Biochemistry Enzymes VI: Regulatory Enzymes

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

Today, our topic is going to be regulatory enzymes; regulatory enzymes do exactly what the name suggest.0004

They regulate the speed of certain processes.0011

Most enzymes participate in metabolic pathways.0016

The product of one enzyme reaction becomes the substrate for the next enzyme reaction and on down the line.0020

One or more of those enzymes in a metabolic pathway is going to regulate the flow of substrates through there.0027

It controls what the body needs when it needs it - very, very important regulatory enzyme activity.0034

OK, let’s see what we have got.0040

Let’s repeat what we just said in writing here.0044

In metabolism, both catabolic and anabolic groups of enzymes, they work in sequences - excuse me - called metabolic pathways to achieve a certain goal, and that goal is some molecule that they need to either completely breakdown or completely synthesize.0048

OK, now, the product of one enzyme in the sequence - excuse me - as we said, becomes the substrate for the next enzyme in the sequence.0097

OK, a good example of this is glycolysis.0137

It is going to be one of the first metabolic pathways that we actually look at when we get to the second part of this class.0141

An example - oops - is glycolysis, and glycolysis is the breakdown of the conversion – I will go ahead and call it the breakdown because it is a catabolic pathway - the breakdown of 1 glucose molecule to 2 molecules of something called pyruvate, 2 molecules of pyruvate.0149

It is a beginning of how the body metabolizes the sugar that you intake in order to produce energy.0191

OK, the sequence goes like this.0199

We have glucose going to glucose 6-phosphate going to fructose 6-phosphate going to fructose 1,6-biphosphate going to glyceraldehyde-3-phosphate and dihydroxyacetone.0203

The dihydroxyacetone is converted to another molecule of glyceraldehyde-3-phosphate.0228

Now, we have 2 glyceraldehyde-3-phosphates.0236

This goes to 1,3-biphosphoglycerate, goes to 3-phosphoglycerate, goes to 2-phosphoglycerate, goes to phosphoenolpyruvate, and then, finally, we have our pyruvate.0242

This is a metabolic pathway; it is glycolysis.0264

These are the individual steps, and each step is catalyzed by a particular enzyme- a separate enzyme.0266

OK, we have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 enzymes in this metabolic pathway.0277

One or more of these enzymes is going to be a regulatory enzyme.0284

It is going to control the flow of glucose through this pathway.0288

I mean, this is the first one; this is the last one.0294

Now, the regulatory enzyme does not have to be the first or the last.0298

It tends to be often, and it can be somewhere in the middle, too; but ultimately, what it is controlling, it is controlling the flow of the initial substrate all the way through product.0302

That is all that is going on here; OK, let’s see.0312

One or more enzymes along a given pathway, they have the capacity to affect the overall rate of the pathway.0317

I will just say the rate at which substrate flows through the pathway - it is probably a little better description - by increasing or decreasing catalytic activity.0352

As you can see, the reason the body works as well as it does is because of this regulation.0386

There are millions of things going on, millions of reactions happening simultaneously.0394

The body needs to maintain a certain steady state, some reasonable degree of equilibrium at all times, but the body is subject to all kinds of external effectors, if you will- temperature, hydrogen ion concentration, the food that we eat, all kinds of things going on, immune stress.0398

The body needs to adjust that- regulatory enzymes, regulatory proteins.0420

That is what they do - OK - in catalytic activity - alright - in response to specific signals, obviously, in response to specific signals.0425

For example, if there is too much of certain molecule in the body and the body needs to, sort of, cut that back so that excess is actually used up, a regulatory enzyme will shut down a particular pathway that is producing that molecule temporarily until that concentration of molecule diminishes, and then it will open up the flood gates again to allow more- that is it.0444

That is all that is going on here- specific signals.0464

OK, those are the regulatory enzymes.0468

Now, these - let me go back to black, I like black, actually, oh, 2, oh, nice - regulatory enzymes do exactly what they are named for.0470

