Biochemistry Enzymes II

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

Today, we are going to continue our discussion of enzymes, and we are going to start off by talking a little bit more about the enzyme substrate complex and how it actually facilitates, how it does what it does, how it contributes to an enzyme doing what it does.0004

Let's just go ahead and get started; we mentioned that you have an enzyme that comes in contact with its particular substrate, and it forms something called the enzyme substrate complex.0018

OK, this enzyme substrate complex is absolutely key to understanding why an enzyme does what it does and how it does what it does.0032

Now, catalysts, we know, they bring about their effects by binding well...catalysts, let's not use the word catalyst.0042

Let's go ahead and just call them enzymes.0066

OK, enzymes bring about their effects - let's start again, shall we, alright - by binding well not to the substrate as is but fitting well or binding well with the transition state.0072

In other words, let me talk about this a little bit.0116

When an enzyme...yes, I mean an enzyme is going to recognize its particular substrate.0120

That is part of the specificity of an enzyme; an enzyme is not just random.0125

It has a particular substrate that it works with and only that substrate, maybe another one, but it is very, very specific for the molecule that it wants to bind.0129

When it binds that molecule, it is not just going to bind the substrate as is.0138

It recognizes the substrate, and then, what happens is, it plays around.0145

It moves a little bit; it twists the substrate around a little bit.0152

The best binding interactions, the optimal binding that takes place between a substrate and an enzyme, in other words, when it is at its most optimal enzyme substrate complex state, it is when the enzyme has actually bound well to the transition state of the substrate.0158

In other words, the substrate, in order for it to actually go through and go from substrate to product, it has to pass through some transition state.0178

Well, the enzyme does what it does not by binding well to the substrate, but the best binding happens because it binds well to the transition state; and we will show what happens energetically in just a minute.0186

Enzymes bring about their effects by binding well not to the substrate as is, but by fitting well, binding well, with the transition state.0199

That is what is important; now, this is actually best explained diagrammatically.0207

I am going to put up an image in just a moment, and then, we will go through that.0215

Let's say that the particular reaction we want to talk about, let's say that our particular reaction is the breaking of a stick.0221

OK, I have this stick, no catalyst, no anything.0247

In order for me to break it, I have to take that stick, and I have to invest a little bit of energy and actually bend that stick.0253

I have to take it to this point, right?0259

I have to take it to a point where it is just about to break and at that point, a little extra push, and you end up getting 2 pieces, right?0261

It breaks right there; now, what a catalyst does is the following.0270

I have to invest a certain amount of energy before boom.0276

That is this; here is the stick.0280

Here is the broken stick; this point right here, the transition state, that is this point right here.0285

I have to put in a bunch of energy in order to get it to that point, and then, after that, boom.0290

I go over, and I end up with my 2 pieces.0296

Now, let's take a look at a diagram what a catalyst actually does.0300

What a catalyst does is it recognizes that stick, and then, what it does is it fiddles with that stick.0306

It twists it; it changes it.0312

What the catalyst does, it actually bends it; the catalyst, the interactions that the stick has with the amino acid residues in the active site of the enzyme, every time it does that, it actually takes it to the transition state for me.0315

That is what a catalyst does.0330

Here is the substrate energy; here is the product energy.0334

Without a catalyst, I have to go up to here, but because the catalyst, what it does, the enzyme, it takes it to the transition state for me.0337

It takes it to the bent position; it is in this position that all of the interactions are really, really tight and strong between the enzyme and the substrate.0348

At that point, it has already raised the energy level up to here, here, here, here, here.0358

Now, the only thing that the reaction has to do is just go over that hump.0365

Now, it has less energy that the actual reaction has to invest in order to go over that hump.0369

That is how an enzyme, a catalyst lowers the activation energy.0375

It is not so much of lowering of the activation energy; that is true.0381

I mean the transition state is a transition state; in order for the stick to go from single stick to a broken stick to pieces, it has to pass through this transition state.0384

Instead of us putting in the energy to actually bend it, the interactions between the amino acids, the weak interactions, the hydrogen bonding, the hydrophobic interactions, the ionic dipole interactions, all of those things, they end up stabilizing the transition state enzyme complex.0395

