Topic 5
2025-09-22 section 1
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You didn't show me in. I.
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Oh, that. I.
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And I even sound like this. I like.
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2000. All right.
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Let's get started. We have. So just so we have finished topic 1 to 4.
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Now we are down to the, uh, the very last topic of unit A.
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Uh, we're going to look at a type of proteins that's, uh, you know, very, very useful in our bodies.
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Uh, we're going to look at enzymes and how enzyme works.
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So, uh, if you think about biochemical reactions, uh, a lot of them actually,
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if you don't, uh, if you just let them happen, they can be very, very soft.
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Uh, for example, um, one of the examples that we are going to look at, we're going to look at how an enzyme digests, uh, proteins.
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Now think about that. Without an enzyme. Um, the digestion can take forever, you know, because we are going to break those, um, peptide bonds.
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And remember, you know, how we, you know, how peptide bonds they are made, you know,
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through the condensation reactions, peptide bonds can be made and then we can synthesize proteins.
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Uh, but, you know, sometimes we need to break the peptide bonds.
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We need to digest peptides for, uh, proteins. For example, if there's no enzyme, we just let the reaction to happen.
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Think about that. If you put a solution of proteins just on the table, um, 20 years later, you come back.
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Half of the peptide bonds will still be intact because stable, you know, peptide bonds are very stable.
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That means the digestion will be very, very efficient.
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So let's see how an enzyme can make these reactions, you know, trillions times faster.
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But before we do that, uh, let's go through the objective.
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So we are going to learn how, uh, enzymes can catalyze, uh, chemical reactions.
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Uh, some terms that we are going to define, you know, uh, in this topic, uh, you need to know the meaning of these terms.
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And then, uh, we're going to use a chemo trypsin, this example to show you how an enzyme can,
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uh, increase the rate of, uh, chemical, uh, specific chemical reaction.
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And then, uh, in the second half of the topic, we are going to look at, you know, the kinetics of, of, uh, chemical, uh, you know,
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enzyme catalysis and also, you know, how to design or how can we, uh, you know, come up with inhibitors that can inhibit enzymes.
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Uh, there are, you know, um, multiple sections in chapters six and seven, uh, that you may want to,
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um, you may want to read in case, you know, there's some concepts not, uh, very clear to you.
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Uh, keep in mind that we're not covering all the materials in, in this two chapters.
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Just, you know, focus on the materials that we cover in the lecture.
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Uh, the lecture slides and, uh, topic notes, uh, and find the corresponding uh, sections that you need to, uh, you want to read on.
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Okay. So what is an enzyme? Uh, you can think of it as a catalyst.
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Um, it can increase, you know, chemical reaction rate, uh, especially biochemical reactions.
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And, um, many of them, I'll, I would say most of the enzymes are proteins.
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You know, just a very small number of them. Uh, they may not be protein.
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That could be RNA. Uh, but most enzymes, they are proteins.
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Now, it's important to realize that enzyme can accelerate chemical reactions, but they are unchanged by the reaction.
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So an enzyme can participate in the reaction.
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But after that the enzymes will be regenerated.
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You will get the enzyme back. We're not going to just use up the enzyme.
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Now, um, if you look at enzymes, you know, they some of their names are very complicated.
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Uh, but many of them end with, you know, ASC, for example, we call, um, you know, enzymes that can cut peptides,
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uh, protease, uh, or that, you know, facilitate oxidation without, uh, reduction reactions.
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We have the reductase, uh, and sometimes the, and, you know, the name of enzyme.
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And with uh, Zeinab Abney, for example, lysozyme here, uh, and also some enzymes, the Nema after the reaction they catalyze.
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So based on the name, you have some idea of what reactions this enzyme is, uh, is catalyzing and also, uh, substrate.
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That means the molecule on which an enzyme acts on.
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So if we say this this one is a substrate of this enzyme means the enzyme will bind to this substrate and convert it into a product.
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So, uh, that's why we call it that. That's that's, you know, that that's why we call it the substrate of an enzyme.
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Now, when it comes to enzyme, you, you may learn, um, in chemistry, uh, you know,
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there are some other, uh, uh, you know, chemicals that can catalyzed reactions,
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but enzymes, if you think about it, compared to other, um, you know, chemical is they are more, uh, they are very amazing.
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Uh, I would say, for example, uh, enzymes can be highly specific in thinking about that.
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You know, we have so many small molecules or substrate.
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Well, chemicals in cells, then enzymes somehow can recognize only one or a few of these hundreds or thousands of molecules in the cell.
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So enzyme the action of enzyme is highly specific.
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It doesn't just randomly bind to different molecules and convert them into, uh, undesired products.
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You know, enzymes are highly specific. And also enzymes.
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They almost never make a mistake. So it's very, very accurate.
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It will bind to the substrate, convert the substrate to product.
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It's not going to be like, well, I don't know.
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Maybe half of the time it will convert it into one chemical and at a time it will convert to something different.
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Enzymes, as you know, almost never make no mistake.
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And then, uh, it's very fast, you know, it can accelerate, uh, reactions by billions or even trillions times over the rate of, um, catalyzed reaction.
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So instead of letting you know, proteins sit in our stomach for, you know,
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20 years in order to digest peptides, uh, it will take the enzyme no time to, to do those digests.
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And also, um, enzymes, they are able to work under mild conditions.
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So in some, you know, chemical reaction, if you have catalysts, uh,
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sometime you need to have the chemical reaction that very high temperature or under very high pressure.
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But we don't need that. You know, we don't want to heat our body up to 100 degree, 200 degrees in order to carry out a chemical reaction.
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And we don't want to live under like very high pressure.
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So, um, enzymes can actually work under very mild conditions in a very fast, very accurate and a very specific way.
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Now, just to give you a couple of examples here. Uh, once again, you don't need to memorize the table.
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Uh, but here I'll show you. The first column here is some, uh, some enzymes and, um, the hop time of the substrate, if, you know,
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let's say if we present a peptide bond to a kind of trypsin, this is an enzyme that can cut the peptide bond.
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If we just lift up the peptide bond there, uh, without an enzyme, it will take about 20 years for half of the peptide funds to disappear, to be cut.
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But then with the enzyme now become much, much faster.
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And if you look at the last column here, the enhancement.
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So basically we are comparing the unfertilized rates to the catalyzed grade.
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And you can see that I can't really see that uh ten to power 11 we are talking about almost a trillion fold increase in the rate.
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So instead of waiting for 20 years, uh, this reaction cutting the peptide bond can happen the very, very short time.
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Now, enzymes can be classified depending on the reactions that, um, they catalyze.
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For example, we have, you know, some enzymes that can increase the the rate of oxidation reduction reactions.
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And we call those enzymes oxygen reductase.