They regulate the overall rate at which substrate and/or product appears or disappears- very, very intuitive notion.0517

There is nothing particularly counterintuitive about regulation; we do it all the time in our daily lives.0540

OK, now, let’s talk about some of the mechanisms of regulation, what do enzymes do to regulate, how do they go about it.0544

OK, let me go to...I have some green ink now- very nice.0551

I will go to blue; OK, the first one we are going to talk about is called allosteric regulatory, an allosteric mechanism, allosteric regulatory enzyme.0560

OK, this is one that requires the reversible - very, very important - binding of a second substrate - actually, I do not want to call it a substrate, I was going to put it in quotes, but I will not do that - the binding of a second molecule at an allosteric site.0582

An allosteric site just means, it is another site on the enzyme.0624

That is not the active site of the enzyme; that is all it means, just a fancy name for another site somewhere else on the enzyme, so it could be...if the substrate binds here, well, the allosteric site might be close by.0629

It might be over here; it might belong to a completely different subunit.0640

If you have a multi-subunit protein, which is often the case, allosteric sites are often found on other subunits of the protein.0643

A regulatory enzyme is one that requires the reversible binding of a second molecule at an allosteric site - OK - not the active site.0651

OK, now, these second molecules are called...we want fancy names for them.0668

They are called allosteric modulators, and again, modulation is just a fancy word for change.0685

They are allosteric changers, allosteric modulators or allosteric effectors.0690

You are going to see all of these words floating around in the literature.0695

Now, they can be metabolites; they can be anything.0702

They can be metabolites; they can be intermediates along the pathway, not necessarily initial product - I am sorry - not necessarily substrate or final product.0707

They could be anyone in the metabolites in between, or they can be metabolites that have molecules that have nothing to do with the actual pathway itself.0719

They can come from completely different source; it can be anything.0729

Just think of it as a random molecule; they can be metabolites.0735

They can be cofactors; you know, we have talked about cofactors.0738

They can be small molecules; they can be the actual substrate itself.0744

It can also act as an allosteric modulator, so the substrate can actually control how that enzyme does what it does; so I will just put etc.0751

OK, that is one process; we are going to be looking at this in detail in just a minute, but I just want to list the process.0765

Allosteric regulation usually involves some other site on the protein.0771

It could be a single subunit protein; it could be a multi-subunit protein.0777

It is just another site that affects how the enzyme does what it does.0781

OK, another process is something called covalent modification, so another way that modification.0787

OK, covalent modification - let me see if I am actually going to be defining it a little bit later - activity is modulated by adding or subtracting.0807

OK, covalent modification, this is the covalent.0813

I am going to talk about this a little bit later, but I might as well go ahead and write it down now.0819

It is the covalent - now, I will write it this way - adding or subtracting some molecular group covalently.0824

Let's say you have a particular enzyme that is doing something.0850

If we add some small molecule to it, let say, a phosphoryl group , a PO₃^2- and attach it covalently somewhere on the protein, now, because that protein has that thing attached to it, it is either going to be more active or less active depending on the nature of the particular enzyme.0855

That is what we mean by covalent modification; we do something to the protein, add or subtract some molecular group.0875

It could be anything; it could be a big group.0880

It could be a small group, and it changes the nature.0881

It changes the catalytic activity of that enzyme, and it is done covalently.0885

OK, now, in either case, in the case of covalent modification or allosteric regulation, the regulatory enzymes, they tend to be multi-subunit proteins.0895

Multi-subunit because it allows for, well, a greater degree of complexity just by nature of the fact that you have multiple subunits, and we already know that proteins, they, sort of, they shift.0929

They move; they breathe, if you will.0942

A change in one part of a protein in a different subunit has a greater degree of control on the shift that takes place near the active site.0945

Regulatory proteins, because of their complex nature, they tend to be multi-subunit because it allows for a greater control, and that is really what we want.0955

The body wants really, really fine-tuned control.0964

It does not want broad strokes control; it wants very, very fine strokes control- detailed, very, very careful.0970

