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Lecture Comments (5)

2 answers

Last reply by: Zachary McCoy
Tue Sep 16, 2014 8:07 AM

Post by Zachary McCoy on September 11, 2014

Just in case anyone is confused, Professor Hovasapian accidentally circled the wrong hydrogen at 23:34. He meant to circle the hydrogen that's a part of the C1 hydroxyl group (non-ring form).

Professor, you're the best!!! And I'm not just saying it.

1 answer

Last reply by: Professor Hovasapian
Wed Sep 11, 2013 8:38 PM

Post by Vinit Shanbhag on September 11, 2013

why do we need phosphatases in cell if we have bifunctional enzymes?

Glycolysis II

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  • Intro 0:00
  • Glycolysis Step 1: The Phosphorylation of Glucose 0:27
    • Glycolysis Step 1: Reaction
    • Hexokinase
    • Glycolysis Step 1: Mechanism-Simple Nucleophilic Substitution
  • Glycolysis Step 2: Conversion of Glucose 6-Phosphate → Fructose 6-Phosphate 11:33
    • Glycolysis Step 2: Reaction
    • Glycolysis Step 2: Mechanism, Part 1
    • Glycolysis Step 2: Mechanism, Part 2
    • Glycolysis Step 2: Mechanism, Part 3
    • Glycolysis Step 2: Mechanism, Part 4 (Ring Closing & Dissociation)
  • Glycolysis Step 3: Conversion of Fructose 6-Phosphate to Fructose 1,6-Biphosphate 24:16
    • Glycolysis Step 3: Reaction
    • Glycolysis Step 3: Mechanism
  • Glycolysis Step 4: Cleavage of Fructose 1,6-Biphosphate 31:10
    • Glycolysis Step 4: Reaction
    • Glycolysis Step 4: Mechanism, Part 1 (Binding & Ring Opening)
    • Glycolysis Step 4: Mechanism, Part 2
    • Glycolysis Step 4: Mechanism, Part 3
    • Glycolysis Step 4: Mechanism, Part 4
    • Glycolysis Step 4: Mechanism, Part 5
    • Glycolysis Step 4: Mechanism, Part 6
    • Glycolysis Step 4: Mechanism, Part 7
    • Hydrolysis of The Imine
  • Glycolysis Step 5: Conversion of Dihydroxyaceton Phosphate to Glyceraldehyde 3-Phosphate 55:38
    • Glycolysis Step 5: Reaction
  • Breakdown and Numbering of Sugar 57:40

Transcription: Glycolysis II

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

In the previous lesson, we did an overview of glycolysis.0004

Now, we are going to actually get into the details of glycolysis, and we are going to discuss each step of it in a reasonable amount of detail, concentrating on mechanism – that is what is going to be important - and a little bit about the enzymes; but mostly, it is about mechanism- how do these transformations take place.0008

Let’s just jump in and get started; OK, let me see.0025

I guess I will stick with black, so step 1 of glycolysis, it is going to be the phosphorylation of glucose.0032

It is going to be the conversion of glucose to glucose-6-phosphate.0041

It is the phosphorylation of glucose0046

In terms of an structures and reaction, you have something like this.0056

Let me do these, actually, in blue; it is a good idea.0063

We have our glucose molecule, and we have the C and the OH; and the reaction that takes place is the following.0069

And, of course, we have these biochemical symbols; ATP comes in.0087

ADP goes out; magnesium ion is required for this, and we usually go ahead and put the enzyme down below, and we said that the enzyme for this transformation is the hexokinase - OK - and we are left with the following.0094

We are left with C, O and a PO32-, the phosphoryl group on the no. 6 carbon of the glucose, so OH, OH and OH.0114

There you go; that is the transformation that takes place.0135

From this to this, this is what we have put on.0138

OK, now recall, oops, let me go back to black here.0142

Recall that a kinase - OK- is an enzyme that facilitates, catalyzes, the transfer of the terminal phosphoryl group, which I will go ahead and draw the picture here, P, O, O.0154

This is a phosphoryl group right here, right- PO32-, not PO43-.0198

It is phosphate that facilitates the transfer of the terminal phosphoryl from ATP to the substrate, and the substrate is the nucleophile.0205

This is actually a nucleophilic substitution reaction, and we will also say N.B. that hexokinase - now, we are getting into all the little details - the enzyme actually requires magnesium ion.0223

Actually, that is right there; it requires the magnesium Mg2+, and the ATP that is bound to the active site of the enzyme is actually Mg ATP 2-, not ATP 4-.0251

