Raffi Hovasapian

Glycolysis III

Slide Duration:

Table of Contents

Section 1: Preliminaries on Aqueous Chemistry

Aqueous Solutions & Concentration

39m 57s

Intro

0:00

Aqueous Solutions and Concentration

0:46

Definition of Solution

1:28

Example: Sugar Dissolved in Water

2:19

Example: Salt Dissolved in Water

3:04

A Solute Does Not Have to Be a Solid

3:37

A Solvent Does Not Have to Be a Liquid

5:02

Covalent Compounds

6:55

Ionic Compounds

7:39

Example: Table Sugar

9:12

Example: MgCl₂

10:40

Expressing Concentration: Molarity

13:42

Example 1

14:47

Example 1: Question

14:50

Example 1: Solution

15:40

Another Way to Express Concentration

22:01

Example 2

24:00

Example 2: Question

24:01

Example 2: Solution

24:49

Some Other Ways of Expressing Concentration

27:52

Example 3

29:30

Example 3: Question

29:31

Example 3: Solution

31:02

Dilution & Osmotic Pressure

38m 53s

Intro

0:00

Dilution

0:45

Definition of Dilution

0:46

Example 1: Question

2:08

Example 1: Basic Dilution Equation

4:20

Example 1: Solution

5:31

Example 2: Alternative Approach

12:05

Osmotic Pressure

14:34

Colligative Properties

15:02

Recall: Covalent Compounds and Soluble Ionic Compounds

17:24

Properties of Pure Water

19:42

Addition of a Solute

21:56

Osmotic Pressure: Conceptual Example

24:00

Equation for Osmotic Pressure

29:30

Example of 'i'

31:38

Example 3

32:50

Biochemistry Glycolysis III

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Section 9: Glycolysis and Gluconeogenesis: Lecture 3 | 59:17 min

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Post by Sara Tee on September 21, 2020

Hello prof Raffi,

I finished watching the entire Metabolism series. I tried to string things together. I have one question regarding the reduction of NAD+. You know how there is always an H+ as the product of NAD+ reduction (NAD+ --> NADH + H+). For some reaction such as reaction 3 in Citric acid cycle (oxidation of Isocitrate to alpha-ketoglutarate), I understand the H+ comes from the H on the O of the HO-C-H (NAD+ get the Hydride attached directly to the C).

However, for reaction 6 of glycolysis, the area that reduce NAD+ was not HO-C-H; it is rather H-C=O. I can see the Hydride on the substrate that reduces NAD+; I don't know where the H+ come from for this particular rxn 6 of Glycolysis. Could you help me clarify?

Many thanks!

1 answer

Last reply by: Professor Hovasapian
Tue Jul 31, 2018 12:30 AM

Post by Swati Sharma on July 27, 2018

Dear Professor,

I carefully studied all the mechanisms as you were writing I was writing too . I was wondering if these mechanisms would be asked in the MCAT Biochemistry section or what kind of questions should I be prepared from on the mechanisms?. I would be grateful if you could just let me know any kind of random questions on mechanisms.

I am preparing for MCAT on my own I just hope I am doing fine. I am preparing from your detailed lectures so that I can deal with any Biochemistry passages.

Regards
Swati

1 answer

Last reply by: Professor Hovasapian
Fri Mar 28, 2014 5:32 PM

Post by Alan Delez on March 26, 2014

Hi Dr. Hovasapian,

On step 10 I was wondering why the carboxylate does not protonate

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Glycolysis III

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

Glycolysis Step 5: Conversion of Dihydroxyaceton Phosphate to Glyceraldehyde 3-Phosphate

Glycolysis Step 6: Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Biphosphoglycerate

Glycolysis Step 7: Phosphoryl Transfer From 1,3-Biphosphoglycerate to ADP to Form ATP

Glycolysis Step 8: Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate

Glycolysis Step 9: Dehydration of 2-Phosphoglycerate to Phosphoenol Pyruvate

Glycolysis Step 10: Transfer of a Phosphoryl Group From Phosphoenol Pyruvate To ADP To Form ATP

Glycolysis Balance Sheet

Intro 0:00
Glycolysis Step 5: Conversion of Dihydroxyaceton Phosphate to Glyceraldehyde 3-Phosphate 0:44

Glycolysis Step 5: Mechanism, Part 1
Glycolysis Step 5: Mechanism, Part 2

Glycolysis Step 6: Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Biphosphoglycerate 5:14

Glycolysis Step 6: Reaction
Glycolysis Step 6: Mechanism, Part 1
Glycolysis Step 6: Mechanism, Part 2
Glycolysis Step 6: Mechanism, Part 3
Glycolysis Step 6: Mechanism, Part 4

Glycolysis Step 7: Phosphoryl Transfer From 1,3-Biphosphoglycerate to ADP to Form ATP 19:08

Glycolysis Step 7: Reaction
Substrate-Level Phosphorylation
Glycolysis Step 7: Mechanism (Nucleophilic Substitution)

Glycolysis Step 8: Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate 28:44

Glycolysis Step 8: Reaction
Glycolysis Step 8: Mechanism, Part 1
Glycolysis Step 8: Mechanism, Part 2
Glycolysis Step 8: Mechanism, Part 3
Catalytic Cycle

Glycolysis Step 9: Dehydration of 2-Phosphoglycerate to Phosphoenol Pyruvate 37:20

Glycolysis Step 9: Reaction
Glycolysis Step 9: Mechanism, Part 1
Glycolysis Step 9: Mechanism, Part 2
Glycolysis Step 9: Mechanism, Part 3

Glycolysis Step 10: Transfer of a Phosphoryl Group From Phosphoenol Pyruvate To ADP To Form ATP 45:16

Glycolysis Step 10: Reaction
Substrate-Level Phosphorylation
Energy Coupling Reaction

Glycolysis Balance Sheet 54:15

Glycolysis Balance Sheet
What Happens to The 6 Carbons of Glucose?
What Happens to 2 ADP & 2 Pi?
What Happens to The 4e⁻ ?

