidative decarboxylation.1971

Let me go ahead and write that in blue; this is an oxidative decarboxylation.1986

Decarboxylation means we are going to lose CO₂, and oxidative means that one of the carbons, either that one or another one, its oxidation state is going to rise.1997

It is going to be oxidized; let me go back to black here.2008

Let’s see, we have isocitrate.2013

Let’s go ahead and do C, C, C and C.2018

Let’s go a little bit higher up here, C, C, C, C.2024

This is going to be CH₂, COO^-.2032

This is COO^-, and the H is here.2036

The OH is here, and the H is here and COO^-.2040

OK, that is our isocitrate, and let me see.2045

Are we going to actually go through...yes, we will go through the mechanism for this one.2053

Alright, we have our NADH, so this is our oxidation.2059

This is our decarboxylation, so CO₂ leaves.2063

NAD⁺ comes in; NADH leaves.2068

It is actually NADH + H⁺ - right - 2 hydrogens that we are actually removing, and this is isocitrate dehydrogenase that catalyzes this reaction, and our final product is our alpha-ketoglutarate.2072

We have got C, C, C - still a 4-carbon - CH₂, COO^-.2096

We have decarboxylated, so now, we have lost a CO₂ group, and let me go ahead and write the carbonyl over on this side, COO^-, so alpha-ketoglutarate.2107

What you have lost is this; let’s go ahead and do red.2122

You have lost this; let’s try this in red- there we go.2125

We have lost this; that is our CO₂ that is left.2133

The NAD oxidized, so it took this hydrogen and this hydrogen away leaving just the carbonyl; and what you are left with is your alpha-ketoglutarate.2138

That is what is happening.2146

A handful of reactions, NAD⁺ oxidizing something, NAD⁺ oxidizing something, it takes the electrons.2152

It takes the 2 hydrogens; it is a handful of reactions that show up over and over and over again.2158

The body does not have 15 different ways of doing the same thing.2163

It usually just has 1 or 2 ways of doing the same thing, and it does it over and over and over again in different circumstances.2167

OK, this is our isocitrate.2174

OK, there you go; that is reaction no. 3.2179

A couple of things to note about this particular reaction.2187

Notes: there are 2 isocitrate dehydrogenase enzymes.2192

OK, one uses the NAD⁺.2213

The other uses the NADP⁺, and the other, not a big deal.2225

It is just the NAD⁺ with an extra phosphate group attached, otherwise, they are identical.2234

Now, also, the enzyme requires manganese.2241

OK, now, let’s go ahead and talk about the mechanism here.2253

Let me see, 1, 2, 3, 4, yes, it is fine; I can do it on this page.2257

Let me go ahead and do the mechanism in blue; we will start in blue.2263

Hello blue, blue; there we go.2269

Now, we have blue; alright, we have got C, C.2272

We have got C, C, C, C.2280

This is CH₂, COO^-, and we have C.2284

I will go ahead and write this one as that, and this is an H over here; and we have our H, and we have our OH over here, and we have our COO^-2290

OK, the first step is going to be the...yes, the dehydrogenation, the oxidation.2308

This one goes that way.2314

The first step is going to be the NAD⁺ to the NADH2318

I will not go ahead and talk about that mechanism- dehydrogenation2323

This is the oxidation; this is the first part that is going to take place before the decarboxylation, and what you are going to end up is the following.2329

This H and this H are actually going to be taken away, and it is going to be turned into the ketone.2335

We have C, C, C and CH₂, COO^-, and this is going to be C.2340

I am going to go ahead and do it this way, and this is a carbonyl now; and this is COO^-.2352

Now, the Mn²⁺ is actually going to be coordinated with those.2360

Electrons on the oxygens, they sort of coordinate with metal ions.2371

OK, now, here is where we have our decarboxylation.2375

Let’s see, we are going to have... let me go ahead and draw an arrow going out what is going to happen.2379

Here is what happens; let me do this in red.2386

These electrons come here to form CO₂.2389

These electrons get pushed there.2395

These electrons get pushed onto oxygen.2400

What you end up getting at this point is the following.2404

I will go ahead and do it right over here; let me go back to blue.2410

OK, I have got C, C.2415

I have got C, C, C and C.2422

I have got CH₂ and COO^-.2426

Now, I have an H group, and I have a double bond here.2430

Now, I have a single bond with the electrons there, and I have COO^-.2435

Now, what happens is these electrons come back down to form the carbonyl, and it pushes these electrons to go ahead and grab an H⁺ from solution, from an environment; and what you end up with is your final molecule, which is C, C, C, C, CH₂, COO^-.2440