It is not just free ATP, not ATP 4-, so it looks like this.0283

We have O, P; actually, I am going to draw the structures in blue.0290

I will try to be as consistent as possible, although, no guarantees.0295

O, P, O, P, O, P, O, ribose and adenine, I think that is right.0300

I have 1; I have 2, and I have 3.0312

That is 1-; that is 2-.0316

That is 3-, and that is the 4-.0319

The Mg, the 2, actually coordinates here.0321

OK, what you are left with, 2+, 2-, you are actually just left with Mg ATP 2-.0326

That is what is in the active site; the magnesium is required by this enzyme to facilitate this process.0332

If you want, you can consider the magnesium as a coenzyme, if you want, but that is perfectly fine.0340

It needs it in order to do what it does.0345

OK, now, let's go ahead and take a look at the mechanism; again, it should reasonably familiar to you from organic chemistry- basic nucleophilic substitution reaction.0349

One last thing I would like to say; so hexokinase, like all of the enzymes for glycolysis, like all of the glycolytic enzymes, is soluble and cytosolic, so this takes place in the cytosol.0359

OK, now let’s take a look at the mechanism here.0394

A mechanism is how this transformation is affected; all the little details, the electron pushing- that is what we are going to be doing.0399

It is a simple nucleophilic substitution, pretty much.0405

You have a nucleophile; you have an electrophile.0418

It comes in, replaces, kicks something off- that is it.0420

Let’s go ahead and draw our glucose here; let me do this in blue.0424

We have our glucose molecule, and we have this C, and we have this OH up here on the…I will go ahead and put the hydrogens in this time.0430

That is that; this is going to be H and OH, and, of course, we have our ATP.0442

This is O, P, O, P, O, P, O, ribose and adenine.0451

We have our double bond to the phosphorus, double bond to the phosphorus.0461

We have that minus; we have that minus.0466

I am going to go ahead and leave the Mg off just for the sake that I do not want to make things too busy- alpha-phosphorus, beta-phosphorus, gamma-phosphorus, the terminal phosphor.0468

This is the phosphoryl group right here, this, this, this, this.0478

This comes in that way, kicks these electrons on to there.0482

What you end up with is your glucose 6-phosphate.0489

That is fine; I will go ahead and redraw it.0496

So, what you end up with is that, C, O; and you have the PO32-.0499

That is going to be attached to that; let me go ahead and finish this glucose off here, and we have this.0507

And, of course, now, we have adenosine diphosphate.0516

We have O, P, O, P, O ribose, adenine, double bond, double bond, there and there and there- that is it.0519

Just comes in, nucleophile kicks that off- very, very simple, very straight forward.0536

Well, we have glucose 6-phosphate.0545

This thing right here, what is actually formed is the glucose 6-phosphate.0551

I am going to actually do this a little bit better.0556

I am going to write Glc, and I am going to actually write that no. 6 carbon, Glc, O, P - so that we actually see the structure altogether, it is always nice to see structures - plus ADP.0560

This is 3- + H+, and this H+, you are wondering where it come s from.0578

You know what, I am sorry; let me do this a little bit differently.0594

Well, actually you know what, that was fine.0613

This right here, let’s try this again, so CH2, O, P.0617

Let’s do this, and this right here is our ADP.0626

This is ADP, and it is 3-; and we have the H+.0632

Now, this H+, this comes from the C6, OH deprotonating- losing a proton.0640

Again, we are just, sort of, keeping track of everything, that every little thing- that is what is going on.0658

When this OH attacks and goes over here, now, what you have is an oxygen that is attached to the carbon.0662

It is attached to the phosphorus, and it is attached to the hydrogen, so it is carrying a positive charge.0669

It releases that hydrogen to become just this because oxygen is divalent.0672

It prefers to have 2 things attached to it; I just wanted to show you where this thing actually comes from.0680

You are going to form glucose 6-phosphate; you are going to form ADP, and you are going to release a proton in the solution- that is it.0685

That is the mechanism; that is step 1 of glycolysis.0691

OK, let’s take a look at step 2; let’s go back to black here.0696

Step 2, this is the conversion of glucose-6-phosphate to fructose 6-phosphate.0703

In terms of structures, I think I will just going to go ahead and keep it black.0723

Let’s see; we have got this C, O and P.0728

I am just going to go ahead and write it as a P; we have this, that, that and that.0736