And now, it is already at its transition state.0416

Now, because it is here, all it has to do is - poop - go over and form product.0420

That is what is happening; the energy that comes from all of the small interactions of binding, those actually lower the activation energy.0425

Really, what you are doing is you are taking this, and you are bringing it closer to this and that.0435

A substrate energy level is not going to change; a substrate is at a certain energy level.0443

The products are at a certain energy level; that does not change.0447

What does change is the amount of energy that we have to invest to make it go over that transition state.0450

The enzyme is there to do most of the work for us; that is why reaction rates proceed faster.0458

In the process of forming this enzyme substrate complex, which is really an enzyme transition state complex because the optimal binding between the 2 happens when the substrate is in its transition from here.0464

It is just ready to go over; now, all the molecules have more than enough energy to go over.0478

All of them will; 100% of them will, not just 3% or 5% or 10%.0483

That is where this comes from; OK, this difference in energy...let me see.0487

Actually, let's do another diagram here; I am going to do another energy diagram.0499

We have something like this; we have our substrate and our product.0508

This is the uncatalyzed reaction- something like that.0514

Here, this is the enzyme substrate complex.0518

This is substrate; this is enzyme substrate.0524

This other one does not matter; because it is in this form, because the substrate is already in its transition state when it is bound to the enzyme, now, this transition state is here.0527

Now, instead of all of this energy, all I have to do is go - poop - a little bit that way, just a little bit of energy in order to get from here all that way to product.0539

That is what is happening; the transition state is easier to reach.0549

The energy for that instead of us putting it in to make it go over this hump, the enzyme with its weak interactions - hydrogen bonding, hydrophobic interactions, ionic interactions - that it has with the substrate, bending it and twisting it and already taking it into the transition state, now, we do not have to work as hard to get over that transition state.0554

All it does is it brings the transition state within reach of us.0581

That is all that is doing; that is how enzyme works.0585

This difference in energy, in other words, the difference in energy from here, now, here, this difference comes from weak interactions between substrate and functional groups on the amino acid residues of the active site.0592

This is called binding energy.0651

OK, now, these weak interactions, I can do a little bit better than that.0662

Actually, it is hydrogen bonding, bonding hydrophobic interactions, ionic interactions, account for an enzyme's specificity.0687

When an enzyme binds to its substrate, every time some weak interaction takes place, it actually acts as a support for the substrate.0734

Every time there is some hydrogen bonding interaction, every time there is some ionic interaction, some hydrophobic interaction, that energy is released as free energy, and what that does is that brings the transition state, the amount of energy that we need to invest.0745

In order to get to the transitions state, it makes it a lot smaller.0759

That is what it is doing.0763

The best way to think about it is let's say you are standing on a stair step or something.0767

You know that if you lean over too far, you are just going to fall over.0773

Gravity is going to pull you; it is just going to pull you down.0777

Well, if you were to lean over a little bit, let's say one of your friends grabs your hand, now, you can, sort of, release yourself.0781

Your friend is supporting you; he has you.0786

Let's say you lean over a little further; now, another friend comes and basically, let's say, grabs your other hand and maybe another friend is holding you from the back.0788

At this point, you can, sort of, let your entire weight fall on your friends.0796

Those friends are the weak interactions; they are supporting the substrate.0801

You are the substrate; they are supporting you.0805

You do not have to expend any energy in order not to fall over; now, they are the ones that are actually taking the energy and supporting you.0809

That is what an enzyme does; an enzyme supports a substrate so that the substrate does not have to support itself, and it takes it to the transitions state.0816

Now, it is very, very simple; now, your friends can just, sort of, take you, lean you down and put you down on the ground, if that is the reaction that needs to take place, and it happens in a very controlled manner as opposed to just - boom - falling over.0825

That is what an enzyme does; well, these interactions, they not only account for the lowering of the transitions state energy, the activation energy.0839

They also account for the specificity of an enzyme.0847

In other words, an enzyme is going to reach out for the substrate that has the most affinity for its particular active site, and the more tightly the substrate binds, the more weak interactions, the more tightly it binds, now, it is more specific.0851