Biochemistry Online Course

Section 1: Preliminaries on Aqueous Chemistry
	Aqueous Solutions & Concentration	39:57
	Dilution & Osmotic Pressure	38:53
	More on Osmosis	29:01
	Acids & Bases	39:11
	Titrations and Buffers	41:33
	Example Problems with Acids, Bases & Buffers	44:19
	Hydrolysis & Condensation Reactions	18:45
Section 2: Amino Acids & Proteins: Primary Structure
	Amino Acids	38:19
	Amino Acids, Continued	27:14
	Acid/Base Behavior of Amino Acids	48:28
	Peptides & Proteins	45:18
	Amino Acid Sequencing of a Peptide Chain	42:47
	Sequencing Larger Peptides & Proteins	1:02:33
	Peptide Synthesis (Merrifield Process)	49:12
	More Examples with Amino Acids & Peptides	54:31
Section 3: Proteins: Secondary, Tertiary, and Quaternary Structure
	Alpha Helix & Beta Conformation	50:52
Section 4: Proteins: Function
	Protein Function I: Ligand Binding & Myoglobin	51:36
	Protein Function II: Hemoglobin	1:03:36
	Protein Function III: More on Hemoglobin	1:07:16
Section 5: Enzymes
	Enzymes I	41:38
	Enzymes II	44:02
	Enzymes III: Kinetics	56:40
	Enzymes IV: Lineweaver-Burk Plots	20:37
	Enzymes V: Enzyme Inhibition	51:37
	Enzymes VI: Regulatory Enzymes	51:23
	Enzymes VII: Km & Kcat	54:49
Section 6: Carbohydrates
	Monosaccharides	1:17:46
	Hexose Derivatives & Reducing Sugars	37:06
	Disaccharides	43:32
	Polysaccharides	39:25
	Polysaccharides, Part 2	44:15
	Glycoconjugates	44:23
	More Example Problems with Carbohydrates	40:22
Section 7: Lipids
	Fatty Acids & Triacylglycerols	54:55
	Membrane Lipids	38:51
	Membrane Lipids, Part 2	38:20
	The Biologically Active Lipids	48:36
Section 8: Energy & Biological Systems (Bioenergetics)
	Thermodynamics, Free Energy & Equilibrium	45:51
	More on Thermodynamics & Free Energy	37:06
	ATP & Other High-Energy Compounds	44:32
	Phosphoryl Group Transfers	30:08
	Oxidation-Reduction Reactions	49:46
	More On Oxidation-Reduction Reactions	56:34
	Example Problems For Bioenergetics	42:12
Section 9: Glycolysis and Gluconeogenesis
	Overview of Glycolysis I	43:32
	Glycolysis II	1:01:47
	Glycolysis III	59:17
	Glycolysis IV	39:47
	Gluconeogenesis I	41:34
	Gluconeogenesis II	34:18
	The Pentose Phosphate Pathway	42:52
Section 10: The Citric Acid Cycle (Krebs Cycle)
	Citric Acid Cycle I	36:10
	Citric Acid Cycle II	49:20
	Citric Acid Cycle III	44:11
Section 11: Catabolism of Fatty Acids
	Fatty Acid Catabolism I	48:11
	Fatty Acid Catabolism II	45:58
	Fatty Acid Catabolism III	33:18
Section 12: Catabolism of Amino Acids and the Urea Cycle
	Overview & The Aminotransferase Reaction	40:59
	Glutamine & Alanine: The Urea Cycle I	39:18
	Glutamine & Alanine: The Urea Cycle II	36:21
	Amino Acid Catabolism	47:58
Section 13: Oxidative Phosphorylation and ATP Synthesis
	Oxidative Phosphorylation I	41:11
	Oxidative Phosphorylation II	36:27

Transcription: Glycolysis III

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

In the last lesson, we talked about the first phase of glycolysis often called the preparatory phase.0004

And today, we are just going to continue on until the end of glycolysis, what they often call the pay-off phase.0010

Again, there is no real reason to break this up into this place or that place.0017

I mean, glycolysis is this pathway with 10 reactions, but sometimes, I think it helps to categorize it in a certain way; but it is not absolutely necessary.0021

Do not feel that you have to know pay-off phase or preparatory phase or things like that.0034

It is the reactions that are important, the mechanisms and the enzymes; that is what we want you to concentrate on.0038

OK, so let’s get started; I am going to do just a little bit of quick recap from the last steps.0044

In the last lesson, I actually left off the mechanism before step 5, which was the conversion of the dihydroxyacetone phosphate to the glyceraldehyde-3-phosphate.0051

I am going to go ahead and do that really quickly, and then we will jump into step 6 through 10.0061

