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Lecture Comments (4)

1 answer

Last reply by: Professor Hovasapian
Mon Feb 8, 2016 1:06 AM

Post by Michael Sramek on February 1, 2016

I am confused as to the location of which lactate is converted into pyruvate. doe this happen in the cytosol while producing NADH?

1 answer

Last reply by: Professor Hovasapian
Wed Jul 17, 2013 4:16 PM

Post by Aimee McGuire on July 17, 2013

15:06, should the reaction be the other way around?

Gluconeogenesis 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
  • Oxaloacetate → Phosphoenolpyruvate (PEP) 0:35
    • Mitochondrial Membrane Does Not Have a Transporter for Oxaloactate
    • Reaction: Oxaloacetate to Phosphoenolpyruvate (PEP)
    • Mechanism: Oxaloacetate to Phosphoenolpyruvate (PEP)
    • Overall Reaction: Pyruvate to Phosphoenolpyruvate
    • Recall The Two Pathways That Pyruvate Can Take to Become Phosphoenolpyruvate
    • NADH in Gluconeogenesis
  • Second Pathway: Lactate → Pyruvate 18:22
    • Cytosolic PEP Carboxykinase, Mitochondrial PEP Carboxykinase, & Isozymes
    • 2nd Bypass Reaction
    • 3rd Bypass Reaction
    • Overall Process
  • Other Feeder Pathways For Gluconeogenesis 26:35
    • Carbon Intermediates of The Citric Acid Cycle
    • Amino Acids & The Gluconeogenic Pathway
    • Glycolysis & Gluconeogenesis Are Reciprocally Regulated

Transcription: Gluconeogenesis II

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

In this particular lesson, we are going to continue our discussion of gluconeogenesis.0004

Now, in the last lesson, we talked about the first bypass step, and that bypass step going from the pyruvate back to phosphoenolpyruvate happens in 2 steps.0010

We convert the pyruvate to oxaloacetate, and the oxaloacetate goes to phosphoenolpyruvate.0021

OK, now, we are just going to continue on with the process.0030

Alright, let's just jump on in.0035

OK, it is reduced to malate.0039

Now, let's continue on; now, the mitochondrial membrane does not have a transporter for the oxaloacetate that we formed.0045

You remember the first thing we did?0074

Where we left of was we actually formed oxaloacetate; that was that mechanism that involved the biotin coenzyme.0075

OK, for oxaloacetate, the oxaloacetate is actually reduced to malate, and a malate is sent back out into the cytosol.0084

It is reduced to malate then transported back to the cytosol, where it is reoxidized to oxaloacetate.0102

Yes, reoxidized to oxaloacetate, all these molecules floating around.0132

OK, this is what actually takes place.0148

We have the oxaloacetate; let me go ahead and do this in blue.0153

We have oxaloacetate plus the NADH + H+.0158

It goes to L-malate + NAD+, and then, the reverse reaction takes place once it actually is sent back out into the cytosol.0179

You have NAD+ plus our malate going back to oxaloacetate, and NADH + H+ is regenerated.0183

This part takes place; this first one takes place in the mitochondrion, and then, this one takes place outside the mitochondrion once that malate is sent back into the cytosol.0203

OK, here is the second reaction; let me go back to black here, so reaction no. 2.0215

We have our oxaloacetate molecule: C, C, C, C.0224

Excuse me.0230

I have that, and I have a carboxyl over here.0235

This is where we are going to use the guanosine triphosphate instead of the adenosine triphosphate, and what ends up happening is, one of the things that leaves is, of course, the GDP; and the other thing is now, the CO2, this right here, is lost.0240

What you end up with is H2, C, C, C, C, O, O, and then, of course, I will go ahead and put the phosphate down here- PO32-.0260

Now, what you have is your phosphoenolpyruvate.0272

This right here is your phosphoenolpyruvate.0276

This is your oxaloacetate.0281

Let me go back to black; OK, here is the mechanism- mechanism, mechanism, mechanism.0284

OK, it is actually a pretty easy mechanism, so it is not going to be that involved.0291

Let me just go ahead and draw...yes, that is fine.0296

I will do C, C, C, C.0303

I will go ahead and draw that - woo, I swear, once you start writing all these things out, it starts to get really, really, kind of crazy - O-.0308

I will go ahead and put my know, that is OK.0319

I will just write H2 here; we have 2 hydrogens attached there.0323

Now, I will go ahead and write my...let me see.0327

I have got my guanosine, ribose.0331

I have got O, P, O, P, O, P, O, here, here, and I have got that.0336

So, this is our guanosine triphosphate; here is what happens.0350

Let me go ahead and do this in red; we said carbon dioxide really likes to form, and if there is something nearby that will allow it to form, it will do so.0355