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And also they are enzymes that can help to transfer functional groups from one molecule to another one.
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Remember one of the uh, post translational modifications that we, uh, we talk about in topic three and four,
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uh, this post transmission modifications, uh, all of them are catalyzed by enzymes.
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For example, kinase is an enzyme that transfer a fossil group from ATP to another molecule.
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Uh, we call this one the phosphorylation of a protein.
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And it's, um, you know, this reaction is accelerated by, uh, an enzyme, uh, by a class of enzyme called kinase.
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Now, if you think about how an enzyme work, if it acts on the substrate, the first thing the enzyme must be able to recognize the substrate.
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So let's go through a couple of definitions here.
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Uh, when we talk about an enzyme, it's not the whole proteins who participate in the chemical reaction.
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A lot of times it will be the active site of an enzyme. So it's a small part of an enzyme that's critical for the for the function of the protein.
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The rest of the proteins may be just there so that it can support the active site, you know, forming uh.
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Forming the right structure for uh, for the enzyme to function.
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You need later on, you will see that the precise structure of an enzyme is very important.
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If the enzyme changing structure is not going to, um, it is not going to be, uh, functional.
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Now, when the substrate bind to the active site, it is very similar to ligand binding that we learn in topic four.
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So that will be multiple. Multiple weak bonds and the binding could be reversible.
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Maybe I would just stand here if I know.
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Um, so we have the substrate, we have the enzyme, and it's going to turn into product.
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So basically we have the substrate comes in bind to the enzyme.
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Um and this one could be reversible just like a ligand protein binding.
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But most likely the enzyme will um convert the substrate into product and then release the product.
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In this case, it's cutting this, uh, substrate into two pieces.
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So a couple of definitions here. Active site.
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That's where all the action is. Uh, um, you know, the action of the chemical reaction.
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And then substrate is the one that binds to the enzyme and later on will be converted into product.
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Now. I mean, one thing to keep in mind is that enzymes, uh, can be regenerated, you know, uh, after the reaction during the reaction,
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because, you know, we don't want the enzyme to just turn into something else because cells,
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usually they just, you know, uh, synthesize a little bit of enzymes, but these enzymes,
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they they are, you know, we can we use the enzymes many, many, many times.
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So basically a, um, a molecule, a, a substrate comes in, bind to the enzyme, form, this enzyme substrate complex.
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And then catalysis, uh, happens, you know, the substrate start to turn in the product.
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And the enzymes will then let go of the product. Now the enzyme, you say the enzyme is regenerated after the reaction.
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Now it can, you know, go back to um, to turn more substrates into products.
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So we don't need to let's say if we have 100 substrate molecules, we don't need 100 enzyme molecules,
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because each enzyme can just go back and and turn more substrates into product.
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Now another key point here is that enzymes, um, they can accelerate the reactions, but they don't change, um, the energy in that landscape,
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you know, they don't uh, in other words, they're not going to make the reactants or products more stable, as you can see here.
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Um, you know, enzymes will not change the delta g of the reaction if the delta g is smaller than zero.
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This is a spontaneous reaction. Uh, having the enzyme will not will not turn it in into a non spontaneous reaction.
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Uh, the same for here. So enzymes will not change the relative, uh, free energies of the reactant and product.
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It only increase the rate of the reaction and make this one happen faster.
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Now if we think about a simple chemical reaction, uh, we can draw out a reaction coordinate.
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For example, there's a covalent bond between A and B.
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Now we add, uh, another chemical and undergo a chemical reaction.
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Then the bond between A and B uh is broken.
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And then B and C they form let's say a covalent bond.
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Here. If we draw the chemical reaction coordinate it could be you know, we start with the reactant and then uh,
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somehow uh, the bond between A and B, they, they break like partially.
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Now we refer the bond between A and b and b and C they start to form, uh, they start to form bonds.
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And this one we call this one the transition state is highly unstable.
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So this transition, say has a very high energy and it doesn't look exactly like the reactant.
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And it doesn't look exactly like the substance, uh, the product.
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But then uh, once the reaction pass through the transition, say it will come to the product or,
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uh, it will just fall back into, you know, fall back to this side and become the reactants again.
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So once they go to the transition, stay as the choice to come back or go to the product.
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And this one, you know, in the reaction profile in the reaction coordinate, the transition say is the highest point of the reaction coordinate.
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And we what we call the difference between, um, the transition state free energy and the reacting, the free energy of the reaction.
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We call this one, uh, the free energy of activation.
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So that delta g double dagger basically is, you know, the hurdle the reactants need to overcome in order to become the product.
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You can see that if this one is very high, you know, let's say that the free energy of the system is very high.
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Then it would take forever, take a very long time for the reaction to overcome the hurdle and come down with the product.
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So the chance to become a product will be very, very low.
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So let's look a little bit into the transition state because it looks like is a very important say because, you know, that's the hurdle.
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Uh, the Brackins they need to overcome. They need to become the chances and say, and then they come to the product.
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So we can think that the transition stay as an intermediate form between the reactants and the product.
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Like I said, it doesn't look like the the exactly like the reacting.
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The also doesn't look like the product is something in between.
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So is that like an intermediate form?
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Uh, but is important to, um, to remember that this is the point of the highest free energy in this simple reaction coordinate.
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This is the highest point. So this is the rate determining step.
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How high is this free energy or how low it is?
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Uh, will decide how fast the reaction can go. Okay.
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So let's say if you need to climb up a really high mountain, it will take you a longer time.
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All right. Um, you can think about that. You know, there's a lot of ball making and bond breaking, uh, during the, you know, in the chances in space.
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And this one is a very short lived state. So a lot of time, you cannot really isolate the chances in space.
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And you can find that, you know, once, you know, go to the chances and say, uh,
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very quickly, either you go back to the reactant or you'll go to the, uh, product side.
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Now, how to catalyze a reaction?
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You know, based on the simple reaction coordinate.
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We know that enzymes are not going, you know, will not change the free energy of the reactant and the product.
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So we're not changing the equilibrium, but somehow, uh, enzymes can lower the free energy of the transition state.
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So instead of, you know, in the UN catalyzed reaction, the chances say has a very, very high free energy here.
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So climbing up to here will take a long time.
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But what happens if the enzyme somehow can lower the energy of the chances and say,
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now the reaction can go faster, but it doesn't change the equilibrium,
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you know, because the free energy difference between the reactant and the products, uh, remains the same, but just this barrier become lower.
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So that's how enzymes can catalyze a reaction without changing the the free energy difference between the reactants and product.
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But you know, but by lowering the activation uh, free energy here and we can describe this one as you know,
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delta delta G double decker uh, basically is, you know, if we have an catalyzed reaction, we have a very high activation free energy.