Let’s see here.0068

In the last lesson, I left off the mechanism for step 5, which was the dihydroxyacetone phosphate to the glyceraldehyde 3-phosphate.0073

Here we go; let’s go ahead and draw our molecule here.0101

Yes, let me see; let me go ahead and do this in blue, I think.0107

OK, we have got C, C, C, and we have got OH.0111

I will put an H there; I will put an H there.0116

We have our carbonyl; let me make that double bond a little bit more clear here.0120

We have our carbonyl there, and we have our C, our O and our P.0125

This is the phosphoryl group, and let me go ahead and draw my enzyme.0130

Actually, you know what, yes, that is fine; I will go ahead and draw my enzyme, something like that, and we have just some B, some general basic group that is going to involve some of this general base catalysis.0136

It is going to abstract the proton, and then, of course, general acid catalysis is the donation of a proton.0154

This is our enzyme, and here is the dihydroxyacetone phosphate inside the pocket of the enzyme.0161

This is going to abstract this proton, and these electrons are going to move over here; and these electrons are going to go ahead and grab a hydrogen ion from the environment, and what you end up getting is this now.0170

We have - let me draw the molecule first - C, a double bond C, single bond C; and then we have our O - let’s see, O - and let me go ahead and put this H here.0185

Let me put this H here; I have an OH here, and I have an O, and I have the phosphate.0201

Let me see if I have forgotten anything; and again, I am not going to put down all of the hydrogens.0211

The ones that are unnecessary I will just leave off just to save a little bit of space.0217

Let me go ahead and mark off the enzyme again, so this is our enzyme.0221

And, of course, now, our base is attached here, so let’s put a little positive charge on there.0226

Now, what is going to happen is these electrons are going to pop down here.0233

These are going to abstract this hydrogen; these electrons are going to move back to the base to recover the base, and what you end up with once you lose this hydrogen ion...0243

When these electrons come down here to form the carbonyl - this oxygen, now, is carrying a positive charge - it is going to release this hydrogen, so that it is left only with the carbonyl.0260

What you end up with - and let’s go ahead and write released by enzyme - and then, what you get is - of course, I will put the carbonyl there, I will put the hydrogen there, I will put the OH group there, put that there, and I will put that like that, I will go ahead and put the H₂ here - what you have is the glyceraldehyde-3- phosphate.0270

This is the mechanism for step 5; OK, let me write this down: glyceraldehyde-3-phosphate.0297

That is the mechanism for step 5; now, we can go ahead and jump into the rest of glycolysis.0306

OK, let me go back to black here.0311

Step 6 is going to be the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate- very, very important step.0320

This is where it actually starts the real process- oxidation of the glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate.0328

Woo, these names are long; OK, here is the over-all reaction.0351

Let’s see; we have got our...let me put the carbonyl up there, H.0357

We have OH, and we have O; and we have P.0366

I will do that; we have H here.0371

We have H₂; this is going to be plus our inorganic phosphate.0374

Let me go there; inorganic phosphate is going to be actually one of the reactants here.0382

I will go ahead and do a double arrow this way, and I will go ahead and do…0387

NAD⁺ comes in, and NADH and H⁺ leave.0395

This is an oxidation; this is oxidized.0403

The NAD⁺ is reduced to NADH, and the enzyme that facilitates this, catalyzes this - let me see - glyceraldehyde-3-phosphate dehydrogenase; and we know what dehydrogenases do.0407

They take away hydrogens; that is what they do, and they use NAD⁺ to do it.0432

OK, now, the final products of this is going to be the 1,3-bisphosphoglycerate.0438

That is going to look like this: C, C and C, and let’s go ahead and leave that carbonyl there.0444

We have gone ahead and attached - let me draw the full structure out, and then, of course, I will do a shorthand notation a little bit later - OH and H there; and we have the O, and we have the PO₃^2-.0452

This PO₃^2- and this PO₃^2-, they are the same thing.0467

It is just here, I have drawn out the structure; here, I have just used the shorthand.0472

And again, I will just go ahead and put 2 hydrogens there; OK, there you go.0475

This is the conversion: glyceraldehyde-3-phosohate to 1,3-bisphosphoglycerate.0479

It is an oxidation; it involves the phosphorylation and oxidation.0484

Notice, it is not just the P; it is the actual O and the P.0490

Everything is attached, so the ΔG for this is 6.3kJ/mol.0494

OK, now, let’s go ahead and run through the mechanism for this.0505

A little bit of an involved mechanism, nothing really strange happening, just, it seems like a lot is going on.0509

Let’s go through this very, very carefully; it is a very important mechanism.0516

Let’s go ahead and do this one in…I think I will go ahead and do this one in blue.0521

Well, you know what, yes, that is fine; I will go ahead and do it in blue.0528

OK, so let me go ahead and write "mechanism" here.0532

It is going to take me a little bit to draw everything out; I think that is going to be the slowest part here.0538

I apologize for that.0543

Again, illustrations are really, really nice, but when doing mechanisms, well, when doing everything, I really, really prefer to write everything out because again, the act of writing things out manually really, sort of, solidifies them in your mind.0548

You have to be able to draw the structures; you have to be able to move electrons from one place to another.0560

It is one thing to understand what is happening just by looking at it passively; it is another to actually do it actively- completely different.0566

OK, let’s go ahead and draw the little enzyme here.0573

OK, we have got NAD⁺ in this little fold, and let’s go ahead and put our glyceraldehyde 3-phosphate molecule in here.0578