It is not just any random molecule that can wander into the active site of an enzyme, but they do.0870

I mean, molecules wander into active site of enzymes all the time, but the enzyme is not going to interact with them because there is no way that the enzyme is going to twist and turn to make that particular substrate, whatever it happens to be, bound tightly; but there is usually 1 substrate, the substrate for that enzyme that binds beautifully, that binds tightly.0876

It is the substrate that the enzyme is designed for, that it evolved for.0896

That is what makes enzymes so amazing.0900

The evolved for specific substrates; it is absolutely fantastic.0904

These weak interactions, they also account for an enzyme's specificity.0909

OK, the key point, the point that we actually want to makes is the following.0914

I will do this in red- the key point.0923

Optimal interactions between substrate and enzyme happen when the substrate is in its transition state.0929

The closer the substrate gets to its normal transition state, it is at that point that it binds very, very, very tightly with the enzyme, and it is at that point that the reaction actually takes place because it is already at the transition state.0969

There is no actual energy; it says "boom"- that is it.0983

It happens; OK, now, let's go back to black.0985

An enzyme is much, much larger than its substrate.0997

OK, now, although substrate molecules tend to be flexible, although substrates can be flexible because of the sheer size of enzymes and the fact that they are made up of amino acids, enzymes enjoy greater flexibility.1010

This is very convenient.1045

Now, since optimal enzyme substrate interactions occur not between enzyme and substrate as is, but between enzyme and transition state for the substrate, the enzyme will often - always actually - undergo changes in conformation to accommodate this.1050

An enzyme is very flexible; it has a certain active site.1137

It recognizes the substrate, starts to bind with it.1142

As the binding in interactions get stronger, it binds more with it, twists and turns it this way.1146

The enzyme will twist; the enzyme will turn.1152

The enzyme will open; the enzyme will close.1155

It will conform to something, so if I want to grab this thing, this is the enzyme, this is my substrate, I do not just...yes, I mean, it fits, but I have to...notice, I am changing my fingers.1158

I am conforming to actually fit the substrate.1175

Now, the binding is really tight; now, the substrate is held really, really well.1180

This is the enzyme that is very, very flexible; there is an induced fit.1184

If this were something else, holding it this way as a pen, it is inducing me to do this.1188

If I wanted to hold it a different way, it would induce me to hold it like this.1197

This and this are 2 different things.1201

This is one enzyme; this is another enzyme.1205

We call it induced fit; that is the whole idea.1208

This changes in conformation that enzymes undergo.1211

It is called induced fit because the fit is induced by the particular substrate.1214

Let me write that in red- very, very important.1227

That is all that it is; it is actually pretty intuitive.1231

There is nothing about this particular nomenclature induced fit that is strange.1234

It is when an enzyme conforms to a substrate when it changes, closes, twists, turns - very, very important, very deep important functionality of an enzyme - and then, later, when we talk about regulatory enzymes, it is going to play an even bigger role.1242

Let's go ahead and let's look at a couple of images of induced fit.1259

They are not the best, but I think it just, sort of, gives you the idea; it is nice to see a couple of pictures at the very least so something like this.1265

Here is your substrate; you have this active site here.1271

It fits in there; when it actually fits, the enzyme closes in on it.1275

It is an induced fit, not just that is it; substrate comes it- that is it.1281

The enzyme just stays there; that is not what happens.1285

This one right here, here we have 2 substrates; we have 1.1288

We have 2; you have an enzyme which is actually open.1291

These need to come in contact with each other in order to react.1297

They come; they enter the space, closes, closes, closes.1302

In this particular case, it actually brings the molecules in close contact, so that the molecule can react again, another way of lowering the activation energy.1307

It goes ahead, the product is formed, and then, the enzyme opens up again- that is it.1317

It is induced fit; it can actually release its particular product and other substrate or whatever it is that it happens to do.1322

OK, now, let's look at...actually, let me go to blue here.1333

Let's look at some catalytic mechanisms.1350

Now, that we have talked a little bit about enzymes generally, let's talk about some catalytic mechanisms.1354

