Biochemistry Phosphoryl Group Transfers

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

Today's topic, we are going to talk about phosphoryl group transfers.0004

We have been talking about how you take some endergonic reaction, and you couple it with an exergonic reaction - like the hydrolysis of ATP - to actually drive the endergonic reaction forward.0008

Well, we are going to talk about how that actually happens- what is it that takes place, what is it that makes this possible.0019

Let's go ahead and get started.0024

OK, let's go ahead and recap one more time; I know that you have heard this several times already.0030

I hope you are not getting sick of it, but it is important to hear things several times before it actually sinks in; and you start to think about it in the background regularly.0037

That is OK; we will go ahead and keep it in black.0047

We saw that the coupling of ATP hydrolysis to the phosphorylation of glucose allowed an otherwise endergonic reaction - I should say an endergonic phosphorylation because that is what is happening - an endergonic phosphorylation to proceed exergonically, right?0050

We had Glc + PI going to G6P + H₂O.0117

And, of course, we had the ATP hydrolysis, which was ATP + H₂O going to ADP plus the PI.0126

And, when we combined that, we ended up with an alternate pathway: Glc + ATP going to ADP plus - well, actually, I should write the G6P first - going to the glucose 6-phosphate plus our ADP.0136

OK, now, the glucose is activated and ready for further reactions.0157

OK, this is what ATP generally does.0181

Now, this is what ATP generally does - excuse me - namely, it provides its energy - that -30.5 that it has - it provides this energy by transferring a phosphoryl group, a pyrophosphoryl or an adenylyl group.0185

OK, this is what ATP generally does; it provides its energy by transferring either a phosphoryl - like in this case, a pyrophosphoryl - or an adenylyl group to a substrate molecule - let's see, you know what, I will talk about the raising of the energy a little bit later - a pyrophosphoryl or adenylyl group to a substrate for further reaction.0243

OK, and often, the next reaction is displacement of the group that was transferred.0286

What happens is the following.0313

We have glucose, which is a specific molecule; and in the process of phosphorylating, what it does is, now that a phosphoryl group is attached to this glucose, now, the energy content, it has invested energy into this glucose molecule.0315

It has raised its free energy content; now, this molecule can go on to react further with something else.0330

And now, the thing that it is going to end up reacting to, now there is a bigger difference.0340

Now, it has raised the energy content; now, that molecule can react and give away its free energy- that is what it is doing.0344

If somebody only has $5, but they need to buy something, I can come along and give them a $100, and now, they have a $105.0352

They can do the thing that they wanted to do, whereas before, they could not do it with the $5; they did not have enough.0359

That is all that is happening; ATP, by transferring its phosphoryl group in the active, putting on a phosphoryl group on that molecule, in this case, on glucose, it has raised the energy content of the glucose.0365

Now, the glucose can go and do whatever it wants; it has invested money into the glucose.0378

That is what ATP does, and this is what it generally does.0383

And usually, what happens in the next step of the reaction, more often than not - or maybe a couple of steps further down the line in a metabolic pathway - is that this phosphoryl group that is now attached to this molecule, is going to end up being displaced.0386

Literally, what you are doing is you are taking a molecule that often does not have a very good leaving group.0402

By attaching a phosphoryl to it, you have converted, you have created a good leaving group; good leaving groups tend to, well, they want to leave, so it is going to pull the reaction forward.0407

Its energy content has been increased, so now, in a reaction, it is going to have a -ΔG when it reacts further.0416

Hopefully, that makes sense, but we will see more of it here.0425

OK, let's see what is going on; let's do an example of this, actually, then I think, of course, it will make more sense.0428

Let's do this in blue.0436

OK, so the example; let's go ahead and do the following reaction.0441

Let's take glutamate, and we are going to ATP, ADP + glutamate + ammonia, I should say.0446

Those are the reactants, and we are going to form glutamine.0464

OK, let's take a look at this reaction; we are going to take glutamate and ammonia, and we are going to produce glutamine, and ATP is going to actually drive this reaction.0471