But, first of all, let’s go ahead and put some…actually yes, let me go ahead and do that now.0587

I have got C - I wonder if…yes it is fine, I am not going to keep going back and forth - C, C, C.0593

And notice, I have actually put the aldehyde down at the bottom; here, I put it up at the top, but I put it down at the bottom to show how the mechanism is actually working.0601

I am going to go ahead and put this H here.0609

I will put this OH here, and I will put O; and I will put the PO₃^2- up here.0613

And again, I am going to leave off some of the hydrogens that are…well, maybe not.0618

I will go ahead and put it there; I will go ahead and put that here.0622

OK, we have got a cysteine residue, and there is an S and an H.0627

Remember cysteine, it has that sulfur.0634

And then, of course, over here, there is a histidine residue, and this is going to be very, very important, this histidine.0638

We have got boom, boom.0645

It has this 5-membered ring with some nitrogens in there and some double bonds.0650

And, of course, this nitrogen has an electron pair on it, so it is going to act as a base.0659

It is going to do some abstraction; here is the first thing that happens.0668

These electrons take this hydrogen.0674

These electrons are pushed, and they attack the carbonyl; and these electrons jump onto the oxygen.0679

OK, so what you end up with is the following.0688

Let me go ahead and redraw, like that.0693

We have our NAD⁺, and now, we have this glyceraldehyde-3-phosphate actually covalently linked to the sulfur, to the enzyme itself- very, very important.0701

That does not always happen.0712

OK, we have got our C, our C and our C; and we have an O^- over here.0716

This is going to be OH; this is going to be O, and this is going to be PO₃^2-.0724

I have H₂; I have H, and this is linked to sulfur, and this is the cysteine.0732

And now, let’s go ahead and have our histidine.0743

Again, we will go ahead and draw our 5-membered ring with a couple of nitrogens in it, that now, has a hydrogen that it abstracted.0746

There is a hydrogen here.0756

There is that, and there is that; and now, because of this hydrogen, there is a positive charge on nitrogen - right - formal charge, positive.0760

OK, and this is going to be coordinated hydrogen bonding, something like that.0771

Now, we have this; now, here is what is going on.0777

Actually, I think I will do this in red; these electrons move down here.0781

OK and there is also - oops, sorry - a hydrogen here that I forgot about.0786

This is actually a very, very important one; now, let me go back to red.0793

These electrons on the oxygen moved down to reform the carbonyl, and they actually pushed this hydride to NAD⁺.0797

Now, what happens, let’s move on to the next page; let’s see what we have got.0807

OK, now, let’s redraw in blue.0815

We have got something like that.0822

Now, the NAD⁺ has been reduced to NADH, and we have our C, C, C.0828

This is there; this is linked to S, which is the cysteine.0836

Let me put the OH there and an H and an H₂ and an O, PO₃^2-; and we still have our histidine - OK - little 5-membered ring, nitrogen, nitrogen.0841

There is an H there, double bond there; there is an H there, and we have a positive charge.0861

OK, from here, here is what happens.0866

Alright, now, NAD⁺ comes in to replace the NADH, just a turnover of the NAD⁺ and NADH.0872

We are trying to recover the enzyme because we are almost finished with the mechanism.0884

Inorganic phosphate comes in- P.0892

That comes in; NADH leaves.0900

OK, let me write that down, red.0906

The NADH leaves, and another NAD⁺ takes its place.0913

Let me go back to blue; OK, this is our inorganic phosphate, remember abbreviated P_i.0929

Here is what happens; when everything comes in...let me redraw this.0937

That is fine; I will go ahead and...that is there.0946

Now, we have an NAD⁺ there.0950

We have our C, our C, our C.0954

We have our carbonyl; it is attached to that.0958

We have our cysteine, OH, H, O, PO₃^2-.0962

This is H₂; we have our histidine.0969

Let me go ahead and draw that out; OK, we have that, that.0974

We have an H; we have an H.0980

We have a positive charge; OK, now, here is what happens.0984

Now, let me go ahead and put my phosphate; I am going to do my inorganic phosphate in red.0988

My phosphate, this is this thing; it comes in, H, O^- minus.0992

OK, this attacks the carbonyl.0999

The carbonyl electrons, I am just going to do it in a circle; they jump up to the oxygen.1005

They jump back down to form the carbonyl; I am going to represent it like this in a single step, and then, this goes and grabs that, and these electrons jump back on to the nitrogen, and what you end up with, after release of the product…1009

Again, inorganic phosphate comes in, nucleophilic attack on the carbon; it attacks the carbonyl.1033

OK, the standard mechanism of a carbonyl reaction jump up to the oxygen, drop back down to form the carbonyl.1039

We often represent it as a circle like this; it pushes these electrons that were bonded to the sulfur of the cysteine residue.1044

They grab another hydrogen breaking that bond to the enzyme, to the sulfur.1050

Once that is done, you can go ahead and release this; now, the phosphate is actually attached here through the O, and what you end up getting - I will go ahead and do that on the next page - is the following.1057

I will do this in blue; what you get is C, C, C.1072

Excuse me; you have your carbonyl, and now, you have your O and PO₃^2-.1077

This stays there; this is O.1084

You raise that; we have PO₃^2-.1089

This is H₂; this is H- there you go.1094

Now, that, and let me go ahead and draw what the enzyme looks like now, after it has released.1102