OK, we have allosteric regulation, and we have covalent modification.0977

I am going to list 2 other mechanisms, and we may talk about them a little bit towards the end.0982

There are 2 other regulatory processes.0991

One is addition or interaction of a regulatory protein.1002

OK, let's make sure that we have everything straight here because we already know that enzymes are proteins.1013

We are using the word protein and enzyme; let’s see if we can straighten this out.1024

You have an enzyme; it needs to be regulated in some capacity.1029

Well, one of the ways that that happens is, you have other proteins that interact with the enzyme in regulatory capacity.1033

In other words, their job is to interact with the enzyme in order to help regulate the enzyme.1042

Those are the regulatory proteins, so we definitely differentiate.1047

In this particular case, the regulatory enzyme itself, we specifically refer to it as an enzyme unless we are talking about it generically as a protein.1054

When we say regulatory protein, we are not talking about the regulatory enzyme itself.1061

We are talking about the protein that interacts with it in order to help regulate the enzyme- very, very important.1066

OK, and another process that occurs is proteolytic cleavage- very fancy name for basically just cutting off a piece of the protein, of the enzyme, to make it active as opposed to its, sort of, being inactive.1073

Proteolytic cleavage is where the enzyme activates or regulates when peptide fragments are removed from the enzyme.1096

Now, it is important to know about proteolytic cleavage, is that proteolytic cleavage is irreversible.1134

Once you cut off a piece of that enzyme to activate it or let it perform its regulatory function, it is not going to go back.1140

Now, if the body needs it, it is going to have to produce a new version of that enzyme.1149

Proteolytic cleavage is irreversible.1155

The other processes, they are reversible.1159

OK, now, very, very important, more than one process can occur for a given regulatory enzyme.1170

A regulatory enzyme does not have to choose between these 4.1180

It can have allosteric modulation; it can enjoy a covalent modulation.1184

It can participate in regulatory protein activity, and/or it can do proteolytic cleavage.1189

It can do all of them or just one of them.1197

It is not limited, and this, again, has to do with really, really fine strokes.1201

This is not a big, broad strokes kind of thing; regulation is very, very important.1208

It has to be able to control it in very, very small amounts, so it uses different mechanisms of regulatory control.1213

More than one process can occur.1221

Sorry; I am going to be a little redundant here.1232

More than one regulatory process can occur for a given enzyme.1238

OK, now, let’s talk about these things in a little bit more detail; let’s begin with allosteric regulation.1252

Let me go ahead and go to red now just for the heck of it, a little change of pace.1259

Excuse me; we said allosteric regulation is where some other molecule binds to another part of the enzyme and controls the activity, either increases the activity or decreases the activity.1269

We will call it positive allosteric modulation, negative allosteric modulation.1282

If it increases catalytic activity, it is positive effection.1287

Now, in allosteric regulation, there may be more than one allosteric regulatory site.1295

There is no law that says you have only one place on that molecule that controls it.1316

You might have 1, 2, 3; you might have 3 on 1 subunit.1320

You might have 3 on another subunit, as many as you need to control that enzyme- that is it.1324

You can have more than 1 allosteric site.1331

Now, if the modulator is the substrate itself, which we said it can be, right - it does not have to be a different molecule, it could be a substrate itself that acts in a regulatory capacity - the enzyme is called homotropic.1336

A homotropic regulatory enzyme is one where the substrate itself controls the rate at which the substrate flows through the metabolic pathway.1377

You can also refer to the modulator itself as a homotropic modulator.1391

It just means that it is the same as the substrate for that enzyme called a homotropic.1397

If the regulatory modulator, if the molecule that binds to the allosteric site, if it is different, well, we call it heterotropic enzyme.1408

If different, then, we call it heterotropic.1421

OK, now, allosteric modulators, they work by stimulating conformational changes in the protein/enzyme to increase or decrease activity- that is it.1435

It will bind to one site; it will cause the protein to shift a little.1485

The shift in one subunit may cause the other subunit to shift a little that will either make the enzyme have more affinity for its substrate or less affinity for its substrate- speed up the reaction, slow down the reaction.1489