This is our glucose 6-phosphate, and this is going to be...0744

OK, and again, we are going to require magnesium for this, and the enzyme that catalyzes this is the isomerase, the hexose isomerase or let’s call it phosphohexose isomerase or isomerase; and the transformation we are affecting is this one: CH2, OH, OH, OH, OH.0751

And, of course, this C over here still has its phosphate group- that is it, glucose-6-phosphate to fructose 6-phosphate.0787

Let me number the carbons in red; this is 1, 2, 3, 4, 5, 6.0799

And now, we have 1, 2, 3, 4, 5, 6.0808

It is still a 6-membered sugar; it is just attached differently- that is all.0814

That is the numbering; this is our glucose 6-phosphate, and this is our fructpse-6-phosphate.0821

Now, the ΔG for this reaction is 1.7kJ/mol.0830

You notice, it is actually pretty small; it is essentially reversible that is why we wrote it this way instead of just in 1 direction like the previous reaction.0837

I apologize; I actually forgot to write the ΔG for that.0846

Let me tell you the ΔG for that reaction was -16.7kJ/mol, so it is highly exergonic.0851

In this particular case, 1.7, just slightly endergonic, but enough to make it reversible- not a problem.0860

OK, now, let’s talk about the mechanism for this reaction.0868

This is going to be our first real, reasonably complicated mechanism; it is not complicated.0872

It is just a little bit involved; let’s see what we can do.0877

I am going to go back to black here, so our mechanism.0882

Now, what is happening ultimately is that the oxygen C1 bond, this bond, is breaking and OC2 bond is forming.0890

OK, so let’s go ahead and start with…yes, I think I have enough room to do it in here.0916

Let’s go ahead and start off with our glucose molecule.0922

We have the C and the O and the P; this is our glucose 6-phosphate.0930

This is OH, OH, OH.0936

Alright, the first thing that is going to happen is going to be binding.0941

It is going to bind to the enzyme, and the ring is going to open- binding and ring opening.0946

We are going to end up with a straight chain version of this carbon; let me go ahead and draw that and I will draw the enzyme around it.0955

I have got 1, 2, 3, 4, 5 and 6.0963

I have got my aldehyde here; I have an OH group and an H group.0972

This is glucose, still; that is there.0979

This OH is there, and this OH is there; and this is going to be O, and this is our phosphate, right?0983

Let’s just make sure that we have everything straight, and if I make a mistake, please watch very, very carefully.0989

Now, this has bound to the enzyme; let me write the word “binding” a little bit better.0995

I get really, really, quick in my writing and I blow over the words.1002

It binds to the enzyme; the ring opens, and now, this is actually in the enzyme, in the active site of the enzyme.1008

I am going to draw the enzyme like this; OK, that is, sort of, the pocket of the enzyme.1016

Now, I am going to put a little B here with a couple of electrons, and this is just some amino acid residue that acts as a general base.1027

In other words, and we know what bases do: bases take protons.1037

Sometimes we are going to be writing it with an A, which is some acidic residue, some amino acid residue in the protein that happens to act as a general acid catalyst.1041

We know what acids do: they actually give protons- that is all.1054

When you see the A and the B- that is what is happening; sometimes, we will specifically list the particular residue.1058

We will actually say that it is a lysine or it is an arginine or whatever it is; but in this case, we are just taking about generic amino acid that acts as a base.1065

So, here is what happens; let’s go ahead and pull off this hydrogen here.1077

This base will pull off this hydrogen here; let me do this in blue actually since we want to see this, or maybe it is a better idea to do this one in red.1084

This will pull off this hydrogen; these electrons will go here, and they will pull an H from solution and attach it to the O.1097

I will go ahead and do that, and then what you end up with is the following.1115

OK, you end up with…let me see.1125

That is OK; I will go ahead and put it over here.1130

Oops, let me go back to black.1135

We have 1, 2, 3, 4, 5 and 6.1140

Now, what we have is an OH and an H.1146

We have a double bond there; we have another OH here, and then, of course, we have the rest of it.1153

We have the H; no, wait a minute.1163

We just pulled that H off; OK, we have our OH here, and then we have our OH here and our OH here.1166

And then we have our O and our P like that, and we have our enzyme, right?1176

This is our enzyme; now, we have this base that has a hydrogen attached to it.1183

So, we will put a little positive charge on there; this base is what took it.1189