These electrons go here to reform that.0365

These electrons come back up here to actually reform the double bond.0371

These electrons come over here on a nucleophilic attack.0376

They push these electrons onto that, and what you are left with is the phosphorylated oxaloacetate that has lost its carboxyl, which is phosphoenolpyruvate, the formation of a double bond.0383

What you are left with is the following.0396

You are left with H2, C, double bond, C, C, O-, O, PO32-.0404

This is what came from the GTP- that is it.0418

The overall reaction here...we started off with our pyruvate.0422

We added ATP; we added GTP.0433

We added bicarbonate,, HCO3 is -1, not -2.0440

Carbonate is -2; we formed PEP.0448

We released ADP; we released GDP.0455

We also released the PI, remember?0459

We invested the energy of ATP; we lost the ADP and the PI in the first reaction.0463

It was the energy that we used, and we also lost a CO2.0468

Now, the ΔG for this reaction - standard ΔG - is 0.9kJ/mol.0473

We said that this is a highly exergonic reaction; that is not highly exergonic.0482

Under physiological conditions, it is highly exergonic.0487

ΔG, under physio conditions, is equal to -25kJ/mol.0492

That is huge; I mean, it is not massive, but it is big- certainly enough to make it virtually irreversible.0498

Now, this PEP - phosphoenolpyruvate - is used in many other reactions.0507

It is a molecule that the body uses for lots of other things.0519

As it is formed, it is actually, well, it is used in other reactions.0522

As it is formed, it becomes depleted because it is being used for other things.0536

As we form the PEP, we are pulling it away and using it for other things.0550

Well, Le Chatelier’s principle, as we pull away a product from a particular reaction, we are messing with the equilibrium, and what that is going to do, it is going to pull the reaction forward.0554

That is what makes this reaction so highly exergonic under physiological conditions because this product, PEP, is being used for other things.0568

It is pulling the reaction forward, under Le Chatelier’s principle.0576

As it is formed, it becomes depleted, thus pulling the overall reaction forward and making it irreversible, giving it that large -ΔG value.0581

OK, now, let's go ahead and I am going to just change pages here, and I think I am going to go back to black.0609

Recall the 2 pathways; I am going to redraw it, but I am going to redraw that thing with the mitochondrion and the 2 pathways, but without all of the information, without the enzymes.0621

Let me go ahead and draw this here; this is a mitochondrion.0630

We had our 1 pathway, which was pyruvate to pyruvate to oxaloacetate, oxaloacetate to malate, malate out to malate, malate back to oxaloacetate and oxaloacetate to phosphoenolpyruvate.0636

Now, the other pathway we had was the following; we had the pyruvate here, but it actually came from lactate, and then, of course, we had pyruvate to pyruvate, and in the mitochondrion, we had the oxaloacetate and the oxaloacetate to directly the phosphoenolpyruvate, and then, of course, that is shipped out into the cytosol to do whatever it does.0662

OK, here is the important step; the oxaloacetate to the malate - let me do this in red - NADH + H+ NAD+.0685

Oxaloacetate is reduced to malate - OK - and then, it is sent out; and then, the malate is reoxidate.0704

NAD+ is our oxidizing agent, and then it, itself, is reduced to NADH + H+.0711

Here, this step right here, this is NAD+ NADH + H+.0720

Notice what is happening; in this particular case, well, here the NAD+ NADH is being produced in the cytosol.0732

Here, NADH is being used up in the mitochondrion, and it is being reproduced in the cytosol.0744

Now, let's talk about what is going on here.0750

The ratio of NADH concentration to NAD+ concentration - OK - in the cytosol is pretty low actually, is quite small, about 8 x 10-4.0754

That is actually quite small; there is not a lot of NADH in the cytosol.0781

OK, now, this is much lower than the NADH to NAD+ ratio in the mitochondrion.0788

In other words, there is a lot more NADH available - about 5 times more - in the mitochondrion than there is in the cytosol.0817

OK, let's take a look at what this means.0826

Now, one of the reactions of gluconeogenesis, in gluconeogenesis, NADH is consumed.0830

OK, in one of the reactions that takes place in the cytosol, NADH is necessary.0850

It is actually consumed in converting the 1,3-biphosphoglycerate to 3-phosphoglycerate.0855

NADH is something that is required for gluconeogenesis to actually continue.0870

This is not one of the bypass reactions; this is just one of the reverse reactions of glycolysis.0875