The hydrolysis of ATP is going to drive this reaction, but we know that when we say that, we do not mean that ATP is actually hydrolyzed.0482

There is no hydrolysis taking place; ATP is going to invest glutamate with a certain amount of energy allowing it to turn into glutamine more easily because it would not, otherwise, happen.0488

Here is how it takes place; let's go ahead and draw our glutamate molecule.0499

We have N, C, C; I will just write OO^- here, and I will go ahead and put that there.0505

We have CH₂, CH₂, C; I will go ahead and do it this way.0515

This is our glutamate molecule; now, let me go ahead and draw the same thing that I just drew.0521

We have ADP + PI, and we have adenosine triphosphate there.0527

Now, glutamine is going to be this.0532

Let me just write NH₃ in red; I might as well take advantage of the fact that I have got these multiple colors here.0538

I often get so wrapped up in it that I forget that I can do that.0544

NH₃⁺, that is C; that is COO^-.0548

That is CH₂; that is CH₂.0553

That is going to be COO, and now, this ammonia is going to end up being attached here to form glutamine.0556

Here is what is going on; Let's do this, well, that is fine.0568

I will just go ahead, and yes, let's go ahead and do this in red.0572

Now, it appears as though this reaction just takes place in one step, but it is actually two.0576

And, this will demonstrate how ATP does what it does; it is actually two.0602

The first step is the following; we have the N.0609

Should I do it on the next page or should I do on the...no, that is OK.0616

I can do it on this page; it is not a problem.0619

Let me go ahead and go back to blue here.0620

So, we have N, C, C,; we have got CH₂, CH₂, COO^-.0624

The first step is the ATP; boy, all these letters - oh my God - I think that is the most frustrating aspect of biochemistry.0635

It is just the sheer, the abbreviations, the letters; woo, it is crazy.0648

This is COO^-; let me just go ahead and make sure I have the structures correct here.0656

We have H₃N; this is C.0663

This is COO^-; we have CH₂.0667

We have CH₂, and we have COOP.0669

What has happened here is that the adenosine triphosphate has transferred its phosphoryl group - the final phosphoryl group - onto this glutamate.0682

That is right here, and now, the next step, what happens is the ammonia comes in, and it kicks off this phosphate.0692

The ammonia comes in, and it displaces the phosphate; now, you have your glutamine.0704

This is a positive charge, by the way; now, you have H₃N⁺, C, COO^-.0709

Now, you have CH₂; you have CH₂.0717

You have COO, and you have NH₂; what looks like a single step process is actually a two-step process.0721

Glutamate reacts with adenosine triphosphate to form this right here.0729

ATP transfers its phosphoryl onto the glutamate.0738

Now, this is not a good leaving group, but now, this is a good leaving group.0743

Now, ammonia can come in and displace this phosphate and attach where the phosphate is attached, and PI goes.0746

Here, you see the PI has left; ATP is converted to ADP + PI.0754

The energy of hydrolysis of ATP is used to drive this reaction, but it is happening this way.0762

It phosphorylates, then something else happens.0770

In the process of phosphorylating this molecule and changing it from this to this, I have actually invested energy into that molecule.0774

I have raised the free energy content of this molecule to a higher level.0783

Now, it has more money that it can spend; in other words, now, a nucleophilic substitution reaction can take place.0788

Well, it is actually not nucleophilic substitution; it is happening at a carbonyl.0796

But now, a substitution reaction can take place; that is what is going on here.0800

OK, let's say a little bit more.0806

Here, a phosphoryl group was put onto the carboxyl glutamate in order to activate the carbonyl group.0814

OK, what these did was raise the free energy content of the glutamate.0856

Now, the glutamate reacts easily because inorganic phosphate is an excellent leaving group.0882

OK, in other words, I will describe it this way; I will do this in red.0914

This is the rest of our protein, and I am just going to be concerned with my carboxyl portion.0921

I am going to draw this a little bit differently; sorry about that.0925

Let's try this again; that is that.0929

I have C; let's draw it this way.0932

OK, now, under normal circumstances, ammonia...I do not want to come in here, do this.0934