Now, what happens is the following; let me go back to red.1194

Now, these electrons over here, they go down this way.1199

This goes here; the electrons hop back on to there.1207

OK, and when that happens, this hydrogen right here, once this binds, this ends up with a positive charge.1212

This is going to give up its hydrogen; this is going to turn into…let me see.1223

Let’s go ahead and do it this way; let me come down here.1233

Actually you know what, I think I am going to go over to this side.1240

I am going to go over here; I am going to write it as C, C, C, C, 1, 2, 3, 4, 5 and 6.1245

So, what I am left with is an OH here, an H there, a double bond there.1254

I have my OH here, my OH here, my OH here, my O and my phosphate group there- good.1264

OK, I have my enzyme, and now, my base is back to where it was because I took its proton, and these electrons ended up on it- not a problem there.1276

I will go ahead and an H leaves.1288

This H is this H, so I will put another 3 lines underneath it.1292

Once this actually forms the double bond, again, then now, it is carrying a positive charge because there are 3 bonds to the oxygen, so it is going to release that as a proton.1297

This is what you end up having; we basically changed.1304

A double bond has moved from the no. 1 carbon to the no. 2 carbon.1309

OK, so now, what happens is the following: 1, 2, 3, 4, 5.1313

Now, you have got the electrons on the hydroxy on the no. 5 carbon, will attack there; and this one will go ahead and pull another some hydrogen ion from the environment.1320

What you end up with is - 1, 2, 3, 4, 5 - a 5-membered ring.1340

The double bond has moved from the no. 1 carbon to the no. 2 carbon, and then the hydroxy closes the ring again.1350

The hydroxyl in the no. 5 carbon re-closes the ring to form the fructose instead of the glucose.1356

Now, what you have is ring closing, and - I do not know what it is, I cannot seem to write very clearly today, sorry about that- dissociation from the actual enzyme.1363

The enzyme will now release the substrate, and what you are left with is our final product, which is boom, boom, boom, boom, boom.1385

What you have is C, OH.1396

Yes, this H is this H, and then you have OH.1407

You have OH; you have OH, and, of course, we still have our C, which is our 6 attached to the phosphate.1414

There you go- from glucose 6-phosphate to fructose 6-phosphate.1422

That is the mechanism; OK, and hopefully, it does not look like I have missed anything here.1428

We have got some general base catalysis; a base takes a hydrogen1433

A base gives the hydrogen back, and now, it is back to where it started.1437

That is what an enzyme does; it always goes back to where it began.1441

OK, so let’s see what we have got here.1446

OK, now, we are on step 3, so step 3.1454

We have the conversion of fructose 6-phosphate to fructose 1,6-biphosphate.1461

OK, that is going to look like this.1479

C, OH, OH, OH, OH, C, O and P, and this is going to go that way.1486

And again, we are going to have this ATP come in.1500

ADP is going to leave; Mg2+ is going to be required, and this is phosphofructokinase-1, better known as PPK-1.1505

And what you are going to end up with is the same molecule, except now, you have a C, an O and a P.1525

You know what, let me make this a little bit smaller so that we have a little bit more room.1533

OK, let me make sure this is properly erased.1544

We have got zero there, there - not zero, O, sorry.1548

We have C, O and our phosphate.1553

We have our C, O and our phosphate there; we have our OH here.1556

We have our OH here, and we have our OH here.1561

This is our fructose 6-phosphate.1565

This is our fructose 1,6-biphosphate.1572

Woo, all these structures, all these names, they will make you crazy.1577

OK, now, let’s go back to black; now, the ΔG for this reaction is equal to -14.2, and this is kJ/mol.1587

OK, now, the mechanism is exactly like it was before.1600

It is just the basic nucleophilic substitution reaction.1605

It is nucleophilic.1610

This, right here, is what accessed the nucleophile, and that is it.1616

It attacks the terminal phosphoryl, the ATP and it is transferred over nucleophilic substitution - OK - at the oxygen of C1; and this is the C1 now.1621

Red, that is the C1; that is all that is happening.1648

OK, let’s go back here; now, let me do this one in red.1654

OK, this step is irreversible - as you can see from the highly -ΔG - under cellular conditions.1662

There are certain reactions in the glycolytic pathway that have high -ΔGs but under standard conditions, under cellular conditions, they can actually be quite reversible.1680

In fact, we will see one in just a minute, but this one, under cellular conditions, is irreversible - OK - and is the step that actually commits to glycolysis.1694