In glycolysis, 3-phosphoglycerate to 1,2-biphosphoglycerate, the other way around, NADH is actually being used up.0880

Well, when it is used up, the concentration is already low in the cytosol.0889

So, in order for gluconeogenesis to continue, there has to be something that actually produces enough NADH to keep it readily supplied in the cytosol.0893

What this particular pathway does is it uses the fact that inside the mitochondrion, there is NADH concentration.0903

There is plenty available, so what it does is it uses that NADH to convert the oxaloacetate to malate, and then, it sends it out into the cytosol; and then, it reuses the NAD+ concentration to reproduce the NADH, which goes on and constantly replenishes the NADH concentration in the cytosol.0910

It is, sort of, taking NADH that is in the mitochondrion and sending it out into the cytosol by doing this oxaloacetate to malate, malate back to oxaloacetate, to make sure that there is enough NADH in the cytosol.0931

That is what is going on; in gluconeogenesis, NADH is consumed in converting the 1,3-biphophoglycerate to 3-phosphoglycerate.0947

The NADH in the cytosol is being depleted.0959

Well, it has to be replenished.0972

Now, by using NADH to reduce the oxaloacetate to malate, sending this malate out to the cytosol, then reforming NADH in the cytosol itself upon reoxidation of malate to oxaloacetate, we replenish the NADH concentration.0978

It is absolutely fantastic that the body has come up with this method of actually making sure that there is enough NADH.1055

By just this one little extra step, we replenish the NADH concentration- absolutely beautiful.1061

It is extraordinary what the body does.1068

We replenish the NADH in the cytosol.1074

OK, there you go; by using the NADH to reduce the oxaloacetate to malate, sending the malate out to the cytosol and reforming the NADH in the cytosol upon reoxidation of malate to oxaloacetate, we make sure that the NADH concentration in the cytosol stays sufficiently high, so the gluconeogenesis does not come to a complete halt- that is what it is doing.1080

Alright, now, let's take a look at the second pathway.1102

Let me go back to black here, now, for the second pathway, the conversion of lactate to pyruvate.1107

Notice, remember, outside the mitochondrion, when lactate is the precursor, it converts it to pyruvate.1131

Well, in that reaction, the conversion of lactate to pyruvate, NADH, the reaction which takes place in the cytosol, before the pyruvate actually enters the mitochondrion, it is already producing NADH.1140

In that particular case, it does not need to form the oxaloacetate, reduce it to malate.1151

It does not need to send it outside to reoxidize it to oxaloacetate to form phosphoenolpyruvate.1156

It can go ahead and do both of those reactions- pyruvate to oxaloacetate, oxaloacetate to phosphoenolpyruvate.1163

It can do it in the mitochondrion because before the pyruvate even enters the mitochondrion, we have already produced NADH in the cytosol.1170

It does not need to do it- absolutely fantastic.1180

Now, in the second pathway, the conversion of lactate to pyruvate already produces the NADH for cytosolic usage.1184

It is amazing that the body has come up with this, that the evolution has taken an enzyme and has designed one enzyme to do the same reaction...2 enzymes, one of them...both enzymes do the same reaction, but one of them does it in the mitochondrion.1208

One of them does it in the cytosol- 2 completely different contexts.1224

The enzymes, themselves, have 2 completely different properties, totally different properties; and yet, they catalyze the same reaction.1228

That is absolutely extraordinary.1234

OK, now, let's talk a little bit about...we will say a couple of words about these things.1237

Let's do this in blue; the cytosolic...well, I guess we are not going to do it in blue.1245

Let's try again; let's do this in blue, how is that?1252

Are we blue; yes, we are.1256

Cytosolic PEP carboxykinase and, of course, the other enzyme is the mitochondrial PEP carboxykinase - OK - these are called isozymes.1260

Isozymes are enzymes that catalyze the same reaction - we have seen them before - but in different contexts and with different properties.1293

That is what is amazing.1316

OK, and the final word on these, let me see.1326

Hey, I am not getting my red; there we go.1336

Isozymes are encoded by different genes, of course, because the amino acid sequence is actually different.1341

That is what is amazing; it is not the same enzyme that just looks a little different or does is an entirely different - not an entirely different - but it is a different amino acid sequence, so obviously it is going to be encoded by different genes.1356

OK, now, let's talk about...say our final words about gluconeogenesis.1370

We will talk about our second bypass and our third bypass reaction- not a big deal for these.1378

Our second bypass reaction, that one is going to be - I am not going to say too much about it - fructose 1,6-biphosphate + H2O.1388