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But when we have a catalyzed reaction, the activation energy come much lower.
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So how much you know, how big the decrease in in you know,
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the activation free energy is this delta delta g here basically shows the efficiency of the of the catalysis.
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You know, on paper it seems working.
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But, you know, actually, how can, um, how can an enzyme lower the activation energy?
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Well, we can think of this simple, um, situation here.
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We have two molecules. They need to come together, or they need to bump into each others in order to, uh, for the reaction to happen.
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Now we have this, you know, these molecules, they are very busy.
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They're just, you know, rotating, uh, you know, uh, diffusing, uh, in different directions for them to come together.
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Um, it may not happen very quickly, but will happen if we can provide a platform for them to react.
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So, uh, for example, this enzyme here, it can bind to both, you know, the molecule in purple and the molecule in yellow.
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And you know, it has a well-defined it, you know, sites for for these two molecules.
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So when they bind to the enzymes, you know, if the enzyme is very high affinities to these molecules,
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then very quickly, uh, we will have, you know, these two molecules sitting at the right positions for the reaction,
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not just a, you know, they now bind an enzyme, but enzymes, they are designed in a way that,
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you know, these two molecules, they will be very close in space.
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And also they have the right orientations for the reactions to happen.
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So this is one way an enzyme can catalyze a chemical reaction.
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But this is not the only way. So there are some other way that enzyme can help to uh, to lower the transition state energy.
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Uh, for example, uh, we saw that, you know, all random molecules so that they can,
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uh, um, they can, uh, you know, carry out their reactions very quickly.
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Uh, sometimes enzyme will change the charge distributions of the substrates to just make the reaction, uh, go faster.
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Uh, when we look at the kind of trypsin, when we look at the example, you will see, you know, how enzymes can help to rearrange.
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We arrange the electrons in the substrate so that, uh, it can make the reactions feel faster, or,
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um, enzymes can, uh, bind to the substrate molecule and force it into a transition stage shape.
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So in this case, it looks like this. So this is not, uh, the substrate, the shape of the substrate.
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But once they bind to the enzyme, uh, the enzyme can stabilize the transition state.
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So it can, uh, force the ends, uh, the substrate to go into, uh, a conformations that,
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you know, that's like the transition state of, uh, of the reaction, uh.
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Now, let's say if we want to study enzymes.
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Uh, the first thing we want to know is, you know, how fast or how good is this enzyme in binding the substrate.
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Now, the enzyme can be very efficient, but if it cannot recognize the substrate,
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cannot find very tightly the substrate to begin with, then that's no use, right.
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Because the substrate will not come to the enzyme, um, to uh, uh, to undergo the chemical reactions.
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So, um, so a lot of time, when we study enzymes, we design molecules that can bind to the enzyme, uh, but it may not undergo the chemical reactions.
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So we call those transition state analogs. So these are the enzymes that we sample the chances and say it has a similar geometry and charge to zeros.
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And just like the transition states but they do not undergo a chemical reactions.
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So you may think you know why we want something like that.
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Let's say if this is an enzyme I want to inhibit, you know, let's say um, the enzyme, uh, uh, the activation of the enzyme can cause a disease.
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So in this case, I want to design a drug molecule that can, uh, inhibit the enzyme.
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One way I can do is I can, you know, design something that looks like the substrate so it can compete with the substrate for binding to enzymes,
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but somehow, uh, the inhibitor cannot convert into the product.
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So basically, just use of the enzymes.
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Now if you think about, uh, how you enzymes to act, uh, accelerate chemical reactions, we say that the enzymes can bind the substrate,
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but also can bind to the chances and say to lower the energy of the chances and stake.
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So it turns out that many drugs and antibiotics are enzyme inhibitors, and many of them are effective because they are transition state analog.
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So the drugs actually look more like the transition state than the substrate, so that it can bind to, uh, the enzyme much more effectively.
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But we are going to look at competitive inhibitor later on, you know, in the second half of the topic.
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But I just want you to, to realize that, uh, actually the transition state is the state that has, um.
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Higher affinity to the to the to the enzyme is even better than the substrate.
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Now a couple more definitions before we move on to, um, to, uh,
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an example that show how an enzyme can lower the activation energy of the, of the reaction.
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So, uh, just, you know, want to spend several minutes to talk about cofactors.
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So some enzymes, they require a partner to function.
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So this sounds familiar. Remember a protein that can find oxygen but only in the presence of an other organic molecule.
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Remember we talked about model globin and hemoglobin without the heme groups.
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You know those proteins cannot buy oxygen. Uh it's that also applied to some enzymes.
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Some enzymes will need cofactors in order to, uh, to carry out a function.
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And you know, these cofactors, they can be metal ions.
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Sometimes the enzyme need, uh, metal ions, you know, magnesium, zinc or some other ions to bind to the protein in order to, um, to become functional.
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And sometimes it will be in self metal ions.
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It could be, uh, organic molecules. And we call those enzymes and Co enzymes can further the, you know, uh we divide into two groups.
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One is called a Co substrates and the other is the fatty foods.
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So basically we the cofactors could be either metal ions or organic molecules.
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And Co substrates are, you know organic molecules that that will also go into the active site.
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Uh, but they will only bind to the enzyme transiently.
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Um, you know, one example will be, let's say if the enzyme is going to accelerate, uh, an oxidation of a substrate.
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So the Co substrate could be something that will bind to the active site and will be reduced.
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So the reduction of the Co substrate can accelerate the oxidation of the substrate.
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So you can think about that way. Uh, but the important thing is that, you know,
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the difference between Co substrate and post that would cause substrate is something that will go into the active site,
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but it will only stay there transiently. And after the reaction Co substrates could be regenerated or it will turn into something else.
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But prosthetic work here, just like the group is not just chance in me, bind to the enzyme.
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It will stay with the enzyme, almost like permanently.
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And then, um, it's important for the for the function of the enzyme.
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So it's not going to come into the active site and live.
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It will just stay there. It will be part of the enzyme. Now, the example that we're going to go through today is um, is an enzyme called hemo trypsin.
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Now this protein or this enzyme is a member of Syrian protease family.
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So now we're talking about protein family. And we know that what that means is there's a group of enzymes.
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They have similar structure similar function.
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And the sequence of these enzymes there will be some residues that are highly conserved because we know that those are the residues.
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That's very important for the function of the protein. Um so later on you will see that what are the important residues in this, uh, kind of trypsin?
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Now each member of this family, why this called the Syrian protease.
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It turns out that each member of this family contains a critical serine residues in the active site.
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So once again, remember what an active site is. It's just a part of the enzyme, not the whole enzyme molecule.
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Just, you know, the part of the enzyme that, um, all the chemical reactions, uh, happens.