OK, the glucose 6-phosphate and the fructose 6-phosphate, they can go down other paths, but the fructose 1,6-biphosphate, it means glycolysis- that is it.1722

It is committed to glycolysis.1752

Let’s make this a little clear just in case.1758

OK, now this PPK-1, it is a regulatory enzyme and is the primary regulation point.1777

This step is the primary regulation point for glycolysis.1797

OK, say just a couple of more things about this particular enzyme here.1816

Low ATP levels, it actually activates this enzyme.1821

Low ATP levels activate the PPK-1, and sufficient ATP levels inhibit shutdown PPK-1.1836

This is a primary control point to tell the body "OK, engage in glycolysis or slow down glycolysis or stop glycolysis".1860

OK, now, let's go ahead and talk about step 4.1871

Step 4 involves the cleavage of the fructose 1,6-biphosphate- very, very important reaction here.1877

This is where we are going to actually break it down; OK, let's go ahead and draw out what this looks like.1895

That is OK; I will go ahead and leave this in black.1904

We have zero - O, sorry, I keep saying zero, I keep thinking about mathematics - C, O and P; and we have OH, and we have OH.1908

We have OH, and we have CO; and we have P.1928

We have our fructose 1,6-biphosphate.1934

I am going to number these, actually, in just a minute.1942

This is going to be the enzyme aldolase or aldolase, and we are going to end up forming...I will go ahead and put them here.1947

C, C, C, that is there, H; and this is going to be O, and this is going to be P.1961

This is going to be glyceraldehyde-3-phosphate + the dihydroxyacetone phosphate.1973

I will go ahead and put the carbonyl over here.1983

I will put the O and the P, and I will go ahead and put the OH here, and I will go ahead and put the H2 over here.1987

Well, this right here is our fructose 1,6-biphosphate.1996

This is our glyceraldehyde-3-phosphate, and this is our dihydroxyacetone phosphate.2004

And now, let's go ahead and number some of the carbons; this is 1, 2, 3, 4, 5, 6.2015

Our glyceraldehyde is going to come from carbon 4.2024

That is 5; that is 6, and again, we will do this a little bit later.2029

It is not a problem, 4, 5, 6; and this is going to be 1, 2, 3, 1 on the phosphate, right?2033

That is correct, 1, 2 and 3.2040

I think I have got that right; now, the ΔG for this particular reaction is equal to 23.8kJ/mol.2046

Now, notice, this is highly endergonic; however, this is standard conditions.2057

Now, under cellular conditions, the ΔG is quite small; and the reaction is quite reversible, which is why we wrote it that way.2062

Again, just because we have a standard ΔG, the standard is what we use general, it is our standard.2094

It is our point of reference; however, under cellular conditions are different- different concentrations.2105

Under cellular conditions, this is actually a reversible reaction, not an endergonic reaction.2111

OK, it is quite reversible; so now, let's get into the mechanism of this thing.2117

This is going to be a little bit long, but it is reasonably straightforward.2122

Let us start; I will go ahead and go back to black.2127

Our mechanism, alright, let's go ahead and start with our fructose molecule.2132

This is CO, and then this is P.2145

We have OH, OH, OH; and we have CO there.2152

We have our fructose 1,6-biphosphate.2158

The first thing that is going to happen is binding to the enzyme and ring opening.2162

And now, I am going to draw out the molecule in its open form and the surrounding enzyme.2175

We have a 6 carbon; we have C, C, C, C, C, and we have C.2183

Now, we have that.2190

Remember, we have our carbonyl on our no. 2 carbon now?2196

This OH group is here; this OH group is here.2201

This OH group is here; and, of course, we have another phosphate group there.2204

Now, let me draw my enzyme around it; this is, now, in the active site of the enzyme, a little pocket.2209

And now, I have got an actual lysine residue; and this one, I will say specifically, this is a lysine residue that is going to be responsible for a lot of the chemistry here.2218

We have another general base catalyst; we have another general base catalyst, and we have an acid catalyst, which acid means, it is actually attached to some H.2229

It has an H to give up, so just some general catalyst.2244

This is our enzyme.2249

There we go; OK, here is what happens first.2259

Let me go ahead and do this in red; these electrons on the nitrogen, they come here.2264

The double bond goes after that, and the electrons are shifted over that way.2270

What happens next?2278

OK, now, what you have is the following.2281

Well, let's go back to black.2289

We have C, C, C, C, C, C.2295

Now, we have O and P.2300

Now, we have an OH on that no. 2.2305

And this is, of course, now, attached covalently to the nitrogen of the lysine.2311

