Biochemistry Overview & The Aminotransferase Reaction

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

Today, we are going to start our discussion of protein and amino acid metabolism and breakdown.0004

Today, I am just going to do a quick overview, talk about the basic reactions, and then, we will start talking about the individual reactions; and, in particular, we are going to talk about this amino transferase reaction.0013

Let's just jump right on in.0024

OK, the proteins that you ingest or the proteins that happen to be inside the cells, anyway, they undergo normal catabolism like anything else.0027

The thing with proteins or the thing with amino acids is they do not just have the carbon skeleton part, they also have that amino group, that NH₃ group or NH₄ group.0040

So, the metabolism happens in 2 ways.0051

The amino group is metabolized, and the carbon skeleton is metabolized.0054

The first thing that we are going to discuss is the metabolism of the amino group, so just a quick breakdown here.0058

We have our proteins from whichever source, and, of course, they are going to be broken down into amino acids.0068

And, so the amino acids, there is one pathway; this is the other pathway here.0078

This becomes NH₄⁺; the amino group is taken away, and this eventually enters the urea cycle.0083

Some of it is used for biosynthesis, this NH₄; the rest of it enters the urea cycle for excretion.0096

Let's go ahead and just put something like that urea there.0103

That is the urea cycle; now, these over here, these are the alpha-keto acids that are left over - OK - what we would call the carbon skeletons without that NH₃ group.0108

OK, and we are going to have...let's go ahead and do...at this point, those are going to enter the citric acid cycle, which we have already discussed, and between them, there is something called the aspartate-argininosuccinate shunt.0125

There is a connection; these amino acids, the amino group is metabolized one way.0161

The carbon skeletons are metabolized another way, and there is a passage between them.0165

This is, sort of, the big picture of what is going on with amino acid metabolism.0172

OK, we are going to talk about this first part right here and in particular, what happens right here; and then, we will go ahead and discuss the urea cycle in subsequent lessons.0177

OK, let's see here.0186

Now, these processes all take place in the liver, in liver cells.0190

What I am about to do here...these processes all take place in the liver.0196

OK, this is going to be the overview here.0212

You know what, let me go ahead and do this in blue, I think.0217

We have C; we have COO.0221

This is R; we have H, and we have our NH₃⁺.0225

This is just some amino acid that is either inside the liver or came from outside from dietary protein- things like that.0230

What happens is the following: C, C, R, O, O^-.0239

I will go ahead and draw this 1, 2, 3, 4, 5, O^-.0250

This is alpha-ketoglutarate, and this is going to be O^- there; and then, we have 1, 2, 3, 4, 5.0256

We have COO^-; we have H.0267

Let me go ahead and put this NH₃⁺ here, CH₂, CH₂, COO^-.0272

This molecule, well, actually, let me go ahead and finish up the drawing here.0279

This is going to be the NH₄⁺.0286

OK, this is going to be excretion, and it is going to be either NH₄⁺.0291

It is going to be urea, or it is going to be uric acid, depending on the particular organism that we are talking about.0299

For us, it is going to be urea; now, let me see here.0307

In red, let me go ahead and do...this is glutamate, this molecule right here.0312

This molecule, right here, is the alpha-ketoglutarate.0321

OK, this is just your any amino acid.0328

Notice that we have an R-group there; it could be anything, and this is your alpha-keto acid that is left over after the amino group is taken off.0333

This amino group is what is taken off; it is transferred to alpha-ketoglutarate, and it is held as glutamate.0341

Glutamate is the central molecule in all of the processes that we are going to be describing that take place in the liver.0350

Glutamate is the place where amino acids are held until they are - I am sorry - further processed.0355

In the process of going from glutamate back to alpha-ketoglutarate, this amino group has released its NH₄.0365

Now, some of these is used for biosynthesis, and the rest is excreted.0373

It enters the urea cycle, which we will discuss in a little bit, so that is all that is going on here.0380

These alpha-keto acids, they are the carbon skeletons that are left over from amino acids that transfer their amino group to alpha-ketoglutarate to form glutamate.0386