These are going to be just simple hydrolysis reactions; let me go ahead and write them out actually.1402

I am not going to draw out any structures- not a big deal.1407

We have fructose 1,6-biphosphate + H2O going to fructose 6-phosphate, and the ΔG on this reaction is going to be -16.3kJ/mol; and the third bypass reaction - in other words the third reaction that is unique to gluconeogenesis, not just a reverse of the glycolytic - is we have glucose 6-phosphate.1411

Again, just another hydrolysis reaction: H2O going to glucose + inorganic phosphate, and then the ΔG on this reaction is equal to -13.8kJ/mol.1458

And again, this one is going to be catalyzed by fructose 1,6-biphosphatase, and this one is glucose 6-phosphatase; and both of these are magnesium dependent.1484

Magnesium is necessary for these things to operate.1514

OK, now, let's go ahead and take a look at the overall process.1518

We have something like this; we have 2 pyruvates + 4 ATPs + 2 GTPs + 2 NADHs + 2H+s + 4 H2Os.1525

This one is going to go to, well, what we are forming; we are forming glucose + 4 ADPs + 2 GDPs + 6 PI + 2 NAD+.1550

Is this 6 correct?1570

It should be; OK, overall ΔG equals -16kJ/mol.1572

Gluconeogenesis is a reasonably exergonic reaction under physiological conditions.1583

It is essentially irreversible just like glycolysis is.1591

OK, now, let's say a final couple of words about pyruvate.1596

Now, pyruvate...I think I will go back to black for this one.1603

Pyruvate, lactate, are not the only feeder pathways for gluconeogenesis.1615

OK, those 2 pathways that I showed you, those are the primary pathways.1640

Other molecules can come into those, but it is going to pass through one of those.1648

OK, now, the 4, 5 and 6-carbon intermediates of the citric acid cycle can become oxidized to oxaloacetate, and, of course, the oxaloacetate can go on to become the phosphoenolpyruvate on to gluconeogenesis.1653

Any of the intermediates, any of the 4, the 5, the 6-carbon intermediates of the citric acid cycle can become oxaloacetate, and you will see how when we actually end up discussing the citric acid cycle next unit - OK - which becomes PEP, well, which becomes PEP.1704

Let me just list some of these intermediates just so you, sort of, see them, and we will see these again, so do not worry about them.1733

You do not necessarily have to know them now; you are going to learn them eventually, you have to.1740

these are the intermediates of the citric acid cycle: citrate, isocitrate, alpha-ketoglutarate - and I am listing them in order of how they appear in the citric acid cycle – succinyl-CoA, succinate.1744

We have fumarate, and we have our famous malate.1771

Now, not only the carbons, the intermediates of the citric acid cycle, can actually enter gluconeogenesis, but most amino acids - this is what is amazing - are metabolized to either pyruvate directly or other molecules of the citric acid cycle - I am going to capitalize that - which then enter the gluconeogenic pathway, so not gluconeogenetic- gluconeogenic pathway.1778

OK, these amino acids, they are actually called gluconeogenic amino acids- that is it.1867

Once the amino group has actually taken off, and we will discuss how that happens later on, these things are actually metabolized to these intermediates, which can become oxaloacetate.1887

Oxaloacetate goes on to become phosphoenolpyruvate onto gluconeogenesis or whatever it is that they are going to use phosphoenolpyruvate for.1899

OK, and one final thing, which we will actually probably discuss a little bit, I have not really decided yet whether we are going to discuss regulation altogether too much, but I will mention it.1910

Glycolysis, the breakdown of glucose and gluconeogenesis are reciprocally regulated.1921

Regulation is an absolutely fan...if you really want to be fascinated by the true amazing power of the body is to see how it actually regulates these reactions.1943

The fact that these reactions take place at all is amazing in and of itself, but the way the body actually regulates these reactions controls the rate at which they happen.1955

One, this happens in here; this happens here to keep track of these hundreds of thousands of reactions that take place in microseconds- absolutely extraordinary.1964

They are reciprocally regulated - excuse me - and what that means is that as the flow of glucose through glycolysis increases, the flow of pyruvate through gluconeogenesis decreases, and we will have more to say about regulation later on.1975

It is not a problem; it is better to discuss it in a specific context.2018

So, what is important, at this point, is just that you realize that they are reciprocally regulated2033

As one increases, the other actually decreases.2039

If they just happen at the same rate, actually, the only thing that would end up happening is you would end up just producing heat.2043

OK, that takes care of gluconeogenesis.2051

Thank you so much for joining us here at; we will see you next time, bye-bye.2055