Let me go ahead and put my OH there, my OH here, my OH here.2319

Let me make sure I have everything drawn out.2325

N, and we have an H there; we have an H there.2329

And this is, of course, attached to lysine; so let me redraw my enzyme.2334

That is like that, and we said we had a base over here.2341

We have a base over here; now, we have an acid, which is carrying a...the electrons are, now, on the acid, so it is like that.2346

What happens next is the following.2356

Let me go ahead and just draw the...this is the enzyme, our pocket.2359

Let me go back to red and talk about what happens next.2369

OK, these electrons on the general base catalyst, they end up taking the...I am going to put this H here.2373

This H is part of that; it actually takes the H on the nitrogen.2386

Let me go back to red; these electrons move over there.2394

And let me go ahead and show my black.2403

Actually, you know what, I am going to keep this as red; I am going to do this, and I am going to go, say, H+, H2O.2408

OK, that way, OK.2423

These electrons on this base go ahead and pull a hydrogen off of the nitrogen.2427

The electrons on the nitrogen go here to form a double bond with the carbon.2432

OK, and then, it is going to push these electrons; these electrons are going to go and take a hydrogen from the environment, and this is where the water comes from.2437

This OH and this H+, water is released from this; and what you end up with is the following.2448

You end with C, C, C, C, C, C.2457

You end up with this amine, this shift base; you have got that.2465

You have got the O, and you have got the P.2469

You have an OH here, an OH here; let me go ahead and make sure all of these are taken cared of before I deal with anything else.2473

Actually, you know what, I am going to have to make this a little bit further to this side because I need more room on the right.2480

My apologies; let me make sure these get erased properly.2487

OK, and make this a little bit closer here, so C, C, C, C, C, C.2493

We have this thing attached to the nitrogen; we have our hydroxy there.2501

We have our hydroxy there, our hydroxy there.2507

We have the oxygen; we have the phosphate.2511

And over here, we still have our O and our phosphate there.2514

Now, let's go ahead and draw in our enzyme.2520

OK, this is, of course, connected to a lysine residue.2525

We have our base that now, has an attached hydrogen, right?2532

We still have our general acid, which is containing its electrons, so it is there; and this base is right there.2537

Now, we still have this hydrogen attached, so this nitrogen is carrying a positive charge.2547

OK, there we go.2555

And now, let me say a couple of things about it; we put this in blue.2560

This nitrogen, double bonded to a carbon, OK, this thing, this linkage, OK, if you do not remember from organic chemistry, it is called an imine or an imine.2567

I say imine; some people say imine.2587

I do not know, or you probably hear it more often referred to as a shift base.2588

It is carbonyl, not carbonyl; it is a carbon double bonded to a nitrogen, and this happens to be a protonated shift base.2595

Protonated just means that the nitrogen is carrying a hydrogen, so it is carrying a positive formal charge- that is all that is going on there.2601

OK, now, the next step is going to be the following, and this is really, really interesting.2608

Let me see if we can actually follow this along.2613

OK, let's go to red; now, these electrons, OK, let's see what we can do.2622

Let me write this H a little bit differently here.2630

Let me actually put the bond there.2635

Let me go back to red; we are going to take this proton.2640

These electrons are going to come here to form a carbonyl.2645

These electrons is going to push these electrons up here - OK - to form a double bond.2650

It is going to push these electrons back on to the nitrogen to take care of that positive charge, and let me see if we have missed anything here.2659

This is the bond that we are breaking right here, right?2670

1, 2, 3, 1, 2, 3, we are going to break it up into 2, 3, carbon fragments- that is what is happening here.2674

Now, let's go ahead and come down here.2682

This is all so reversible, so let's rewrite what it is that we have got.2688

Let me actually draw the individual fragments.2696

We have C, C and C; and we have C, C and C.2700

We have our O, and we have our phosphate.2708

We have, now, a single bond to the nitrogen, H; and this is attached to the lysine.2712

We actually have a double bond there, right?2720

We actually formed a double bond right there, and we still have our OH group there.2722

Here, we have our H; this double bond that we formed, this is an H.2729

We have our hydroxy, and then we have our O and our phosphate.2737

Hopefully, I have not missed anything here; now, let me go ahead and draw my enzyme- there we go.2744

OK, now, our base actually has an attached hydrogen, so that is going to be that.2757