Now, we have an H here and an H here.2468

It has been decarboxylated there; it is now a ketone group there, and you have a carboxylic acid there.2473

That is our alpha-ketoglutarate; this is alpha-ketoglutarate.2479

That is our mechanism for that particular conversion, so dehydrogenation first, the oxidation and then the decarboxylation.2486

Again, it is just the movement of electrons.2494

OK, now, let’s take a look at reaction no. 4.2498

Let’s go back to black.; let’s go ahead and draw a little line here, so reaction no. 4.2503

This is going to be the oxidation of the alpha-ketoglutarate to the succinyl-CoA, coenzyme A, releasing CO₂.2512

Now, we are going to release another molecule of CO₂.2537

This is another oxidative decarboxylation.2542

OK, and again, this step, irreversible under physiological conditions.2562

Let’s go ahead and do this in blue here for the molecules.2567

This one is going to look like this.2572

We have C, C, C and C.2576

We have CH₂ and COO^-.2581

I will go ahead and put the Hs here like that.2587

We have our ketone, and we have that, so this is our alpha-ketoglutarate.2591

Now, what happens is the following: what comes in, comes in and leaves and what leaves.2595

What comes in is coenzyme A.2605

What also comes in is, of course, the NAD⁺.2614

What leaves is the NADH, and what ends up leaving is CO₂.2618

CoA comes in; CO₂ leaves.2622

That is the decarboxylation; NAD⁺ comes in.2626

NADH leaves; that is the dehydrogenation.2629

That is the oxidation; what you end up with is the following molecule.2631

Let’s go back to blue, which is succinate; you have C, C and C.2635

Let’s go ahead and write it this way: H₂, COO^-.2643

This is H; this is H.2648

And then, over here, what we have is - yes, it is fine, I will just go ahead and write it this way - S-CoA.2652

This is the CO₂ group that ends up leaving.2667

The CoA-SH is attached, now, to the carbonyl, so all I have done is that I have written this ketone vertically instead of horizontally, so here.2673

Now, the decarboxylation is the loss of the CO₂.2680

The oxidative decarboxylation, the oxidation part is...well, what we have done is this carbon is double bonded to an electronegative atom.2684

Now, it is double bonded to oxygen but also single bonded to S, so it has been oxidized further.2692

That is the oxidative part of the oxidative decarboxylation; OK, now, the enzyme that catalyzes this - let me go ahead and do this in red - this is called the alpha-ketoglutarate dehydrogenase complex.2699

This is not a single enzyme; this alpha-ketoglutarate dehydrogenase complex is very, very, very similar to the pyruvate dehydrogenase complex that we talked about previously for the conversion of the pyruvate to the acetyl-CoA.2723

It also consists of its own E1, E2, E3 in multiple copies.2739

It also uses all 5 of those coenzymes that that enzyme used.2745

They are very, very similar; they are not the same enzyme, but they do have a common ancestor.2751

So, evolutionary, they are from the same place; they do the same thing.2756

They do it the same way; I am not going to go through the mechanism for this one.2760

If you want to see the mechanism for this one, just go back one lesson, and take a look at that mechanism.2764

Remember when we had that arm swinging around, E1 to E2 to E3, and that is what is going on there.2768

OK, alpha-ketoglutarate dehydrogenase complex converts the alpha-ketoglutarate to our succinyl-CoA.2776

Let’s go ahead and say a couple of words about this - just some notes here - and we will go ahead and close out this lesson with this reaction and pick up the rest of the citric acid cycle in the next lesson.2790

This reaction is essentially the same as the PDH reaction, the pyruvate dehydrogenase.2808

Again, it is decarboxylation + oxidation of the carbonyl of the ketone to a thioester-S-CoA.2825

That is what you are doing.2851

This enzyme is very much like the PDH.2856

I am sorry; this complex is very much like the PDH complex in both structure and function.2868

OK, it has its own enzyme 1, enzyme 2, enzyme 3 in multiple copies.2888

It uses the same 5 coenzymes.2906

They are not the same enzyme, but they do come from a same evolutionary ancestor because they do the same thing.2920

They do it the same way; they are arranged the same way, but they catalyze, sort of, the same reaction as far as the class of reaction, but of, course, they have a different substrate, but we do not call the isozymes or isozymal complexes.2926

It is just a different complex; the alpha-ketoglutarate dehydrogenase complex, the pyruvate dehydrogenase complex.2940

Those are the first 4 reactions of the citric acid cycle; we will see you next time for a discussion of the final 4 reactions of the citric acid cycle.2950

Take care; thank you for joining us here at Educator.com, bye-bye.2958