How do enzymes mechanistically, specifically do what they do?1361

OK, now, in this particular case, I am going to be introducing some mechanisms and using some examples of some enzymes that do what it is that they do.1367

You do not have to know these enzyme names; at this stage, it is only general.1378

We want you to have a general idea of acid-base catalysis, metal ion catalysis, covalent catalysis.1382

Later, when we start talking about the metabolic pathways, that is when we are going to get into the specifics, and these enzymes that we talk about here, you are going to see them again; so do not worry about it.1389

Here, we are concerned about just general notions.1398

Later, we will get into actual specific mechanisms.1402

The basic catalytic mechanisms that we are going to be concerned with most are going to be general acid-base catalysis, covalent catalysis and metal ion catalysis.1407

OK, let's talk first about general acid-base catalysis.1435

Well, acid-base catalysis is exactly what you think it is.1452

An acid is something that donates protons, and a base is something that takes protons.1459

I do not like to call it "accepts a proton"'; I like the idea of "taking" better because it just seems to be more appropriate, but that is fine.1463

You can look at it however you want to look at it, and that is it.1469

General acid-base catalysis is when specific functional groups on the amino acid residues that are lining the active site when they act as proton donors or proton acceptors, proton takers.1474

Catalysis, when specific functional groups on amino acid side chains donate or - that is fine, we will just go ahead and say "accept" because that is the accepted standard nomenclature - accept hydrogen ions.1491

OK, now, there are...I am going to go through a little bit of a list here of amino acids- their acid form and their base form.1522

Particular amino acids, let's just go ahead, amino acid, let's see.1538

Its acid form, in which form it actually has hydrogen ion to give away, and its base form, it is the form of the functional group where it is not going to give a proton.1546

It is going to actually take a hydrogen ion from a substrate of another amino acid.1560

Let me see; we have glutamate, and we have aspartate.1567

This is the acid form; this H is available for donation.1576

Its base form, this site, the O^-, is available of protonation.1580

It can take an H; I will see this one again in just a minute.1587

Lysine, arginine RNH₃⁺.1593

This has a proton that it can donate.1601

RNH₂, this has a site that can actually accept a proton or take a proton.1605

Let's see; let's have cysteine, RSH, RS-- proton to give, proton to accept.1612

Actually, let me go to the next page here; we have histidine.1626

Histidine will show up all over the place.1631

Let me go ahead and write the top again, acid form, base form.1636

OK, we have histidine; we have R, C, C, N, C, N.1651

Let's see; it is there.1660

This is there; this is H.1665

This is H; let me see.1668

This is H, and that is a plus; yes, I do not think I have missed anything.1670

Let's go ahead and put that H there; that is the acid form.1674

It has an H that it can give up, and the base form is...it has a site that it can accept a proton; and let me see.1677

Let's go ahead and have...another possibility is serine.1698

We have ROH, RO^-; it has an H to give up.1703

It has an H that it can accept, and let's just go ahead and do one more, maybe a tyrosine, where we have R.1707

Let's make this a little bit nicer, shall we.1722

Yes, there we go, ROH.1727

And again, we have this not unlike this one, O^-.1731

It has an H to give up; it has an O^- that can accept an H.1736

OK, now, let's go ahead and do an example of an acid-base catalysis mechanism just to show you how this thing actually works.1741

Let me go ahead and do this in blue; now, let me...I wonder if I should do it here.1753

Yes, that is fine; I guess I can do it here.1758

Our example, we are going to be using...this enzyme that we are going to be talking about as an example of general acid-base catalysis is phosphohexose isomerase.1762

It is going to be involved in the glycolytic pathway, which we will talk about in the second half of the course.1785

Let me go ahead and draw a little something here.1791

I will go ahead and draw this, and let me go ahead and...that is OK.1795

I will go ahead and do it here, so 1, 2, 3, 4, 5, 6.1800

I will go ahead and put my aldehyde group here.1809

Let me see my OH, my H, OH, H, OH, OH, O, PO₃, yes PO₃^2-.1814

OK, now, I have got a glut C, O^-.1826

Alright, again, general acid-base catalysis, all that it means is that this is the enzyme, and here, we have this glutamate residue; and we said that glutamate, when it acts as a base, it has the COO^-.1840