OK, let’s see.1504

When the enzyme is inhibited - negative - by the end product of a metabolic pathway, we call it feedback inhibition.1511

OK, an example of this would be the conversion of L-threonine to L-isoleucine.1564

OK, L-threonine, one product, 2 intermediate - I am sorry - 1 intermediate, 2 intermediate, 3 intermediate, 4 intermediate and convert to L-isoleucine, so 1, 2, 3, 4, 5 steps.1582

What happens is, the isoleucine, the product of this particular metabolic pathway in the conversion of threonine to isoleucine, it acts as an allosteric modulator.1605

It will actually inhibit this enzyme, which is the first step in the metabolic pathway.1617

What it ends up doing is it actually slows it down.1622

When the end product is the allosteric modulator, we call it feedback inhibition.1636

It is a special case; OK, now, let’s talk about covalent modification.1643

Let’s go back to blue.1649

Covalent modification, this is where regulatory enzyme activity is modulated - which again, is a fancy word for "changed" - effected by adding or subtracting - or you might say attaching/de-attaching because that is what we are doing - some molecular group covalently.1653

OK, now, the group which is attached/de-attached...actually let me, I need a...OK, the group, this group, is attached or de-attached by a separate enzyme, which should not come as a surprise because every biological process in the body, virtually all of them, are somehow catalyzed.1708

There is some enzyme that does that task, so it does not just happen; some enzyme makes it happens.1755

The group is attached or de-attached by a separate enzyme.1761

OK, now, of the several molecular groups that act as covalent modulators or covalent regulators - I will say modulators - the phosphoryl, the PO₃^2- appears to be the most common.1771

For a particular enzyme that is regulated by covalent modification, some PO₃^- group is attached to the enzyme to affect it some way, and it is de-attached to affect it in another way.1814

The phosphoryl group appears to be the most common, so you are going to have something like this.1831

You have the enzyme and then, ATP, ADP, and now, you have enzyme, and attached to it, you have a phosphoryl group attached to it.1836

Now, it has been covalently modified; there is this PO₃^2- that is attached to it.1855

OK, now, serine, tyrosine, threonine and histidine are common amino acids on the regulatory enzyme to which the PO₃^2- attaches, right?1862

It is going to attach to some oxygen or nitrogen.1900

That is what is going on here; serine, tyrosine, threonine and histidine, they tend to be 4 very, very common amino acids that you find on an enzyme to which the PO₃^2- is attached.1905

OK, now, enzymes which actually attach the PO₃^2- are called protein kinases or kinases- again, pronunciation, irrelevant.1920

When we talk about a kinase, we are talking about an enzyme whose purpose is to attach phosphoryl groups to some other enzyme or regulatory enzyme.1950

The protein kinase is not a regulatory enzyme; its function is to attach a phosphoryl to the regulatory enzyme.1961

Now, enzymes which dephosphorylate or de-attach, the enzymes which de-attach the PO₃^2- - excuse me - are called phosphatases.1968

I will write protein phosphatase.1998

A kinase attaches phosphoryl group.2005

A phosphatase de-attaches, removes phosphoryl group.2010

OK, and then a final word: addition or removal of this PO₃^2-, it can affect the regulatory enzyme in an infinite number of ways.2012

I will just write many ways; there is not only 1 way that it affects it, so lots of things can happen.2049

Let’s go ahead and do an example of this.2054

An example would be the enzyme, the regulatory enzyme glycogen phosphorylase.2058

OK, the reaction that this particular enzyme catalyzes is the following.2081

We have multiple glucose units.2087

I will just put glucose_n, which is actually glycogen.2092

What it does is it adds an inorganic phosphate.2097

It breaks off one of those glucoses, and it adds a phosphate to it.2102

So, what you end up getting is a glycogen unit, which is missing 1 glucose unit, plus you get glucose 1-phosphate- that is it.2106