Again, you have recovered your initial state of the enzyme; you have NAD⁺ in that fold.1107

You have your cysteine residue with the sulfur that is, now, protonated again.1114

And then, of course, you have your histidine residue with that really, really interesting thing, and you have this.1119

And, of course, there is some coordination here- the electrons on the nitrogen and the proton on the cysteine residue.1131

And now, it is ready for another catalytic cycle- that is it.1138

There you go; that is the mechanism for step 6.1143

OK, now, let’s move on to step 7.1148

Step 7 is the phosphoryl transfer - excuse me - from what we just made, which is the 1,3-bisphosphoglycerate.1155

I am just going to call it BPG to ADP to form ATP.1159

This is one of the reactions where we are actually using the…this is a coupling reaction.1161

A 1,3-bisphophoglycerate - if you remember - was one of the molecules that has a very, very high free negative free energy of hydrolysis, much higher than the free energy of a hydrolysis of ATP.1169

So, we can actually reverse the ATP reaction, couple it with this reaction and still have enough energy left over for it to actually move forward and still be negative.1201

We are using this to form ATP to recover some of the ATP that we invested in the preparatory phase.1210

OK, let’s go ahead and draw out the general reaction here.1217

We have C, C, C, and I will go ahead and flip it back up, so it looks like this: CO₃, PO₃^2-.1221

We have OH; we have H.1229

We have O, and we have our PO₃^2-.1233

This time, the other molecule here is going to be adenosine diphosphate.1237

We will go O, P, O, P, O, and we have our ribose; and we have our adenine, and let’s go ahead and put our double bonds on our phosphorus, make sure that we have everything here.1244

OK, that looks good; now, this is going to be - go ahead and draw that and that - slightly reversible.1256

We are going to need magnesium ion, and the enzyme that catalyzes this reaction is phosphoglycerate kinase or kinase, depending what you want.1265

Now, notice, kinase, as we know in general, are enzymes that catalyze the transfer of the terminal phosphoryl group from adenosine triphosphate to some substrate.1283

Here, it is going in reverse; it is actually taking some phosphoryl group that is on a substrate, and it is giving it back to ADP to actually form ADP, and that is absolutely fine because we know enzymes catalyze not only the forward reaction but the reverse reaction.1296

All enzymes do that; OK, the products of this reaction are going to be…let’s see, phosphoglycerate.1310

Let’s see; this is going to be C, C, C.1324

I will put the carbonyl up here, there and OH and H, and we have H₂.1328

We have O and PO₃^2- + adenosine triphosphate.1337

This is going to be O, P, O, P, O, P, O with ribose and with adenosine, and there we go.1345

Let's make sure we have everything; there we go.1359

What I transferred was this right here- that, OK - not the whole thing.1362

Notice this O^- that stayed behind; a phosphoryl and a phosphate are not the same thing.1367

Phosphate is PO₄^3-; a phosphoryl is PO₃^2-.1373

This is what I transferred back to the ADP to form the ATP; it came off from here.1379

OK, the overall ΔG for this reaction is -18.5kJ/mol- plenty of energy left over.1385

Now, here is - let me go ahead and do this in red now - the formation of ATP - excuse me - by transfer of a phosphoryl group from a molecule, a substrate from a molecule - whatever molecule is involved - to ADP to form ATP is called substrate level phosphorylation.1398

It is called substrate level; you will hear this a lot.1447

Now, we distinguish this from something called respiration linked phosphorylation - OK - which is the mechanism by which ATP is formed during - woo, getting a little fast here - the final phase of oxidative phosphorylation, when we oxidized glucose under aerobic conditions all the way to carbon dioxide and water by which ATP is formed during the final phase of oxidative phosphorylation; and we will be taking about this later on on the course- phosphorylation along the electron transport chain.1458

The NADH that was formed in the reaction that we just did, in the conversion of the 3-phosphoglycerate to the 1,3-bisphosphoglycerate to the 3-phosphoglycerate, those electrons in a mitochondria are given over to the electron transport chain, and ultimately, they reach oxygen.1560

It is oxygen that ends up being reduced, and the final phase about the oxidative phosphorylation ends up creating a whole bunch of adenosine triphosphate.1581

We have that respiration linked formation, respiration linked phosphorylation, which forms adenosine triphosphate in that mechanism; and we have this, which is substrate level phosphorylation, where a molecule actually gives up its phosphoryl group to ADP to form ATP- that is all that is going on here.1591

OK, now, let’s go ahead and do a quick mechanism for this; it is a simple nucleophilic substitution, so nothing complicated here.1610

Let’s see; let’s go ahead and do that in blue.1618

Mechanism is nucleophilic substitution.1627

OK, let’s draw our C, C, C, and a P.1637

I will go ahead and actually draw everything out: OH, and H, H₂O, PO₃^2-.1651

And then, of course, we have our ADP; we also have the O, P, O, P, O, ribose, adenine.1667

I know I could probably use shorthand for this; sorry about that.1681

OK, we have that, and we have that- that is it.1686

That is all that is going on, and that will give you your product- that is it.1691

The phosphoryl group, the PO₃^2- is just transferred over to the ADP, but it is the ADP that actually does the attacking.1694

To me, it has always been a little weird; when they are talking about transferring a group from something to something, but it is that something that is actually doing the attacking, that never really made sense to me, but that is what is happening.1704