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Now, um, you know, where is this enzyme produced?
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So it's, you know, it's synthesized in pancreas, and it gets secreted to allow the breakdown of, uh, proteins, uh, during digestion in the intestine.
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Now, think about it. If this enzyme is so good that it can, um, accelerate, uh, protein digestion, what happened if it just synthesized in pancreas?
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It was thought to cut the proteins there, which is not a good thing.
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But remember, we have a way to switch on and switch off proteins.
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Later on we will see how the proteins can be regulated up before but but before we move on to that point,
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let's just look at how the proteins how the enzyme carry out the enzymatic functions.
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Now the function is uh is uh the hydrolysis of peptide bonds.
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So remember when two residues, two amino acids, uh they come together to form a peptide bond.
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We're talking about the protein synthesis. Uh, we have this peptide bonds formed between two, uh, amino acids.
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And we call this one a condensation reaction because a water molecules, uh,
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will be removed when these, uh, two amino acids, they form the peptide bond.
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Now we need to do the opposite because we need to digest the peptides.
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We need to digest proteins.
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So we're going to add water back to the, uh, to the, you know, uh, to the system so that these, uh, the peptide bonds will be broken.
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So now we have, uh, separate amino acids.
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Seems like a very, uh, simple reaction. But because the, you know, the peptide bond is so stable.
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And that's why, you know, if we just put, you know, proteins in water, it will take forever for, for this reaction to happen.
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Now, what are the key residues in this protein family.
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It turns out that they are free of residues that are very important for, uh, for the catalytic reaction.
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And they are called a catalytic trial because that's free residues here.
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And you can see that, um, we have, uh, aspartic acid 1O2, histidine 57 and serine 195.
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The numbers, you know, tell us you know where these residues are located in the primary sequence.
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So this one will be the, you know, 190 residue, 195 in the protein sequence.
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Now you can see that they are very far away in the protein sequence.
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And the one is 57. And that is almost 50 residues apart from this one, and almost 100 residues from this one to this one.
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But somehow, because of the folding of the protein, they are very close in space, in the protein structure, in the enzyme structure.
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And they form this catalytic quiet here. So that's one thing to keep in mind.
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And that also tell us how important is, um, how useful is the protein structure.
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Because without the correct fold these three residues, they would not be close in space to carry out a chemical reaction.
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Now, I think the next slide usually is the slide that most students they hate, because that's the catalytic mechanism that's kind of threatening.
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And usually the first questions from students will be do I need to memorize the reactions?
36:02
And you may not like the the answer. You do need to know the mechanisms, but you will not be asked to draw the mechanism.
36:12
Okay. And you may find that while that's, you know, too much, but let's go through it one by one.
36:24
And you will find that that's actually not too bad. And also I want to point out the first half of the mechanism is all I should say.
36:31
The second half of the mechanism is very similar to the first half.
36:43
So if you understand the first half, the second half actually is not that bad.
36:47
Another thing I want you to focus on. You know, this is not a, uh, organic chemistry class.
36:52
I want you to focus on how the enzyme, uh, lower the activation free energy, how the enzyme makes the reaction go faster.
36:58
But you also need to know the reaction, because otherwise you cannot tell.
37:09
How would how the enzyme to get faster. So focus on why the reaction can go faster.
37:12
What are the contributions you know, to lower the activation free energy.
37:18
So let's start. Let's define the problems that we have first.
37:23
So this is uh hydrolysis reaction. We want to break a peptide bond.
37:31
So we want to uh, separate uh, you know, break this top tie bond.
37:36
And um, here is the peptide bond.
37:41
Uh, we call this one the sizzle bond because you think that, you know, a CStr is used to cut off, you know, something.
37:45
So we're going to cut this bond so that we are going to separate, you know, this polypeptide chain to two half.
37:52
So this is the N-terminal half of and terminal part of the polypeptide chain.
37:59
So let's say if this is the peptide bond between uh residue ten and 11 in the polypeptide chain,
38:04
then I'll n will be residue 1 to 10 of the polypeptide chain.
38:12
Because that and turn those uh it is N-terminal uh to the sizzle bond here.
38:20
And then I'll see this will be residue 11 to the end of the protein.
38:25
Okay. So this is the bond that we want to cut.
38:31
Now this is the catalytic file. We don't show the rest of the protein because we assume that, you know,
38:36
the enzyme will fold in a way so that these free residues, they are in the right positions for the catalysis.
38:43
Now the substrate will bind to the active site and it will position here.
38:52
And that's why the shape of the of the active site is very important.
39:01
You know, imagine that the substrate cannot even come close here.
39:07
Then the chemical reactions will not happen. So this that the structure of the enzyme is very important.
39:12
So now we want to focus on what are these three amino acids.
39:18
What are these residues. What do they do. You know how can they accelerate the chemical reactions.
39:24
Substrate come in sitting very close to the serine 195.
39:29
And now histidine here will take the proton from serine so that the oxygen become a much better, uh, nucleophilic group.
39:35
So that means the oxygen is going to attack this carbonyl carbon.
39:47
But if we make this one more negative then it will be more effective.
39:52
So nitrogen histidine is going to use the nitrogen to extract the proton from serine so that oxygen can attack the carbonyl carbon.
39:57
What happened is that once the proton got, you know, taken away from Sirin.
40:08
And you know, now on the on the histidine, the histidine will become positive charge.
40:13
How to make the positive charge cysteine a little bit more stable because we want the the transition state to be more stable.
40:20
So it turns out that aspartic acid 1O2,
40:28
this negative charge two amino acid is going to form a very strong hydrogen bond with histidine to stabilize the positive charge of histidine.
40:33
So now the energy of this transition state is not very high.
40:42
But you can see that now the carbon is going to turn into, uh, tetrahedral form.
40:47
And we call this one a tetrahedral intermediate.
40:54
So a covalent bond now formed between serine 195, the oxygen with the uh, the substrate, the carbonyl carbon here.
40:57
And then um, that's also NH four now that the proton is transferred to the to the histidine.
41:07
So this. Form is not a very stable form and we call this one the tetrahedral intermediate.
41:14
Is actually, uh, you can think of it as the transition state, so very high in energy,
41:21
but without the enzyme, this one will not, you know, it will be very hard.
41:26
So the carbons suddenly convert into this tetrahedral form.
41:32
So the enzymes actually help to stabilize this, uh, this hydro form here.
41:36
Now. Once we've got the attack residual from here,
41:44
the next thing the histidine is going to do is it actually just take the proton from from Syrian and then pass it to the nitrogen here.
41:49
And what you can see is that once this happen, the C-terminal part of the substrate, uh, can leave the active site of the enzyme.