OK, that is one of the processes that takes place.0401

OK, now, let's go ahead...another process that takes place is this one.0406

Let's do this one; let’s go back to blue and our molecules.0414

C, C, C, C, C, O^-, H, NH₃⁺, CH₂, CH₂ - oops - 1, 2, 3, 4, 1, 2, 3, 4, 5, yes, there we go.0418

1, 2, 3, 4, 5, yes, here we go, NH₂.0441

This goes to our C, C, C, C, COO^-.0447

This is H; this is NH₃⁺, CH₂, CH₂.0460

This is there, and, of course, NH₄⁺, which goes onto release either the NH₄⁺, the urea or the uric acid.0465

OK, this one, here, we have glutamine.0482

The excess ammonia that is produced in other tissues, tissues that are outside the liver, that ammonia is tied up as glutamine.0490

So, that is this right here; OK, it is tied up that way.0503

What you have is you have glutamate that actually ties it up as the NH₂ group.0508

So, this NH₂ group on the glutamine are the ammonias that are produced by the metabolism of other proteins that are not in the liver cells, and they are transported to the liver cells, at which they give up this particular ammonia group, this NH₂.0512

They give it up as the NH₄, and that goes on to either be used in biosynthesis or excreted as urea, uric acid or NH₄, and this right here, when it gives it up, it turns into this molecule glutamate.0531

And again, as we saw, glutamate is the central molecule in this amino group metabolism.0546

Glutamate- very, very important molecule, and this glutamate, of course, goes on to release this one in another step, which we just saw a minute ago, in its conversion to alpha-ketoglutarate to free up the alpha-ketoglutarate to take more amino groups from other amino acids that are already in the liver.0555

This, right here, is the glutamine from other tissues, OK?0575

OK, now, the excess NH₃ produced in other tissues, as we just said, is tied up as the NH₂ group of glutamine.0584

It is very, very important; it is this NH₂ that is the excess ammonia from other tissues, not this.0613

It is this, which enters the liver and undergoes the above process.0622

Again, glutamate is very, very central here.0650

OK, now, let's take a look at the excess ammonia that is produced in muscle tissue.0654

In this particular case, we have the following; let me go back to blue.0663

We have C, C and CH₃, H, and we have the NH₃⁺, COO^-.0667

This is alanine; OK, this amino acid.0678

The excess ammonia that is formed in muscle tissue is actually tied up with pyruvate to become alanine, and this alanine that is transported to the liver, and it gives up its amino group to form glutamate.0682

This is alanine; actually, I will go back.0701

Let me go ahead and do the... you know what, I am going to erase this, and I am going to do it a little bit lower because I want to do my alpha-ketoglutarate vertically.0706

Let me go ahead and put it over here; this is C, C, C.0715

No, OK, C, C, C, H, OO^-.0720

This is CH₃; this is our NH₃⁺.0730

What we have here is C, C, C, O, O^-.0735

This is that; this is that.0744

Again, another alpha-keto acid; what we have here is the C, 2, 3, 4, 5.0747

This is that one; this is that one.0756

This is CH₂, CH₂, COO^-, and then, of course, we have our glutamate, 1, 2, 3, 4 and 5.0759

We have our carboxylic acid; we have our NH₃ that has been transferred to alpha-ketoglutarate to form glutamate, and let me go ahead and finish these off with a couple of hydrogens and another carboxyl group.0769

OK, now, this is alanine from muscle tissue, OK?0785

OK, the excess ammonia that is formed in muscle tissue is tied up as the amino acid alanine.0798

Alanine is carried to the liver, where it transfers its amino group to alpha-ketoglutarate.0806

This is alpha-ketoglutarate.0812

It transfers its amino to alpha-ketoglutarate, and in the process, it forms glutamate; and now, glutamate can go ahead and do what it does giving up...this is that.0817

In that process of glutamate back to alpha-ketoglutarate, it gives up the NH₃.0829

It gives it up as NH₄, ammonium, and the ammonium is either excreted directly, or it enters the urea cycle to be turned into urea.0834

This alanine, the carbon skeleton of alanine, ends up becoming the pyruvate, and the pyruvate does whatever it does.0842