The ADP is acting as the nucleophile, and it is literary taking the group.1714

It is not really being transferred to the group; it is taking the group.1718

OK, now, let’s go and do step 8.1723

Let’s see; we are almost there.1728

Step 8 is the conversion of 3-phosphoglycerate to 2-phosphoglycerate.1732

We are just going to move this phosphoryl group up to the no. 2 carbon up there, so conversion of 3…I will just say 3-PG to 2-PG.1737

OK, the reaction is the following; we have C, C, C.1752

Excuse me; this is OH, H, O and PO₃^2-.1757

I will go ahead and put that there, and let’s see.1766

This is going to be there; magnesium is required.1769

The enzyme is phosphoglycerate mutase, and it looks like this: C, C, C.1774

That carboxyl group stays the same; now, the phosphoryl group is on here and CH₂, and the hydroxy, the alcohol group, is now, moved to the no. 3 carbon, so 3-phosphoglycerate to 2-phosphoglycerate.1791

OK, alright; let’s go ahead and do the mechanism for this.1808

It is essentially 2 nucleophilic substitution reactions.1814

Let me go ahead and just write that, and then, I will start the mechanism on the next page.1818

Mechanism- it is essentially 2 nucleophilic substitution reactions.1826

OK, let’s start this on the next page.1845

That is fine; I am just going to list these as steps.1853

I wonder if I should do it vertically; yes, that is fine.1857

I will do it vertically; alright, so we have got C, C and C.1860

We have that; we have OH, and we have our PO₃^2-, and we have our H₂, and we have…let me go ahead and do it this way: 1, 2, 3, 4, 5.1864

I will go ahead and do histidine, is connected, of course, to the enzyme; and we have got that.1885

I will go ahead and put N there, and I will go ahead and put my nitrogen there; and I have got P.1895

I will go ahead and draw this out; there we go, and we have a positive, and we have an H here.1909

OK, on the enzyme, there is a histidine residue, and that histidine residue happens to be phosphorylated.1920

What is going to happen is this phosphoryl group is going to end up here, and then this phosphoryl group is going to end up going back there.1929

It is going to be 2 steps, so let’s go ahead and do that.1938

What you are going to end up with is, once this thing ends up over here…and again, it is going to be nucleophilic attack, this thing on the phosphorus kicking the electrons over there.1945

Let me go ahead and write C.1960

You know what, I am going to need a little bit more room; I am going to make this a little bit smaller.1965

I want to do this on one page; let me just go here and go C, C, C.1968

OK, this is going to be O, and this is going to be PO₃^2-.1976

This is going to be O, and this is going to be PO₃^2-.1981

I am going to ignore the hydrogens for now; excuse me.1985

We have got that, N, N; now, there is an H on there.1991

I have got that, that, and I have got histidine; and it is attached to the enzyme.1999

There is an H here and a positive charge; OK, now, actually, you know what, I am going to do this a little bit differently.2007

Let me redraw this; I want to show the distribution.2016

I have got 1, 2, 3, 4, 5.2023

That is N; that is N.2028

There is that hydrogen, and now, there is this hydrogen; and, of course, the positive charge is shared between those 2 nitrogens now.2031

OK, this is 3-phosphoglycerate.2041

This is 2,3-bisphosphoglycerate.2047

And, of course, our final step, when this is going to be transferred over to here, we are going to end up with C, C, C.2051

This goes there; this is going to be PO₃^2-.2062

This is going to be OH; that is fine.2068

I will go ahead and put the hydrogens in, and it is going to be back to where we were before, so N, N.2073

I will just put PO₃^2-; it is going to be like that.2084

There is going to be H, histidine, enzyme, positive charge- there we go.2090

These are the steps; nucleophilic attack here to take this phosphoryl group to become 2,3-bisphosphoglycerate.2100

It becomes 2,3-BPG.2109

OK, this is 3-PG, 3-phosphoglycerate to 2,3bisphosphoglycerate.2113

And then, of course, you have got attack here; go ahead and take this one, transfer of some protons, and what you end up with is the 2-phosphoglycerate and recovery of this, recovery of the enzyme and the histidine residue with the phosphorylated imidazole ring on the, well, on the ring.2117

OK, now, let’s talk a little bit about this.2143

Now, the catalytic cycle, this particular catalytic cycle - excuse me - begins when 2,3-BPG, which is already present in the cell in very trace amounts - OK - phosphorylates the imidazole or imidazole ring of histidine.2147

There is 2,3-bisphosphoglycerate already in the cell in very trace amounts.2212

It goes ahead and phosphorylates the ring on histidine, and that begins the cycle, the step 1 of what we just did- that is it.2216

That is how it keeps it going; that is how the ring actually ends up being phosphorylated to begin with because of 2,3-PG that is there already in tiny amounts.2227

OK, now, let’s go to step 9.2239

This is going to be the dehydration of 2-phosphoglycerate to phosphoenolpyruvate- very, very, very important.2245

Phosphoenolpyruvate- this is PEP, so the dehydration of 2-PG to phosphoenolpyruvate2262

Dehydration means you are removing the element of water; you are taking away water.2276

OK, let’s go ahead and write out the structures here.2281

There is that; there is O, and there is PO₃^2-.2289

There is C, and there is OH; I am actually going to draw this OH on the other side just so you can see what it is that is actually being taken away.2294