42:01
I know that is not easy to follow a chemical reaction, just, you know, looking at the site.
42:10
But when you study, you will see, you know, these arrows, you know, uh, tell you how the action they go.
42:16
But the important thing is that, you know, you are not asked to draw the mechanisms.
42:22
You just need to know, uh, what the transition states they look like and how they get stabilized by the enzyme.
42:27
So what happened is that, you know, this one, you know, the nitrogen is going to pick up the proton from the histidine here,
42:35
and then, uh, there will be, uh, breakage of this peptide bond here.
42:42
Now we have the carbonyl part, um, you know, separated from, uh, from the rest of the substrate.
42:48
And then you can just leave the active site. But the thing is that because the oxygen here of carbon 95 is forming a covalent
42:55
bond with the carbonyl carbon because they attack the carbonyl carbon here.
43:03
Now we have the enzyme, but it is covalently linked to the N-terminal part of the substrate.
43:08
Okay. So the enzyme is still covalently linked to the enzyme no part of the substrate.
43:15
And we call this one the acyl enzyme intermediate. So it's a covalent intermediate.
43:22
Um it's quite stable. And that's why sometimes that to that this one can be isolated.
43:27
But this is not what we want. Because remember at the end we want to regenerate enzymes.
43:33
We don't want the enzyme just, you know, covalently linked to, um, linked to uh, half of the protein, uh, you know, half of the substrates.
43:39
So what will be the next? We already pass through half of the reactions.
43:48
So it is actually not so bad. Now the second half is water molecules will come in because this is a hydrolysis reaction right.
43:55
So far we haven't seen the water yet. So here is where the you know here's the you know the water.
44:05
So now we have the enzymes. We have the covalent bond between serine 195 oxygen and the carbon.
44:12
You uh carbon. Now water molecule will just come in and then sit in between histidine and Sirin.
44:19
195 once again is so important.
44:27
The structure of the enzyme is right. Otherwise, you know, we will not have something that the water molecules just sit here.
44:31
Now instead of taking the hydrogen from serine, just like the first half of the reaction now is taking the hydrogen from this water molecule.
44:39
So the histidine is going to take the nitrogen from this water molecule so that the oxygen can attack the carbonyl carbon here.
44:52
Now this is going to lead into um a tetrahedral intermediate like this.
45:00
So we're going to have, you know, nitrogen, take the protons from the water molecules.
45:09
Oxygen form a covalent bond with, uh, carbonyl carbon.
45:15
And we're going to end up with this intermediate again. But this is a different intermediates.
45:20
It's also in the tetrahedral form but only have the N-terminal part of the of the substrate.
45:25
Now this is and other chances and say so that means this mechanism actually has two chances in stage two uh, very high energy states.
45:32
And once again uh, the histidine, because you take the hydrogen from a proton, from the water molecules, it become positive charge.
45:40
But that's okay because it has a supporting, uh, um, a friend that's very supportive because it's going to hydrogen bond with the histidine,
45:48
the aspartic acid, 1O2 to stabilize the positive charge of the histidine.
45:58
Now, um, because this tetrahedral in the media is not very stable, it will convert into the product.
46:04
Now, uh, we'll see that, um, the top, you know, that will be, uh, you know,
46:13
bond breaking and bond mixing and we will see that, um, actually the oxygen,
46:20
um, okay, the proton will be returned to the Syrian here and then is going to, uh, let go of the, uh, the essential part of the substrate.
46:27
Now we have the, uh, the enzyme back.
46:38
So once again, it's, you know, you just need to go through the mechanism,
46:42
make sure you, you, you know, at least have an idea how the electrons, how they go.
46:49
But the most important thing is that realizing this mechanisms have two charges and states that, uh, very high in energies.
46:54
And also the enzymes have three, uh, critical amino acids.
47:03
One is, you know, carry out uh, uh, nucleophilic attack to the carbonyl carbon,
47:08
and one is to help to take the proton or donate the proton back to serine.
47:13
And then aspartate 1O2 is the one that support the action of histidine 57.
47:18
And like I said, you know, if you understand the first half,
47:27
the second half actually is quite straightforward because, uh, we're just replacing the serine with the oxygen, uh,
47:30
with the water molecules in the second half, but basically go through the tetrahedral intermediate and then,
47:38
um, you know, convert the ratio intermediate to the final product.
47:44
Now, if I ask you to draw the reaction coordinates of this one.
47:52
Of course, this will be more complicated than a simple reaction, you know, from from A to B, but how are you going to draw this reaction coordinate?
47:56
We always can start with let's say um, a reaction let's say has a free energy here.
48:08
Then will the energy goes up or will the energy goes down when it goes to the, the tetrahedral intermediate.
48:15
The first sector intermediate. So the reactants will not have a higher free energy than the transition states because by definition transition states,
48:22
uh, you know, is the higher energy uh, in the reaction coordinate.
48:34
So what happens is we start with the reaction at, uh, you know,
48:39
certain free energy and then will go up the free energy will goes up, uh, because of this tetrahedral intermediate.
48:43
But the nice thing is that, you know, the enzyme will help to lower this activation energy a little bit.
48:50
So instead of going, you know, very, very high, it will go up, but is manageable for the reactants to, uh, to pass this hurdle.
48:57
And they will come some because of the acyl enzyme intermediate.
49:06
But the enzyme intermediate is not as stable as the product.
49:11
Otherwise we'll just think that, you know, stuck there. So we start with the reactant goes up because of the transition.
49:14
Say it may come down a little bit in the free energy because you know this enzyme intermediate.
49:20
And then it will go up again because now it needs to pass through a second chances and stay,
49:27
which is also very high in energy because before it finally come down in energy to become a product.
49:33
So you will see something like rollercoasters, uh, going up those transitions, they're coming down a little bit.
49:40
Intermediate goes up again the second transition states and then come to the product.
49:47
All right. No. Uh.
49:53
That's nice. You know, the transitions, they just stabilized by those, uh, Akinci, uh, residues.
49:57
But that's actually not enough to, uh, to accelerate.
50:05
Uh, uh, the reaction. There are a couple of other mechanisms that help to stabilize the chances and stay.
50:09
One is call this oxy anion hall. Now, we you see the substrate here.
50:16
That's before any reactions. It binds to the enzyme states very close to the Syrian residues.
50:22
And then, um, so this is a plane, uh, you know, uh, the carbon is, you know, following the peptide.
50:29
So this is a plane, uh, uh, uh, structure here because of the past of the bubble on character here.
50:37
Now, in this structure, the substrate cannot form any hydrogen bonds with the Syrian, uh, a my hydrogen.
50:44
And also, uh, this applies in, uh, two residues away from Syrian.
50:54
Uh, it also is very close in, um, close to the active site.