This is from the alanine.0851

Notice, in all of these transformations of an amino acid to the carbon skeleton, these alpha-ketoacids, this amino group is replaced by a double bonded oxygen, by a carbonyl group.0855

It is called oxidative deamination; Deamination means we remove the N, the nitrogen.0867

The amino group, oxidative means we actually also take away some hydrogens.0872

We take away electrons, and we give them up to oxygen, so the carbon becomes oxidized.0876

It is deaminated, and it is also oxidized, so that is what happens there, again, glutamate, central molecule.0881

OK, now, let's go ahead, and I am going to draw, sort of, a big picture of all three in one place, so that you see that.0890

I am not going to use structures; I am just going to use their names, again, just to get an idea of what is happening here.0900

Let me go back to blue; let's go ahead and put...we have a particular amino acid, whatever amino acid happens to come from intracellular protein breakdown or dietary protein breakdown, and I am just going to go ahead and attach an NH₃ group there.0909

OK, this becomes your alpha-keto acid.0930

Now, this is going to go that way; this is the alpha-ketoglutarate, and this is going to be the glutamate.0938

The glutamate has the attached NH₃, and I will circle that, go back to blue here.0952

OK, now, the glutamate is, of course, converted back to that, and it releases our NH₄.0961

This ends up excretion or maybe some biosynthesis, something like that.0970

OK, now, we have something else.0979

The second reaction that we discussed was our glutamine, right?0983

We had that NH₃ group on glutamine, but we also had that NH₂ group.0991

It is this NH₂ that ends up as...well, actually, this ends up here.1003

So, let me go ahead and do this.1011

This NH₂ is that NH₄.1017

This NH₃, right here, is the NH₃ of glutamate.1022

OK, very, very important, this is that.1029

This ends up becoming this.1036

This is this; it is the glutamine that loses the NH₂ group.1040

It does not lose the NH₃; glutamine loses the NH₂ to become glutamate.1045

Glutamate, then, loses its NH₃, well, to become alpha-ketoglutarate.1049

OK, and one final transformation, the third one that we mentioned here, this was the alanine, and I will go ahead and attach the NH₃⁺ group there.1056

Alpha-ketoglutarate, it transfers this NH₃ group to the alpha-ketoglutarate to become glutamate, and it turns into pyruvate.1074

This is, sort of, the big picture of what is happening; this is what is happening in the liver cell.1088

This NH₃ becomes that NH₃.1096

Alanine from muscle tissue, glutamine from other tissues outside the liver and the amino acids that are either intracellular to liver cells and/or the amino acids that come from dietary protein breakdown, the protein that you ingest is transported into the liver.1101

All of those are converted to glutamate- this glutamate, this glutamate, this glutamate.1122

Glutamate is our central molecule for amino acid metabolism.1130

Glutamate gives up its NH₃, its NH₄, and then, it enters the urea cycle, or some of it goes to biosynthesis.1136

OK, now, let's look at this first transformation in some detail.1144

Let me draw it out again; this time, I think I am going to go ahead and go with black.1153

We have got our amino acids, R, H.1159

We have NH₃, COO^-.1165

We have that, and we are left with our carbon skeleton, alpha-ketoacid.1171

A process is this one, 1, 2, 3, 4, 5, COO^-.1180

This is going to be...you know what, I am actually going to...I think I am...well, that is fine.1190

I will go ahead and leave it on this side; it is not a problem.1194

This is 2, COO^-, and this becomes 1, 2, 3, 4, 5, COO^-.1198

This is H; this is NH₃, H₂, H₂ and COO^- - there we go - NH₃⁺.1207

This NH₃ is transferred to alpha-ketoglutarate here.1220

So, let me go ahead and put a 1 here, and I will also put a PLP.1224

This enzyme, I will go ahead and write this one also, but I am not going to talk necessarily about this one just yet.1231

Here we go; now, the enzyme that catalyzes this transformation of an amino acid transferring its amino group to alpha-ketoglutarate to have it convert into glutamate - the first reaction - this is called an aminotransferase.1242

Oops, let me do this in a different color.1259

Let me do these as red, so 1 and PLP.1265

The enzyme is called an aminotransferase; it makes sense, right?1270

We are transferring an amino group; aminotransferase and/or they are also called a transaminase.1275