Glycogen is, it is just a bunch of glucose units, and what this glycogen phosphorylates does is it pulls off one of those glucose units and attaches a PI to it.2124

This enzyme catalyzes this reaction.2137

Now, what we have is the following.2142

Let's go ahead...yes, it is OK; I can draw it over here.2148

OK, we have a serine, and we have a serine.2153

This glycogen phosphorylates something like this, 2 units, and at each unit, there is a serine residue.2160

Serine has an OH group; serine has an OH group, and what happens is the following.2168

Let's write serine O, PO₃^2-.2180

So, kinase activity, the protein kinase that acts on this particular enzyme, what it does is it attaches phosphoryl groups to both serine residues.2191

Now, you have a different form of this glycogen phosphorylase.2202

Now, the phosphatase, as we said, removes these PO₃ groups.2208

This particular version of it is called glycogen phosphorylase a.2214

It is more active.2224

By phosphorylating this, we have actually, positively modulated it.2228

We have actually made the enzyme more active; we have increased the rate at which glucose units are being taken off this glycogen molecule.2234

That is phosphorylase a.2244

Without the phosphoryl groups, you have phosphorylase b.2246

This is less active.2255

It is just a question of perspective; it is not a question of "it starts here and it becomes this" or "it starts here and it becomes this".2259

There is 2 forms of it; it just depends on what you want to take as your starting point.2266

If you want to take this as your starting point, it is becoming phosphorylated.2270

If you want to take this as your starting point, it is becoming dephosphorylated.2274

A kinase phosphorylates; A phosphatase dephosphorylates.2278

One is less active; one is more active.2282

Again, this idea of less and more, these are relative terms.2286

You have to choose one as your starting point and decide which is less or more.2290

If you choose this as your starting point, this is more active; if you choose this as your starting point, this is less active.2294

That is what is important; it is not as if there is 1 degree of activity, and then, everything else is measured from there.2300

It is relative; it does not matter which one you are starting off with.2308

OK, that is an example of a covalent modification.2314

Again, you want to keep a completely open mind with respect to any of this.2336

Anything can happen; it is not just 1 side that it phosphorylates.2343

It could be 2 sites; it could be 5 sites, 10 sites, 30 sites.2347

That is what gives it the kind of control that enzymes need.2353

Perhaps, the phosphorylation of 1 or 2 sites changes it a little bit but not quite enough.2357

It actually induces the phosphorylation of, maybe, 5 other sites.2363

Now, the enzyme is exactly at the degree of control where it needs for that particular set of circumstances at that given moment in that context.2367

That is what we are talking about; phosphorylation of a regulatory enzyme can take place at 1 site, multiple sites or very, very many sites- very many.2377

OK, and often, 1 site must be phosphorylated before another site can actually be phosphorylated.2399

That "can" is the most important word.2425

If a particular enzyme requires, let say, site 5 to be phosphorylated, well, in order for site 5 to be phosphorylated, first, sites, 1, 2, 3 have to be phosphorylated.2429

Let’s just keep 4 out of it for right now.2442

The idea is, not only does it allow it for really, really, fine tuning of the type of control, but it actually controls the degree to which accidental regulation.2446

If it just randomly attaches to no. 5, and all of a sudden, it changes the activity of that enzyme, maybe that is not what is required at that particular moment.2456

This allows, sort of, a safety valve.2465

It has to go ahead and phosphorylate 1, 2 and 3 before 5 is phosphorylated, and then, when 5 is phosphorylated - boom - now, the enzyme can go ahead and regulate the activity the way it is supposed to.2469

It allows not only regulatory control.2481

It allows for the elimination of random regulatory control, things that should not happen- very, very important.2485

OK, now, let’s go ahead and talk about proteolytic cleavage, and then, we will close it off that way.2496

Let me see; I think I will go ahead and go to the next page here, and let me go ahead and do this in red proteolytic cleavage.2504

OK, now, some regulatory enzymes, they demonstrate regulation by becoming active only when 1 or more small peptide fragments are removed from the inactive enzyme.2519