Our acid is still there; our base still has its hydrogen attached.2765

Let's go ahead and put that, and now, the lysine is, of course, attached that way; so it looks like we have got everything.2772

Notice, now, we have a 3-carbon fragment that is still attached to the enzyme through this nitrogen bond, and we have this 3-carbon fragment, which is the glyceraldehyde-3-phosphate, which is entirely free.2778

At this point, the enzyme releases the glyceraldehyde-3-phosphate.2790

I will go ahead and write that; this molecule, right here - let me do this in blue - is released by the enzyme.2796

This is going to be our C, C, C; and this is going to be our glyceraldehyde-3-phosphate.2806

OK, now, what we are left with is just this species that is covalently attached to the enzyme, still, through this nitrogen bond; and let's see what we can do with that one.2817

Oh, I guess I should probably say, you see this linkage right here, this double bond N?2832

Just so you know, this linkage is called an enamine.2843

OK, that right there, you have and en, an alkene, and an amine' so this is called an enamine.2849

Let me see if there is anything else that we want to say about this; we have released our glyceraldehyde-3-phosphate.2857

This is glyceraldehyde-3-phosphate, and now, let's take care of the next species.2862

Let's go ahead and draw - let's go back to that - out our 3-carbon fragment.2870

This is actually a double bond; this is an N.2882

This is lysine.2888

We still have our enzyme here.2893

We have our base there with the plus charge.2897

We have our base here with this plus charge.2904

We have our A, the minus charge, electron because now, it is carrying the electron.2909

I will go ahead and do that; this is O, and this is a phosphate.2917

This is H, and this is OH, correct?2923

So far, so good; OK, here is what happens.2927

Oops, I forgot that, and, of course, I have the electrons here; now, let me go to red.2931

Let me see; this is going to go and grab...alright.2936

Now, these electrons on the nitrogen are going to move over here.2941

It is going to push these electrons to come and grab that, and it is going to push this back on to that.2946

What you end up with is the following arrangement; you are going to end up back with your imine, your shift base linkage.2953

Let me go back to black here; we have C, C and C.2963

Let's go ahead and do that, H.2969

Let's go ahead and put a positive charge there; it is connected to our lysine.2973

This is O, and this is P; and we have an H here, and we have our hydroxy there.2978

Our enzyme is, now, this way.2985

Now, we have recovered that base there.2990

We have recovered the base here, and we still have our A with that.2996

Now, let's see what happens.3003

Wait, let me make sure I have got everything written out, plus charge, carbon.3008

We have our imine; OK, we have our enzyme.3013

It looks like everything is there; it does not look like I have forgotten anything.3018

OK, now, what we are left with is the following.3021

Now, what happens...OK, from organic chemistry, hopefully, you remember; if not, it is not a problem.3026

I will actually go ahead and do the mechanism in just a minute, but I am going to draw it a little bit differently here.3033

What happens is the following.3040

Water comes in to actually break this bond, to hydrolyze this bond.3045

When water hydrolyzes this bond, it releases this dihydroxyacetone phosphate, so this is what goes away.3052

What ends up going away is this molecule, and that is going to be the C, C, C, O, phosphate, hydroxy.3062

I will draw it exactly as is; water is going to come in.3075

It is going to break this imine bond, and it is going to release this second substrate; and it is going to leave the enzyme as lysine, NH2, B, B, and H, A.3078

The enzyme goes back to what it goes to, and now, what I am going to do is in the next page, I am going to actually draw out the mechanism for this cleavage just so you know.3107

You can see it in any organic chemistry textbook; I do not think this particular mechanism is in your biochemistry textbook, but you can find it in your OCAM textbook, but I will go ahead and go through it.3117

Let me just write out what happens with this one.3127

H2O, it hydrolyzes the NC imine bond.3131

It breaks that bond right there, and it releases everything.3149

OK, let's go ahead and take a look at how that happens.3153

Again, it is nice to have as much detail as possible; let me go ahead and do this one in black.3157

Hydrolysis of the imine goes like this.3164

OK, we have our lysine; we have our N.3181

We have our double bonded to the C, C, C; I am not going to draw everything out because we are just concerned with this linkage, right?3185

OK, H2O, couple of electrons, comes in, attacks right there; that pushes that double bond.3196

It grabs a hydrogen ion from the environment, and it turns into the following.3207

It turns into lysine, N, H, C, H + C, C, O, H.3213

This is H2O; H2O comes in.3238

Well, now, when H2O is attached to this carbon, it is going to be like this.3242

It is going to release one of those into solution, that is why I have it this way.3246