You will see both of them; now, PLP is the coenzyme pyridoxal phosphate.1287

PLP is the coenzyme for this reaction, for this particular enzyme, is the coenzyme pyridoxal phosphate.1295

OK, now, a couple of words about this, so reaction no. 1, it takes place in the cytosol.1310

This direction, it takes place in the cytosol of liver cells, and aminotransferases, they are named for the amino acid giving up/donating its amino group.1321

Some examples would be alanine aminotransferase if alanine were the amino acid, tyrosine aminotransferase, aspartate aminotransferase.1371

It is named for the amino acid that is actually giving up its amino group to the alpha-ketoglutarate.1381

It is giving up this to the alpha-ketoglutarate, and it is being held as the NH₃ group of glutamate.1387

That is what is going on; now, all amino transferases, they have the same mechanism, and they have the same prosthetic group.1396

The prosthetic group is the PLP, the same coenzyme.1408

All aminotransferases have the same mechanism and the same prosthetic group, which is PLP.1412

That is what the pyridoxal phosphate does.1429

It actually carries the amino group temporarily.1433

This amino group, as you will see in a minute when we do the mechanism in detail, this amino group is transferred to pyridoxal phosphate, and then, when we run the mechanism backwards, the pyridoxal phosphate gives it over to alpha-ketoglutarate.1437

So, it is, sort of, a 2-step thing; it is an intermediate carrier of the amino groups.1449

The amino groups is not directly transferred to the alpha-ketoglutarate.1452

It is given the pyridoxal phosphate, and then, pyridoxal phosphate gives it over to the alpha-ketoglutarate.1457

The prosthetic group have the same mechanism and the same prosthetic group, PLP.1462

OK, let's take a look at PLP a little bit before we actually do the mechanism in detail.1471

OK, let's see what it is that we are looking at; let me go ahead and do this in black.1478

OK, we have got boom, boom, boom, boom, N here, 1, 2, 3.1484

Let's go ahead and put a plus there.1496

We have our CH₃ group here; we have an OH here.1500

We have an aldehyde group here, and of course, we have our O, P, O, O^-.1504

This is the pyridoxal phosphate; it has the aldehyde group.1511

This is where the chemistry is going to take place; this is our PLP.1515

This is pyridoxal phosphate.1524

OK, when the amino group has been transferred from the particular amino acid to the pyridoxal phosphate, it ends up becoming a pyridoxamine phosphate.1529

It is going to look like this, 1, 2, 3, 4.1540

Let's go ahead and put a nitrogen there, there, there.1544

Let's go ahead and put our CH₃ group, our hydroxy, and this time, what we have is our C; and we have our NH₃⁺, H and H.1548

Now, this is not an aldehyde; this is a CN bond.1562

It is an amine; it is pyridoxamine.1567

Let me go ahead and put that there, and leave that plus charge there, and, of course, we still have our phosphate group, so O, P, double bond O, O^-, O^-.1570

This is pyridoxamine phosphate, and this amino group - OK - that is what comes from the amino acid.1579

This is pyridoxamine phosphate; OK, now, this comes from the vitamin pyridoxine, vitamin B-6, just so you know what it looks like.1596

CH₃, we have an OH; we have a COH, and we have a COH.1613

OK, this is pyridoxine, which is vitamin B-6.1621

OK, now, just some words again, PLP carries the NH₃⁺ group, again, as it is transferred from the amino acid to alpha-ketoglutarate.1632

The amino acid becomes an alpha-ketoacid, then, in a reversal of the mechanism steps, alpha-ketoglutarate becomes glutamate, and you will see that in just a minute.1668

Alpha-ketoglutarate becomes glutamate- that is it.1704

It is just a coenzyme; it is a carrier for this amino group as it is transferred.1711

It turns into this, then, it turns back into that, back and forth, back and forth, back and forth, ping pong mechanism, if you want to think about it.1715

OK, alright, now, let's go ahead and actually take a look at an example of this in a rather detailed mechanism.1724

Most parts of this mechanism are actually not altogether that important in the formation of this thing called a Schiff base, a carbon double bonded to a nitrogen, but I will go through it rather carefully just to make sure that everything is understood.1734