OK, the inactive form of an enzyme, the inactive precursor - let’s call it that - is called zymogen, in other words, something that is going to generate an enzyme- zymogen.2580

OK, now, the proteases, protease is an enzyme that cuts proteins at specific points.2608

The proteases chymotrypsin and trypsin are 2 good examples.2621

The zymogens chymotrypsin and trypsin are active enzymes.2642

They are activated by the removal of certain pieces of the zymogens that give birth to the chymotrypsin and trypsin.2650

Well, the zymogens are chymotrypsinogen and trypsinogen- that is it.2657

We just add the GEN to the end; let’s see if we can write this out- chymotrypsinogen and trypsinogen.2664

Let’s go ahead and draw these out just to show you.2680

In the case of chymotrypsinogen - let’s go ahead and just draw this out here, something like that - we have the terminal amino end.2685

We have the terminal carboxyl end; it has 245 amino acid residues through a series of steps.2703

We end up with 3 fragments; this is 1 long peptide chain of 245 amino acids.2714

We cut some pieces out of it, so what we end up with is the following.2722

We end up with some Ps that includes 1 to 13.2726

The amino acids 14 and 15 are removed, and we end up with 16-146, and 147 and 148, those amino acids are cut out.2734

Those are removed, so we end up with 149 to 245.2749

Now, these 3 pieces, they are going to be attached by disulfide bridges, and those disulfide bridges, when they come together or when the protein actually folds, that is when you have your chymotrypsin.2759

Your chymotrypsinogen is just a single long peptide chain.2773

The enzyme itself has had 2 pieces cut out and the connections.2779

Let me go ahead and just actually put in the disulfide bridges.2783

Let's go here - OK - and then, we will go...let’s see.2793

Let’s do 1 here S, S, and then, there is another S, S; and then, we have that one right there S, S.2800

This is your active chymotrypsin from the chymotrypsinogen- that is it.2816

That is proteolytic cleavage activation of an enzyme.2824

Let’s just do one more; we might as well do trypsin and trypsinogen while we are here.2828

Let’s see, we have trypsinogen.2833

The zymogen is some single peptide chain, 1-245, and what ends up leaving is a val, 4 asps and a lys; and what you end up getting is 7-245.2840

The first 6 amino acids are just cut off, and what you end up with now, is your active trypsin.2870

This is the one that is active; this trypsinogen, it is inactive.2878

OK, now, there are - excuse me - many enzymes that use more than one of these mechanisms, more than one regulatory process.2885

That is fine; I will call it a process instead of a mechanism.2912

There are many enzymes that use more than 1 regulatory process.2916

I think we have mentioned it before; an enzyme does not have to choose between allosteric regulation, covalent modification.2920

It can be one of them; it can be all of them.2927

All of these things allow for a very, very, very fine tuning ability for regulatory control.2931

An example of this would be bacterial glutamine synthase.2940

It uses multiple allostery.2954

In other words, it has more than 1 allosteric sites.2962

It also uses covalent modification and something that we did not actually talk about in detail.2967

It has associated regulatory proteins.2980

You know what, I will write it this way; I will call it regulatory protein association.2990

And again, there are these things called regulatory proteins.3004

Their only task is to interact with the regulatory enzyme in order to help it control its catalytic activity.3008

OK, it is a separate protein whose job is to function as a regulator by interacting with it- yes, that is it.3016

OK, the big question is, why the complexity or regulatory activity?3028

The answer is very, very obvious.3036

Very careful control- that is what you want.3040

And again, it is the difference between very, very broad strokes kind of action, very, very fine strokes action.3046

The control needs to be very carefully done.3055

That is why you have this degree of complexity- absolutely fantastic, absolutely beautiful.3059

One of the most beautiful areas of biochemistry is regulatory enzyme activity and absolutely a fertile, fertile area of research for any of you that are considering what am I interested in, what would I like to do.3064

There is so much that we just do not know.3077

Thank you for joining us here at Educator.com; we will see you next time, bye-bye.3081