OK, well, there is another H here too, so I guess, I will put that there.2309

What is being taken away is this: dehydration, the H and the OH- that is what is going away.2313

Let’s go ahead and draw that, and, of course, the elements of water.2325

Water is leaving, and the enzyme that catalyzes this is called enolase, and a magnesium ion is required for this.2330

As you can see, magnesium ion is required for almost all of these reactions.2339

Definitely make sure you have enough magnesium in your body.2345

Our final product, is going to be C, C, C.2349

We have that, and we have this; and we have our O and our PO₃^2-, and let’s go ahead and put an H₂ right there.2355

OK, the ΔG for this reaction is 7.5kJ/mol, and do not worry about that endothermic because as you will see in a minute, the next reaction is highly exergonic.2369

No, not endothermic- endergonic.2382

The next reaction is highly exergonic, so it will pull that forward- not a problem.2386

The dehydration is 2-PG to phosphoenolpyruvate.2390

Dehydration- removing the elements of water, catalyzing; this is our PEP- very, very, very important molecule.2394

OK, let’s go ahead and run through the mechanism here.2401

Let’s see.2405

Yes, let me go ahead and start on the next page; I will do the mechanism in blue.2409

OK, let me go ahead and draw this out; I am going to draw it out this way.2419

I have got C, C; I do not know how big I…no, that is OK.2428

Let me see; Should I do it here, there?2436

That is OK; I will do it vertically- not a problem.2439

OK, C, C, C, we have that, and we have that; and we have our O and our PO₃^2-, and we have our H, and we have our H, our H and our OH there.2442

Now, let me go ahead and draw my enzyme around it.2466

OK, and now, I have got coordination to 2 magnesiums.2473

There is coordination on that oxygen, and there is coordination on that oxygen; and there is coordination on that oxygen.2481

And then, of course, I have a lysine residue, which is lysine 3,45.2489

Excuse me, and we have the nitrogen there; and we have our Glc-2,11, C, carbonyl and an O and an H to get started.2496

OK, this is what is going to happen; let me do this in red: abstraction of that hydrogen, movement there, movement there.2518

OK, now, let’s go ahead and see what this looks like now.2533

I am going to go back to blue; now, I have got my C, my C, my C.2541

I have got O^-; I have got O^- there.2547

This is now, a double bond here.2553

OK, let me go ahead and draw everything in.2557

I have got my O and my PO₃^2-.2561

I have got my OH here; I have my…no, I just - wait a minute - took that hydrogen away.2565

OK, let me make sure I have got everything, my H.2573

OK, I have my magnesium ion, and I have my other magnesium ion.2577

I am still coordinated, like that- very, very important.2585

And now, let me redraw my enzyme.2589

OK, and I have my lysine 3,45-nitrogen.2595

Now, I have a hydrogen; I have a hydrogen, and I have a hydrogen.2604

The nitrogen is actually carrying a positive charge, and I have my Glc-2,11.2608

OK, I have got C; I have got that.2616

I have got that, and I have got that; OK, here is what happens.2619

This is a negative charge; this is a negative charge.2624

Let me go back to red; OK, this drops down to reform the carbonyl.2629

This moves over there, and it pushes this over here to grab this; and it pushes these electrons on to here.2637

Then what you have is released by the enzyme, and, of course, H₂O, this; and that is that.2652

That is the dehydration; OK, and what you end up with, your final product is - I will draw this in blue - you end up with C, C, C, double bond.2670

Let me go ahead and put that double bond there, leave that a minus charge.2687

I will go ahead and put the PO₃^2- there.2692

We have H, and we have H; this is phosphoenolpyruvate.2697

This is our PEP molecule- there you go.2702

I hope that made sense, OK, now, step 10, the final step.2708

OK, step 10 is going to be the transfer of a phosphoryl group from the phosphoenolpyruvate to ADP to form ATP.2718

Again, another substrate level phosphorylation, the creation of ATP.2746

The reaction looks like this; I will draw this vertically: C, H₂, O, PO₃^2-.2752

That is that, so it is going to be + O, P, O, P, O, ribose and adenosine.2765

This goes there; this goes there.2781

This goes there, so this, that is the group that is going to end up over here.2783

OK, this is our PEP; this is our ADP.2793

I wonder if I should draw it on this page or…yes it is fine.2801

I can draw it on this page- not a problem.2807

OK, this requires magnesium; it requires potassium, and it is catalyzed by pyruvate kinase, and you end up with the final product of C, C, C.2814

We have a carboxyl there; we have a ketone there, and we have H³+.2834

Then, what you have is O, P, O, P, O, P, O, ribose, adenine, double bond, double bond, double bond.2844

That is O^-; that is O^-.2860

That is O^-; that is O^-.2863

This PO₃ is that PO₃ right there.2865

It is moved from here to here, and this is the final product.2869

Notice now, we have carboxyl group, and we have a carbonyl that is alpha.2874

This is an alpha-keto acid because you have a ketone group that is alpha to the main carbonyl, which is the carboxylic acid group- very, very important.2880

OK, the ΔG for this reaction, -31.4kJ/mol- highly, highly, highly exergonic, virtually irreversible under cellular conditions.2889

This is it; once it gets to this point, it just pulls it forward.2904

OK, now, let’s go ahead; let’s see.2909

A couple of things we want to say about this; this is, again, a substrate level phosphorylation.2913

Let me do this in red; this, again, is a substrate level phosphorylation to form ATP, to form adenosine triphosphate.2919