It is in a position to accept a proton, which might make something easier for the particular substrate.1856

In this case, we have this aldehyde here.1862

General acid-base catalysis is just that.1867

These functional groups along the...this is the enzyme, and this happens to be an amino acid residue in the active site of the enzyme.1871

They can accept protons or donate protons, whatever is needed in order to make the reaction go- that is it.1882

In this particular case, here is what happens; it actually takes this proton.1891

These electrons move here; let me do this in red actually.1896

Let me show the movements of electrons in red; my apologies.1899

It is probably better that way; it takes or it accepts that proton.1902

These electrons move here; these electrons go there and pull some other hydrogen from solution.1907

What you end up with is the following.1914

Again, we have this glutamate, which is, now, glutamic acid, OH.1918

Now, we have C, C, C, C, C, C.1928

Now, this is H, and this has become OH.1939

This is now a double bond; this is also OH.1943

This is OH; this is OH.1948

This is OH, and this is O, PO₄, PO₃ - sorry about that - PO₃^2-, right.1951

In this particular case, it acted as a base; it took this proton so that electrons can move to form something else.1962

Now, something else happens; now, it is going to act...I am sorry.1970

Yes, it acted as a base; now, the next step of this particular mechanism, and again, we will revisit it in the future, it is going to act as an acid.1973

It is going to give back a proton, so here is how that works.1981

Let me go ahead and write this as this way.1986

These electrons move here; these electrons grab this, and they push the bond back to there.1992

Now, it is acting as an acid; now, it is going to give this hydrogen back to this, but this time, instead of attaching to this carbon, it is going to attach to the carbon right above it.2000

All we have done is we have actually shifted something.2012

Well, we are not going to worry about what is actually happening here.2016

We are changing the carbonyl; we are moving it to this carbon and putting it on the second carbon, but all we have done is this amino acid has acted as a base by taking a proton facilitating this reaction, and then, now, it is going to act as an acid by giving up its proton back to this particular molecule.2020

This is an example of general acid-base catalysis; that is all it is.2035

It is the movement of protons; that is all.2038

That is all; that is all.2041

This new name, general acid-base catalysis, it sounds really complicated- it is not.2044

It is just some amino acid residue, some functional group on there that is giving up a proton, taking a proton, giving up a proton, taking a proton- that is it.2048

It is interacting with the substrate.2056

OK, let's take a look at, now, a covalent catalysis.2060

Let's do this in blue; let's try again, blue.2067

Now, covalent catalysis is where the enzyme or the coenzyme - sometimes both - forms a transient covalent bond with the substrate.2078

Remember we said that the interactions that exist between enzyme and substrate are weak interactions- hydrogen bonding, hydrophobic, ionic dipole and ion.2110

There is no covalent interaction, but in the actual catalytic mechanism, in order for the reaction to move forward, it might have to bind with the substrate; or the coenzyme might have to bind with the substrate temporarily in order for it to do what it needs to do.2119

Let's see an example of that.2135

An example of this, we are going to use the glyceraldehyde-3-phosphate dehydrogenase.2141

An again, you are going to see all of these again; this is also another glycolytic enzyme, one of the enzymes involved in glycolysis.2158

OK, let's see if we can draw this out here.2166

Let's see; I am going to go ahead and do it this way, and I will go ahead and put a cysteine residue here, and it has an S, and it has an H, and over here, I am going to go ahead and put a histidine residue, boom, boom, boom, boom, boom.2170

I will just go ahead and put my nitrogens there and there, double bond, double bond.2186

This is there; this is H.2193

OK, now, we have our substrate; let me go ahead and do the substrate in red.2195

I have got C, C and C.2200

This is O, PO₃^2-.2205

This is here; this is H, and this is just H₂.2209

OK, here is the following.2214

Now, what happens is this; this is actually a combination of acid-base catalysis and covalent catalysis.2218