50:59
But when the substrate is in this conformation, it cannot form hydrogen bonds with the MI hydrogen here and the MI hydrogen here.
51:04
But once it turns into this tetrahedral form,
51:12
now the oxygen will be able to the Austrian will be in the right orientation so that it can form hydrogen bonds with,
51:16
uh, with Syrian and also with the Kleist in 193.
51:26
So we call this one the oxygen on hold, because only when the oxygen, you know, attached to this carbon, uh,
51:29
turns into, well, only when the carbon turns into this tetrahedral form, the oxygen carry a negative charge.
51:37
They can form a very nice hydrogen bonding with, um, with these two residues.
51:44
So if that's the case, this is an other mechanism that can stabilize the transition state,
51:50
because the textbook usual, uh, conformation is very high in energy.
51:56
But now because you can form hydrogen bonds.
52:01
So the energy actually is a little bit lower. Maybe I would just stop here.
52:04
Um, next time we are going to talk about how we can make the enzyme more specific.
52:13
Oh, yeah. Text, uh, and then attack certain now. So.
52:18
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2280A Fall 2025
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2025-09-24 section 1
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You know, I went on. Uh, so tonight we, uh.
0:01
Uh, but for some other women. All right. Well, the, uh.
0:20
Means that. My.
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0:33
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I was like, I. What?
1:09
Monday. I'm 20.
1:19
Nine. I don't like it.
1:30
I. And you have.
1:37
Will. What do you mean you want to do?
1:52
Um, but. Yeah. It's, um.
2:05
Yeah. We went on.
2:15
20. I just.
2:53
Us. Like.
3:20
I thought I was. You can pretend that you're right.
3:44
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3:50
The other. My secret life.
3:54
To myself. Not. But when do call?
4:02
Do talk. I just.
4:08
My. And it's knowing that I'm.
4:12
I'm almost. I.
4:32
The way I love.
4:42
Know where I need to go. So he to have my, my.
4:56
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5:10
I mean. I did.
5:22
We. I really.
5:41
Yes, that. All right.
6:55
Let's catch up. Uh, so we're moving on to the very last lecture of unit A.
7:08
Um, so I'm going to, uh, go through some, uh, enzyme kinetics in this topic.
7:15
Uh, in this lecture now, uh, I think last time, uh, we started to look at time of trypsin,
7:20
uh, uh, the complicated enzymatic, uh, mechanism of chemo drugs.
7:30
And, uh, I hope that you, uh, you have already recovered from that.
7:35
Now, uh, I want to look at another thing about this Syrian protease.
7:39
So we said that, uh, serine protease is a family of protease.
7:46
That means in this family, we have, uh, multiple, um, enzymes that have very similar functions.
7:51
Uh, if you compare their structures, they look very similar, expressed with the active site, but somehow,
7:59
uh, different, uh, enzymes in this family, they have preference for, uh, different peptide bonds.
8:06
So some of them will prefer to cut, um, peptide bonds.
8:13
You know, for example, this one, uh,
8:17
chemo trypsin like to cut the peptide bond that has the N-terminal residue is a very bulky, uh, amino acid residues.
8:19
And then, uh, trypsin will like to have, you know, this residues to be, uh, positive charge amino acids.
8:28
So in that case, uh, trypsin will, uh, will cut this one, uh, much better.
8:34
And then we have the Elisa case.
8:41
Uh, this enzyme in the same family, but it preferred to have, you know, to cut the peptide bonds with residues in the and turn those.
8:43
I is a small, very small amino acid. So where does the specificity come from?
8:52
If you look at, uh, the structure of the protein, that's actually, uh, specificity of pocket, uh, sitting right next to the active site.
8:58
So this pocket is not part of the active site, but, uh, it is the pocket that binds to,
9:07
uh, the side chain of, you know, that's N-terminal to the peptide bond.
9:15
So if this is the peptide. Um, this is the size of bond.
9:20
Um, then this side chain is going to bind to this specificity podcast next to the active site.
9:23
Now if you look at chemo trypsin, uh, the podcast looks like this.
9:30
You know, a very wide pocket, uh, very deep pocket.
9:34
And it can interact with it can bind to a very bulky side chain, uh, like fentanyl, alanine, for example.
9:38
Because when you look at this pocket here, uh, it has glycine residues that's, you know, amino acid side, uh, without any side chain.
9:47
And also, uh, various more serine residues at the bottom of the pocket.
9:56
So now you can see, uh, lots side chain can fit into this pocket very nicely.
10:00
The important thing is that once you know the side chain here fit into the pocket, it can position the peptide bond.
10:06
Uh, that need to be, uh, that need to be cut, uh, very close to the active site and also in the right orientation.
10:14
Thinking about that, if this pocket is not, uh, good for this amino acid residue here, uh,
10:22
then we can expect that the size of the bond that's going to be cut will not be in the right orientation or very close to the active site.
10:29
Uh, for the for the chemical reaction.
10:38
Now for trypsin, even though the active site of these three enzymes, they look very similar or almost identical.
10:40
But for trypsin, the specificity pocket is, um, the bottom of the pocket actually has a negative charge,
10:49
uh, residues here that is aspartic active 189.
10:57
So in this case, if the side chain here is positive charge, then it will interact, uh, nicely with this pocket.
11:01
And that will place the peptide bond in the right orientation.
11:11
Now think about that. If we put a, uh, fennel island in here, the interaction may not be very tight.
11:15
In that case, you can think that the substrate start to wobble.
11:21
So it cannot be, uh, you know, uh, very nicely, uh, uh, sitting in the active site, the peptide bond that's to be cut.
11:25
And for this one, you can see that the pocket actually is fairly, uh, shallow.
11:34
Uh, you we have, you know, this pocket has the balian and also the theanine side changes pointing into the pocket.
11:40
Now, only very small, uh, side chains can fit into this pocket.
11:46
And that's why, uh, for this enzyme, it will prefer, uh, to cut peptide bonds that has a,
11:52
you know, the n channel side chain is a very small side chain.
11:58
So that define the. Capacity of, uh, of these enzymes, even though they're in the same family.
12:02
Uh, but they prefer to cut different peptide bonds.
12:08
Now, let's go back to how these enzymes work.
12:13
Uh, is an enzyme that can digest proteins.
12:19
Uh, it can cut, you know, uh, peptide bonds. But when it first synthesized in the pancreas, we don't want this enzyme to be active,
12:22
because that's not the place that where the enzyme supposed to work.
12:31
And we want it to only, uh, start to have this activity when it gets secreted into the intestine so that it will start to digest protein.
12:35
So how to make the protein inactive to begin with?
12:44
Because, you know, I think I mentioned in the last lecture, you know,
12:48
it doesn't make sense to have a big meal and then, you know, a lot of proteins in the intestines.