There is a lot of hydrogens moving around, a lot of electrons moving around.1747

Again, if you are reasonably comfortable with mechanisms, at least, sort of, seeing them with electron movement, they should not really present any problems.1751

Let's see what we have got; let's see, the aspartate aminotransferase mechanism.1761

OK, here we are; we are going to start out over here, and we are going to work our way that way.1767

Alright, let me go ahead and use...I, myself, am actually going to go ahead and use a red if I need to write anything here.1776

Notice what is happening here; now, the enzyme, this pyridoxal phosphate, is just as is.1785

A lysine 258 with its NH₂ group, an actual lysine residue on the enzyme - OK - on this amino tranferase, it is going to be covalently bonded to this free pyridoxal phosphate.1792

So, this nitrogen ends up attacking this carbonyl group, and you know what happens when a nitrogen attacks a carbonyl group.1807

Electrons move up here; you end up with this tetrahedral intermediate, and then, this hydrogen ends up leaving.1813

These electrons move up here; they kick off the hydroxide.1820

The hydroxide either goes away as regular hydroxide, usually picks up some hydrogen as water, and you end up with this Schiff base or what they call an aldimine.1824

Again, names do not really matter here.1835

What matters is movement of electrons; do not worry about...unless your teacher really, really wants you to learn specific names, ketamine, external aldimine, internal aldimine, these things are not important.1839

What is important is the mechanism; now, you have this pyridoxal phosphate bonded to the enzyme itself, covalently bonded in this Schiff base - OK - carbon double bonded to a nitrogen.1851

In this particular case, the amino acid that is coming in is going to be aspartate.1870

So, this mechanism is the aspartate aminotransferase mechanism.1875

This particular enzyme, the amino acid aspartate, and it is going to be this NH₂ group that is going to end up being transferred right there.1879

Now, what it does, it does the same thing that the N from the lysine residue did.1888

It is going to attack this double bond; OK, it is going to kick the electrons over to here.1893

Now, again, they have this carbon that has 4 bonds to it.1900

It is bonded to this nitrogen; it is bonded to this nitrogen.1905

OK, this hydrogen goes away; these electrons jump over here.1908

OK, and then, these electrons from the nitrogen, they jumped over here to form a double bond, and it breaks the bond that the pyridoxal phosphate has with the enzyme.1915

That is what is really happening; it is the aspartate, the amino acid, that happens to be involved in this reaction, is going to take the place, the NH₂ group is going to take the place of the NH₂ group from the lysine.1928

Now, it is going to attach to it, and that is what gives us this right here.1942

Now, we have the aspartate that is covalently bonded to the pyridoxal phosphate.1946

And again, there is going to be some shift of electrons; an electron from a hydrogen bond is going to go here.1952

It is going to form a double bond; it is going to push these electrons here.1957

It is going to push these here, and it is going to push these electrons here.1960

Remember this nitrogen here has a positive charge, so it is an electron sink.1964

It, sort of, pulls electrons toward it, which is what makes all of these transformations possible.1968

Now that you have that - OK - now, these electrons, they push back.1975

They come up here; they push this way.1982

They push the double bond out to grab another hydrogen from solution, and now, you end up with this thing.1984

You have a carbon, a couple of hydrogen, a couple of hydrogens bonded to it.1991

Now, you have it single bonded to a nitrogen, which is double bonded here.1995

Now, what happens is water comes in, and it hydrolyzes this bond.2000

It breaks the bond that the NH₂ group has to its alpha-carbon of aspartate, and that is replaced with this; and now, the amine group is, now, attached to the pyridoxal phosphate.2009

At this point, the alpha-keto acid that is formed, which in this particular case happens to be oxaloacetate - it does not really matter what it is, it is an alpha-keto acid - it leaves.2029

Alpha-ketoglutarate comes in, and then, we reverse the mechanism; and we end up releasing glutamate.2039

This pyridoxal phosphate is converted to pyridoxine phosphate.2046

Alpha-ketoglutarate comes in; pyridoxamine phosphate gives its amino group.2052

It releases glutamate here, and it is turned into pyridoxal phosphate to start the catalytic cycle all over again.2058