I will do this electron movement in black; this goes ahead and takes that.2224

It pushes these electrons up to here to attack the carbon, and it actually ends up going that way; and what you end up getting is the following.2230

You end up getting cysteine, S, connected to a C, O^-, H, C, C, O, PO₃^2-.2247

Now, the enzyme, itself, this is the enzyme here.2260

This is the enzyme; this is actually covalently attached to the glyceraldehyde-3-phosphate, and it is going to go on and do whatever it does.2265

And now, the histidine residue...where are my double bonds?2274

There they are; now, this is protonated.2282

This is a nitrogen, plus charge; this is protonated.2286

Now, that is it; there is a base catalysis happening here.2290

This acts as a base, takes this proton; it pushes these electrons to form a covalent bond with the glyceraldehyde-3-phosphate.2294

This is just temporary; it is going to go on to do something else.2300

These electrons are going to come back down; this is going to go back and grab another...it is going to just do whatever it is...another phosphate is actually going to come in here.2303

That is what is going to happen; we will worry about that mechanism later, but just general covalent catalysis, it where the enzyme or the coenzyme temporarily attaches itself covalently to the substrate.2311

OK, and let's go ahead and finish this off with metal ion catalysis.2324

Metal ion catalysis and the...this is, sort of, self-evident.2332

It is where a metal ion is involved in the catalytic process- that is it.2340

I am going to use isocitrate dehydrogenase.2345

This is one of the enzymes in the citric acid cycle, which we will get to again.2355

Let's go ahead and see, 1, 2, 3, 4, 5.2360

Let's see what we have got here, 1, 2, 3, 4, 5.2365

That is that, COOO^-, H, OH, O^-.2372

I guess it does not really matter where we put it; here, what happens is the following.2385

Here, what we have is isocitrate.2396

The reaction that is going to take place is like this.2400

NAD⁺ goes to NADH + H⁺.2405

What we end up with is the following: 1, 2, 3, O^-.2411

Yes, that is correct, and then, of course, we have our carbonyl, COO dehydrogenase; and we have CO^-, O, O.2429

What happens here is the following; now, I am going to go ahead and use my Mn.2442

Let me do the metal in red.2451

This dehydrogenase, what it does, dehydrogenase does 1 thing; it actually pulls hydrogens away, and it oxidizes.2456

This hydrogen and this hydrogen, they go away; that is where that hydrogen and that hydrogen come from.2461

This oxidizes this molecule, and it turns this alcohol into a carbonyl.2468

This carbon is that carbon; now, it turns it into a carbonyl.2473

Well, this carbonyl is actually coordinated to manganese, and what ends up happening, this manganese is actually, it is a positive charge.2476

It is actually pulling electrons this way, so it is making these electrons here...it is creating, sort of, a positive charge on oxygen.2485

It is actually pulling these electrons that way; it is actually facilitating the next step of the reaction, which is this one, electrons here, electrons here, electrons here.2492

This CO₂ group is about to go away; that is what is happening.2505

That is the next step; this metal ion, which tends to coordinate with negative groups strategically placed to support them, to stabilize them, to make the movement of electrons a little bit easier, whatever it is that it can do to lower the activation energy and to make this reaction move forward in the easiest, quickest, most convenient way possible- that is it.2510

Mn²⁺ helps by interacting with the oxygens shown as well as increasing the electron withdrawing power of that carbonyl.2538

It is, now, the carbonyl wants the electrons more; well, because the electrons wants the electrons more, it is going to pull on these electrons.2588

These electrons are going to pull on these electrons, and it is going to make it easier for this carbon dioxide to go away.2593

This bond is what we end up actually breaking carbonyl, thus, facilitating what we call a decarboxylation.2598

There you have it- general acid-base catalysis, covalent catalysis, metal ion catalysis.2621

These 3 mechanisms are going to account for the majority of the mechanisms that we see in all of the pathways that we discuss when we start discussing metabolism.2628

Thank you so much for joining us here at Educator.com.2636

We will see you next time for a further discussion of enzymes, bye-bye.2640