OK, now, the initial product is not the pyruvate.2947

It is - well, I will show you in a second - the initial product after the nucleophilic substitution because again, we are just transferring the phosphoryl group, so this is a basic nucleophilic substitution reaction.2954

Nucleophilic substitution- the attack of the ADP on the PEP.2980

The initial product after the nucleophilic substitution is this.2987

It is actually going to be - let me do this in blue - C, C, C.2992

This is going to be here; this is going to be O^-.3002

This is actually going to be an OH; OK, it is actually going to be OH, and this is the double bond that is actually going to be there because you remember, that is where it is, so we have H₂.3004

Now, what happens is tautomerization.3015

When you have something like this, when you have an enol, which is an alkene and an alcohol attached, it actually prefers to be the carbonyl instead of the alkene.3021

The double bond prefers to be carbon oxygen instead of carbon-carbon3031

In fact, that is what actually pulls the reaction forward.3035

It really, really, really wants to be a carbonyl; that is what actually gives it the high negative free energy for this reaction.3039

It tautomerizes, and that is when it becomes C, C, C, the alpha-keto acid that we know of; and this is going to be C.3048

This is CH₃ now; this is the enol form, and this is the keto form.3061

You will see this a lot in biochemistry; anytime you have something like this, where you have an alkene and an alcohol attached, generally under cellular conditions of pH7, somewhere around there, the keto form is going to be the one that is most stable.3068

OK, now, this is another energy coupling reaction.3083

We have actually seen it before when we are talking about bioenergetics.3097

OK, the hydrolysis of the phosphoenolpyruvate, which has a very high free energy of hydrolysis, is coupled to the synthesis of ATP because there is more than enough energy to actually accomplish this synthesis, the synthesis of ATP.3103

And the best part is, there is still more than enough free energy left over to make this step irreversible, and you saw that already - very, very high ΔG, high negative, irreversible.3138

I hope I spelled that correctly; OK, let’s just go ahead and write out what we have got here.3171

We have PEP + H₂O - just reminding you of how we put this together - goes to pyruvate plus that.3179

The ΔG for this is -61.9kJ/mol.3193

The hydrolysis of PEP releases 61.9kJ of free energy.3199

Well, the synthesis of ATP from ADP and inorganic phosphate, ATP + H₂O - woo crazy, all these symbols, all these names, how do we keep it all straight - it is +30.5.3204

When we add these together, H₂O, H₂O, PI, PI, we have our reaction that we just ran.3226

The phosphoenolpyruvate + ADP goes to pyruvate + adenosine triphosphate.3233

Substrate level phosphorylation, ΔG for the overall reaction is -31.4kJ/mol- incredible, absolutely incredible.3244

OK, now, let’s go back to black, and let’s do a glycolysis balance sheet.3254

What happened to the carbons?3266

What happened to the phosphoryl groups?3268

What happened to the electrons?3269

OK, let me see; yes, that is fine.3271

We have another page; we have got glucose + 2 ATP.3277

This is all the things that were invested in glycolysis, and you are going to see all the thing that came out of glycolysis; and then we are going to go ahead and cancel and see what the net reaction is.3284

We invested 2 ATP; we invested 2 NAD⁺.3292

We invested 4 ADPs, and we invested 2 inorganic phosphates.3298

What we got out was 2 pyruvate, 2 ADP, 2NADH, 2 hydrogen ion, 4 ATP and 2 molecules of water.3305

Now, when we go ahead and we cancel common terms on the left right side of the arrow, we are left with the following.3329

let me see, cancelling common terms.3336

We are left with glucose + 2 NAD⁺ + 2 ADPs + inorganic phosphates, gives us 2 molecules of pyruvate, 2 molecules of NADH, 2 hydrogen ion, a net gain of 2 adenosine triphosphate - very, very important - and 2 molecules of water.3344

This is what is important; this is what happened.3377

This is what glycolysis did, right there; OK, now, 1. the 6 carbons of glucose.3381

I am going to write that out; I am just going to write C.3392

The 6 carbons of glucose became the 2 x 3 carbons of pyruvate.3400

Let’s go ahead and go to the next page here.3415

OK, no. 2, the 2 ADP and the 2 inorganic phosphates, they became the 2 molecules of adenosine triphosphate.3420

And last, but certainly not least, the 4 electrons has 2 hydrides taken from the glyceraldehyde-3-phosphate.3437

Remember that reaction, NAD⁺ to NADH?3459

We pulled away hydrogens from there, so the 4 electrons, which left as 2 hydrides, 1, 2, and then, 3, 4.3462

There is 4 electrons, those electrons are energetic.3470

Those are what are going to end up in the electron transport chain for respiration linked phosphorylation.3474

So, the 4 electrons - as the 2 hydrogens taken from the glyceraldehyde-3-phosphate - are transferred to NAD⁺, to the 2 NAD⁺; and they become the 2 NADH, ultimately being transferred.3478

NADH goes on to give up its hydrides, to give up these 4 electrons to the electron transport chain.3518

Ultimately, they end up with oxygen being transferred from, they are ultimately being transferred from the 2 NADH to the electron transport chain.3526

We have accounted for the carbons; we have accounted for the phosphoryls, and we have accounted for the electrons.3545

This is glycolysis; OK, thank you so much for joining us here at Educator.com.3550

We will see you next time, bye-bye.3555

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