The important parts of this mechanism are the NH₂ group on a lysine residue of the enzyme attaching covalently to pyridoxal phosphate in the form of a Schiff base, an imine or imine.2068

I do not know; different people pronounce them differently.2081

The amino acid, its NH₂ group, it replaces the NH₂ from the enzyme.2086

Now, you have the actual amino acid covalently bonded to the pyridoxal phosphate, and then, water comes in to hydrolyze that bond between the amino acid and its amino group, so that the amino group gets stuck to the pyridoxal phosphate.2094

And now, it is pyridoxamine phosphate, and the amino acid goes away as an alpha-keto acid.2113

That is what is important: this step, this part, this part and the final part.2120

I hope that makes sense; OK, now, let's go ahead and take a look at the reverse reaction.2129

We have taken some amino acid; we have converted.2139

We have used alpha-ketoglutarate; we have transferred our amino group from that amino acid to the alpha-ketoglutarate to form our glutamate.2143

Now, glutamate is going to release that NH₃ group as ammonium ion and turned back into alpha-ketoglutarate.2152

Let's take a look at that reaction.2160

Let me draw it out one more time here; let me go ahead and use black.2164

We have our C, C.2167

Actually, this is going to be an R, not a C; we are not doing alanine.2171

This is any amino acid, COO^-, and this is NH₃⁺, goes there.2176

This is going to form our alpha-keto acid, whatever it happens to be, and, of course, we had this transformation, 1, 2, 3, 4, 5, COO^-.2185

This is alpha-ketoglutarate, so H₂, H₂, COO^-, and we had 1, 2, 3, 4, 5, COO^-.2199

This is our glutamate, which is the carrier of this amino group, H₂, H₂, and this is OO^-.2213

Now, what we are concerned with is this reaction.2223

It is going to release NH₄⁺.2229

OK, this, right here, let me go ahead and give some names to it.2233

This is glutamate; this is our central black.2238

Let me put this charge here; this NH₃ group is transferred to alpha-ketoglutarate, and it is right here.2244

Glutamate releases it as NH₄, and it goes back to alpha-ketoglutarate.2250

OK, now, this reaction, right here, which is its coenzyme is NAD⁺ or NADP.2261

In this particular case, NAD or NADP⁺, either one of those can be used as a coenzyme for this particular enzyme.2272

This reaction, L-glutamate, well, we already know.2281

When we see NAD and NADP, it is a dehydrogenase- that is it.2288

And, well, the enzyme that catalyzes the conversion of the glutamate back to alpha-ketoglutarate is the L-glutamate dehydrogenase, and it can use either NAD⁺ or NADP⁺.2297

Either one is fine; let's see.2310

The newly formed glutamate, we said that this first transformation, this transformation takes place in the cytosol.2324

Now, this newly formed glutamate is actually transported into the mitochondrion of liver cells and undergoes oxidative deamination.2330

All that means, deamination, we remove this thing.2369

Oxidative, well, we do not just leave it as an H; it is actually an oxygen that is attached to it.2374

It turns back into the carbonyl group of the alpha-ketoglutarate-that is it.2379

And, there you have it, amino acid.2387

A particular amino transferase using pyridoxal phosphate as a coenzyme will convert that amino acid to some alpha-keto acid, a carbon skeleton.2392

That amino group will be transferred to alpha-ketoglutarate, and it will turn into newly formed glutamate.2400

This takes place in the cytosol; glutamate is transported into the mitochondrion.2408

This is the first one; this is from amino acids that are either intracellular, amino acids or amino acids that come from dietary protein breakdown.2412

This glutamate, now, by the action of L-glutamate dehydrogenase under coenzyme NAD⁺ or NADP⁺, it releases its amino group as ammonium ion; and it converts it back to alpha-ketoglutarate to continue the cycle or to do whatever alpha-ketoglutarate does, and, of course, this NH₄ is either used for...some of it is used for biosynthesis.2421

The rest goes to the urea cycle, which we will discuss in subsequent lessons.2445

There you have it; thank you for joining us here at Educator.com.2450

We will see you next time for a further discussion of amino acid metabolism, bye-bye.2455