So, if you want, I can put a little -H+ there to let you know that the water is actually going to release one of its hydrogen ions.3250

Well, this actually grabs a hydrogen ion; it is not the same hydrogen ion.3258

This one released; it is not the one that is attached.3260

This happens to be hydrogen ion that is available in the particular medium that is taking place.3263

What you are left with is this; now, what happens is it continues on.3269

You have an electron on the oxygen, which comes here, and it actually kicks this off to quench this positive charge.3274

Now, you end up releasing; now, you have your lysine, and you have your NH2 that is recovered.3281

And then, of course, you have your C, C, C.3290

And again, we are going to lose a hydrogen ion; once these electrons come here, now, you are going to have an oxygen that has 3 bonds.3295

It is going to release this hydrogen into solution leaving just the carbonyl.3302

And now, you have your O and your P; and, of course, you have your OH and your H, and that is the dihydroxyacetone phosphate.3308

This is the mechanism for imine hydrolysis, for shift base hydrolysis.3317

OK, now, one last thing and I think we should be OK.3324

I wonder if I should do this on the next page.3332

No, you know what, I can do it on this page; that is not a problem.3336

That was step 4; now, we will do step 5 to close off the preparatory phase of glycolysis.3340

Step 5 is conversion of the dihydroxyacetone phosphate to the glyceraldehyde-3-phosphate.3346

OK, we have a 3-carbon fragment; we have C, C and C.3377

This is there; this is O.3384

Excuse me; I will go ahead and put the hydroxy on this side for now.3387

And this is going to be a reversible reaction, and we are going to form C, C, C.3393

And we are going to form the aldehyde down here, OH, and we have O; and we have our P.3403

This is our dihydroxyacetone phosphate; this is our glyceraldehyde-3-phosphate.3415

1, 2, 3 and the ΔG for the O; let's go ahead and write down the...this is triosephosphate isomerase or isomerase, and the ΔG for this reaction equals 7.5kJ/mol.3422

OK, let's see; let's go ahead and leave that there.3456

Alright; OK, now, the final thing that I am going to talk about is just the actual breakdown of the sugar and the numbering of the carbons just so we see what it is that we are actually looking at.3460

Let me go ahead and do this in red just for a little change of pace.3472

We have got 1, 2, 3, 4, 5, 6, 1, 2, 3, 4, 5, 6.3477

OK, we have this, and we have our phosphate.3484

We have our carbonyl there; we have a hydroxy there.3489

We have a hydroxy there; we have a hydroxy there, and we have that one.3494

Now, let's go ahead number the carbons; I am going to number them 1, 2, 3, 4, 5 and 6 in blue.3502

We have taken this fructose 1,6-biphosphate and we split it right after the no. 3 carbon.3520

OK, now, this part, that became the following: C, C, C, O, P, right?3526

That is the carbonyl; that is the hydroxy.3544

This became the dihydroxyacetone phosphate.3548

the 1, 2, 3 carbon, that became the dihydroxyacetone phosphate.3552

The 4, 5, 6 carbon, it turned into C, O, H.3558

This O becomes oxidized to the carbonyl; this C retains its hydroxy, and this C retains its phosphate group.3571

This is 4.3582

Let's go to black; this is 1.3589

This is 2; this is 3.3591

This is 4; this is 5, and this is 6.3594

Now, this right here turns into C, C, C.3598

It is the no. 3 carbon that ends up getting oxidized to the carbonyl.3611

This hydroxy stays the same, and this stays the same.3615

Now, we have the 1, the 2 and the 3.3619

Now, if I were to flip this - OK, let me flip this over - I have got C, C, C.3626

I have 3; let me do the numbers afterwards- 3, 2, 1.3640

In our final products of the glyceraldehyde-3-phosphate, there is 1 molecule of it.3660

There is another molecule of it; one of those molecules comes from the 1, 2, 3 carbon.3669

It is the 3 that ends up with the aldehyde group; another one comes from the 4, 5, 6.3674

It is the no. 4 carbon that is carrying the aldehyde group.3679

So, this is an accounting of what carbons turn into what, and what functional groups are attached to what carbon.3683

There you go; this is a detailed discussion of the preparatory of glycolysis- the first 5 steps.3692

In the next lesson, we will, of course, discuss the last 5 steps- the conversion to pyruvate.3698

Thank you so much for joining us here at Educator.com3703

We will see you next time, bye-bye.3707