12:52
And then, uh, we need to wait until, you know, the production of all these enzymes.
12:59
It will be nice to have these enzymes already produced.
13:04
And once we need them, uh, we can switch on the function of this, uh, these enzymes very quickly.
13:08
So how to do that? It turns out that when, uh, chemo fits in, is first, uh, synthesize is in this inactive form.
13:14
So whenever you see, uh, similar genes, so you the, you know, chemo trypsin.
13:22
But now n with uh o g and so this means the enzyme is in the inactive form.
13:28
That's the name of the inactive form of an enzyme.
13:34
And now um, this inactive form can be activated, can turn into an active form if that's a cliff edge here.
13:37
So and the other enzyme will come in to Cup at this site 15 between 15 and 16.
13:47
That will make the chemo trypsin more active. So now if you think about it, um, trypsin uh, in the beginning it's not active,
13:55
but that's an enzyme in the intestine that can specifically activate trypsin.
14:07
And then trypsin can start to activate chemo trypsin.
14:12
And the interesting thing is that once common trypsin is active, it can also start to do this.
14:16
Uh uh, cleave like here, um, in different positions of common trypsin.
14:22
So common trypsin can activate itself or activate other time on trypsin molecules.
14:27
Now, if you think about it, when all these things first thought, uh, we only have, let's say, one trypsin that's in the active form.
14:35
But because the enzyme can regenerate, right. It's not like you can only use the one enzyme for one reaction, the enzyme after one reaction,
14:44
they can go back and activate another substrate or turn and add substrate into the product.
14:53
So this enzyme you know we have one trypsin. It's going to activate let's say ten times trypsin and 110 times trypsin uh got activated.
14:58
Uh now they can act way more kind of trypsin.
15:08
So we have one trypsin turn on ten of trypsin.
15:12
Now we have 100 common trypsin. Thousand of them. Like it can be.
15:16
You know, uh, we can go up the common trypsin, very, very active common trips and very, very quickly.
15:20
And that's the way to, um, to actually the enzymes.
15:26
So we they will be synthesized first and then uh, in the inactive form,
15:29
they can quickly turn into active form by, uh, sometime by itself or sometime by, by some other enzymes.
15:34
Okay. Now let's move on to another, um, topic that, uh, we want to look into.
15:44
Now we have an example how enzyme works.
15:50
But the most important thing to know is, um, how enzymes in general axle accelerate biochemical reactions.
15:54
You know, we look at it and say, because it can lower the activation energy, you can stabilize the transition stage.
16:03
Uh, but now let's say if there are two enzymes, we want to compare the activity of these two enzymes.
16:11
Which one works better? Which one is less efficient?
16:17
Let's say, um, you want to design an other enzyme.
16:21
I want to make it even better than the existing one. How can I do it?
16:25
And after you generate a new enzyme, how do you compare the activities with the existing one?
16:30
So we're going to look at, you know, how we can study the rates of reactions and what factors can, uh, can change the rate of a reaction.
16:36
It could be the substrate concentrations. It could be there's an inhibitor for the enzyme that will, uh, suppress the activity of any of the enzyme.
16:45
So we're going to look into, uh, enzyme kinetics.
16:55
And also we're going to look at the affinity of enzyme for.
16:59
It's crazy because think about that.
17:03
If an enzyme doesn't have a very good affinity for the substrate, then it's not going to be able to turn into the product,
17:05
because the first thing will be the enzyme need to recognize the substrate, bind to the substrate before we can turn it into a product.
17:13
So an enzyme must have a very good affinity for the substrate to begin with.
17:21
Okay, let's make the you know.
17:28
Make the scenario simpler. So we have substrate.
17:34
And that's enzyme molecules that will turn the substrate into product.
17:38
And we can think that, you know, once the enzyme bind to the substrate is going to form this enzyme substrate complex.
17:43
And will it depends on the affinity.
17:51
You know, uh, the enzyme substrate complex can go back into three enzyme and substrate if the binding affinity is not very tight,
17:53
but also it can turn into the substrate, can be converted into product.
18:02
So we have a situation like this.
18:08
Now let's say if I have the substrate of a solution of substrate, I start to add a little bit enzyme to um, to the solution.
18:11
Now I want to measure the concentration of substrate product over time.
18:20
And what we expect to see is that, you know, in the very beginning, uh,
18:26
enzymes start to bind to the substrate and then start to turn the substrate into product.
18:30
So, uh, and then once it reach, you know, certain steady state, we're going to see the substrate just decrease, uh, very quickly.
18:36
But then if you look at the product, of course, if the substrate decrease, that means, you know, it's turning into product.
18:46
And that's why we see increase in the product concentration. But how about the enzymes now when there's not much substrate.
18:52
When the substrate. Well let's say in the beginning of the reaction when we just, you know, add the uh enzymes to the substrate,
19:01
it may take a little bit time for the substrate, uh, for the enzyme to bind to the substrate and start to convert into product.
19:08
And that's why, you know, in the very beginning, uh, we may see, you know, the rate start to go, uh, the countries of uh,
19:15
uh, enzyme substrate complex start to go up because that take time for the enzymes to, to bind the substrate.
19:23
But then very soon we will find that, uh, most of the enzymes are doing the work.
19:30
You know, the enzyme is going to, uh, pick up substrates and convert them into, uh, into product.
19:35
And depending on the binding affinities, if the binding affinity is not very,
19:42
very high, uh, we may have some enzymes just floating around not doing the job.
19:48
Or it could be just in between when the enzyme, you know, turn one substrate into a product,
19:54
it takes time for it to pick up another substrate and then, uh, and then become enzyme substrate complex.
19:59
But the take home message here is that after the reaction, go for a little bit of time.
20:06
We expect that to see, uh, the enzyme substrate, uh complex, the concentration become uh, become constant.
20:12
Okay. So that's the that's the assumption here.
20:20
Now, if that's, uh, you know, based on this assumption, uh, we can design experiments, for example, here, um,
20:25
we ask the question, what happens if, uh, we have a same amount of enzyme, but then we change the substrate concentrations.
20:34
So in each of these six test tubes, we have the same amount of enzyme in the test tube.
20:45
But then uh, each test tube has a different concentration of substrate.
20:51
This one has way more. This one has, uh, no substrate or very low concentration of substrate, for example.
20:55
Now we take, you know, we take this test tube, uh, and then we measure the rate of the reaction.
21:03
So how fast the substrate get converted into product now with a low substrate concentration.
21:11
Uh, we do expect that, you know, the, the velocity, you know, the reaction rate will not be very high.
21:18
So we're measuring the initial velocity here.