This is not going to happen; OK, this is not going to happen, and it is not going to happen because this is not a good leaving group.0943

This is a very, very stable molecule; it is not going to leave.0951

However, if I phosphorylate that carboxyl group, if I stick on a phosphoryl group onto this, now - this group, the inorganic phosphate is an excellent leaving group - now, NH₃ happens easily, very easily, in fact.0956

It wants to happen; it comes in here.0980

That goes there; that ends up leaving.0983

It facilitates it; the energy from ATP hydrolysis, the energy that would, otherwise, come from the breaking off of that final phosphoryl group of the ATP, that energy is put into this particular glutamate molecule.0987

Now, it has more money; it has a higher free energy content.1003

Now, it will react really easily with this particular nucleophile that it would not have reacted easily with beforehand.1007

That is how ATP coupling works- that is it.1014

OK, now, let's talk about...earlier we mentioned that it not only transfers a phosphoryl group, like you just saw, but it also transfers a pyrophosphoryl and an adenylyl group.1021

ATP, no, let's leave it as, you know what, let's go blue; I love blue.1034

ATP not only transfers phosphoryl but also pyrophosphoryl and adenylyl groups.1042

OK, let's draw this out; we have our - let’s do this in black - O, P, O, P, O, P.1069

And we have ribose, and we have adenosine.1080

Let's go ahead and put our double bonds in, and make sure we have everything here.1086

OK, now, there are 3 places where a particular nucleophile - like in the previous example, the nucleophile was the glutamate - where nucleophile can come in and take a particular group.1092

Now, let me do this one in red.1108

OK, a nucleophile just happens to be our substrate molecule - that is it - whatever it is that we are phosphorylating, whatever it is that ATP is transferring a phosphoryl or a pyrophosphoryl or an adenylyl group to.1114

There are 3 places it can attack; it can attack that phosphorus.1127

It can attack that phosphorus, or it can attack that phosphorus.1132

This is the alpha-phosphorus; this is the beta, and this is the gamma.1138

OK, attack at the gamma-phosphorus transfers a phosphoryl group.1143

If a nucleophile attacks this phosphorus, what happens is, the electrons move here; and this POOO, this group right there, that is what is transferred over.1160

OK, attack at the gamma-phosphorus transfers a phosphoryl.1175

That was what happened in the example that we just did.1185

You end up with the following; you end up with the nucleophile being attached to the phosphoryl.1189

This is the phosphoryl group; notice, it is PO₃^-.1198

Note, very, very important- this is not phosphate.1204

Phosphate is PO₄^3-.1213

So, very, very important, phosphoryl transfer means this part- the P, the O, the O, the O.1219

These electrons end up going here, so it is just this P that is attached to this nucleophile.1228

In the previous example, really quickly, you remember you had the carboxyl like that.1233

OK, this was the nucleophile; the R-group is right there.1241

This is the nucleophile; to this was attached the P, the O, O, and O.1246

This is the phosphoryl; what ends up leaving is this whole thing, which is the phosphate.1255

Very, very important to distinguish between those two; they are not the same thing.1262

OK, and if so, what you end up with when you do it, is you end up with a nucleophile attached to the phosphoryl group plus your ADP, your adenosine diphosphate 1, 2 , so, basically this part of the molecule, right?1267

This adenosine diphosphate- that is what you get.1284

OK, now, I wonder if I should redraw; you know what, I am just going to redraw the adenosine.1288

Yes, that is fine; yes, that is good.1296

I have got an extra page here, so let me go O, P, O, P.1299

Sorry about that; I just want to make sure we have O, P, O, P.1305

No, O, P, O, P, O, ribose.1310

And, I sure hope that you guys are checking my structure; I tend to make mistakes every once in a while, actually, more than just every once in a while.1317

O, P, O, P, O, P, O^-- there we go.1325

That is that; that is that, O^-, O^-, O^-, O^-.1329

Now, let's go to black; oh, what happened?1335

I lost my...OK, oh well; now, this is my alpha-phosphorus.1340

This is my...oops, let me see.1348

Let's try this again; let's try this in black.1351

I have my alpha-phosphorus; I have my beta-phosphorus, and I have my gamma-phosphorus.1354

Now, nucleophilic attack on that does that.1357

Attack at the beta-phosphorus gives pyrophosphoryl transfer.1367

And what you end up with is the following: nucleophile attached to...now, it is this bond that is broken.1389

This right here, that is your pyrophosphoryl group; it is not pyrophosphate.1405

Pyrophosphate has one more oxygen, and we will show you in a minute.1409

So, what you are left with is this nucleophile plus adenosine monophosphate.1412

The adenosine, the ribose, and just 1 phosphate- that is what you get there.1421

Now, let's go ahead and draw.1428

OK, actually you know what, let me say a couple more words here just to make this clear.1433

What we have right here, this is our pyrophosphoryl.1441

This is not pyrophosphate, OK?1450

Pyrophosphate is often abbreviated as PP_i; pyrophosphate is the following.1457

It is O, P, O, P, O; OK, definitely distinguish between the two.1465

This is pyrophosphate right here; that is pyrophosphate.1476

This is the pyrophosphoryl group; it is a group that is attached to the nucleophile.1481

Now, the nucleophile may have brought an oxygen with it, so it might look like there is a pyrophosphate attached to it, but you have to understand where the oxygens came from.1486

So, in this particular case, it is going to be pyrophosphate that is going to end up being the leaving group once this nucleophile phosphoryl reacts further.1498

It may actually end up losing, so we may end up with something like this: R, O, P, O, P, O.1507

In the reaction itself, what was transferred was, this group was transferred to the nucleophile, which is this thing.1520

Now, when this new activated molecule goes on to react, the leaving group is this thing.1530

Pyrophosphate ends up leaving, but it is the phosphoryl group that is transferred to it.1538

This oxygen that ends up going away, eventually, comes from the nucleophile; it does not come from the phosphoryl transfer.1544

Bear that in mind; it is actually very, very important.1550

These little details are what make the difference; OK, and I think we have one more here.1554

Let's see; let's do this in black.1561

Let's draw our ATP again: O, P, O, P, O, P, O.1565

And, we have ribose, and we have adenosine, O^-.1573

This is that; O^-, this is that, and this is that.1578

Now, we have, of course, our alpha, beta and gamma.1584

Now, when we have nucleophilic attack at the alpha-carbon, we are going to release pyrophosphate, and we are going to have adenylyl transfer.1590

We are going to attach an adenylyl group to the nucleophile, so what you get is the following.1602

You get the nucleophile attached to P, O, O, O, ribose, and that plus inorganic phosphate.1610

OK, so this thing, now, you have the O, P, O, P, O.1625

OK, this is the pyrophosphoryl, so that is what you get here.1627

Let's go ahead and say this is the adenylyl group- there you go.1633

And this is our inorganic phosphate, and you know, it does not hurt to draw it out.1645

Again, repetition is the best way to make sure things stay in the brain.1652

OK, in each case, the transfer of one of these groups to some molecule - which will be the nucleophile - raises the free energy content of that molecule, free energy, which the molecule can now spend in other reactions.1662

I am going to say it one more time; forgive me.1745

A particular reaction is coupled to ATP hydrolysis, either the hydrolysis of a phosphoryl, of a pyrophosphoryl or the adenylyl- whichever these bonds the water actually breaks.1749

That energy is coupled to an endergonic reaction to raise the free energy content of that particular molecule.1762

So, now that molecule is activated, and it has enough energy, has enough money to go spend and do whatever it needs to do.1770

That is how this coupling process works; it actually happens by transferring a phosphoryl group to activate the molecule, transferring a pyrophosphoryl group or transferring an adenylyl group.1776

Hydrolysis of ATP does not actually takes place; it is just a terminology that we use to talk about the transfer of energy, the transfer of money.1788

Somebody only has $5; I am going to give them $100.1796

Now that they have enough to do what they need to do, they are going to go and do what they need to do- that is all that is going on here.1799

Thank you for joining us here at Educator.com.1804

We will see you next time, bye-bye.1807