21:25
That means, you know, once we're at, you know, you slowly we will prepare all the solutions with, uh, different substrate concentrations.
21:27
Then we will quickly add the enzyme into the solution, and then we will measure the rate.
21:36
And we want to measure it as fast as possible, because we don't want a lot of products to be to be generated already.
21:42
We want to measure, you know, when the substrate is still in, uh, you know,
21:50
in the concentrations that we, we set it to be and then we measure the rate.
21:55
So each data point here is, you know, the rates that, you know, we measured, um, of these six test tubes.
21:59
Of the seven data points here. So you can see that when the substrate concentration is low, the velocity is not very high.
22:09
And when we increase the substrate, uh, substrate concentrations.
22:18
And then to a point that we start to, uh, fine, you know, the rates will not increase more.
22:22
So usually as you increase the, uh, the substrate concentration, it become faster and faster.
22:28
But to a point they did become, uh, saturated. And we call this one the maximum, uh, velocity.
22:34
So with the same amount of enzyme, uh, if we we increase the substrate concentration, we are going to increase the velocity.
22:41
But that will be to a point that even you put more substrate in, the reaction rate will still be the same.
22:49
And because, you know, if you think about it, uh, machines in the factories, um, you know, this is the maximum capacity.
22:56
The machine cannot work faster because it takes time to convert the substrate to, to product.
23:05
So if you have a constant amount of enzyme, it just reach the maximum, uh, uh, speed of the, of the enzymes.
23:10
Now that's another constant here is called uh KB this uh, um Kellis.
23:19
Uh menten. Uh constant. We will come back to this one later on.
23:25
But when you look at this hyperbolic curve, there's actually only two important parameters that we need to figure out.
23:29
So what is the Michaelis Menten, uh, constant?
23:38
Uh, this is the equations that divide by, um, two, uh, biochemist, uh, more than 100 years ago.
23:42
But one of them, uh, was a Canadian.
23:50
So this is, uh, doctor. Uh, so she was a biochemist and also a physician.
23:54
So, uh, Doctor Mantas, who was born in Port Lambton.
24:02
So this is about 45 minutes drive from London.
24:06
Not not too far. Uh, and she was among the first women in Canada to have an MBA degree.
24:10
So she got her M.D. degree from U of T in 1911.
24:18
But she was very interested in, uh, biomedical research.
24:23
But unfortunately, at that time, women did not do it.
24:28
Not allowed to do research in Canada. It sounds, you know, very strange now, but at that time that was the case.
24:31
So, uh, she decided to move to to Germany, uh, to work with, uh, uh, Doctor Michaelis.
24:38
And then they developed the equation in 1913 to describe this enzyme kinetics.
24:45
Uh, we are still using these equations today.
24:50
And, uh, it's a very, very useful equations in describing enzyme kinetics.
24:55
Uh, and then, uh, she continued her work, you know, and then she did a PhD and then, uh, she became a faculty member in the phase.
25:01
Uh, but later on, she returned to Canada after her retirement in the States.
25:10
And then she continued to, uh, to do research at ABC.
25:15
And that's why, I mean, we're really proud to have a Canadian, you know, like Doctor Menten and,
25:20
you know, this department, biochemistry department, we actually establish,
25:27
uh, uh, lecture series to on her scientific legacy of excellence, but also,
25:30
more importantly, the obstacles we face as a female scientist at that time.
25:36
So, um, now let's look at the equations that Sue established.
25:41
Now, this is not a very complicated equation.
25:47
Uh, if you go to the tax code, you can see how this equation is derived.
25:50
But in this course, we're not going to ask you to memorize how to derive this equation.
25:56
It's more on how to use this equation. And it's nice that there's only two parameters that we need to, uh, we need to consider.
26:02
Now the first one look at the left hand side. Here we have the velocity of the reaction.
26:12
That's when we have the test tube. We add enzyme and we start to measure how fast the, uh, the product, uh, can be produced.
26:17
And that's the initial velocity. So we max is the maximal velocity when, you know, we have a lot of substrates,
26:25
when the enzyme they are so busy, they are all in this enzyme substrate complex form.
26:34
And that's the maximum philosophy that we can um, the enzyme can uh, can achieve.
26:40
And then we have the substrate concentration. That's the substrate, the concentration of the substrate that we put in attached to.
26:46
And then here is the Michaelis constant. Now uh, let's see what this constant is like.
26:53
If we look at this hyperbolic curve, it looks very similar to some binding curves that we saw.
27:04
Uh, in topic four, for example, we look at uh, uh, the offshoot miles of in binding we also see this hyperbolic curve.
27:12
So it turns out that the Michaelis uh, constant here is equal to the substrate concentration.
27:20
And then half of that we max this, you know, again, is very, uh,
27:26
similar to when we talk about the CD and always when the CD is equal to the free ligand concentrations, when half of the protein is in the bound form.
27:32
But here is, you know, if we, uh, look at, uh, the enzyme kinetics, uh,
27:43
we put a lot of substrates in the test tube and we measure the rate and we can have a good estimation what the Vmax, uh, will be.
27:49
And then we look at this curve and find out the velocity which is half of the Vmax.
27:57
And then we can read out, um, the curve here.
28:03
That's the that's equal to the substrate concentration. Uh, when, when the velocity is half the maximum uh, is half of the Vmax.
28:06
So what does this come telling us? You know, remember the CD?
28:17
The CD is the binding affinity. It tells us how good, uh, a protein can bind to, uh, bind to the liquid, how tightly can bind to the ligand.
28:21
And here, uh, the k m, you can think that it reflects the affinity of the enzyme for substrate.
28:31
So very, very similar. But I want you to keep in mind that it's not just that because, you know, enzyme doesn't just just find to the substrate.
28:37
It also converts the substrate into product.
28:47
So uh, but for this course, you can think that, uh, k m reflects the affinity of the enzyme for the substrate.
28:50
Now, this one is the property of the enzyme.
28:58
Uh, so it's independent of the enzyme concentration. It's just like, you know, the KD will not change when you change the concentration of a protein.
29:02
So the KD will remain the same. And that's the same for k m.
29:11
Uh, that's the definition of the enzyme for us for the substrate.
29:15
Now, like I said, you know, for this, first, we don't expect you to derive the equation,
29:21
but we expect that, you know, uh, you know, an enzyme kinetics can be described by this equation.
29:26
And, uh, how can we extract these parameters?
29:33
Okay. So there's only two parameters that we need to know.
29:37
We max and kb because the substrate concentration, when we set up the experiment,
29:41
we know exactly how much substrate we use for that for the experiment.
29:46
And we can use, uh, you know, some methods to measure the, uh, the rate of the reaction and how fast the products get produced.
29:50
Now, if I want to find out Vmax and and KB, I can do a whole bunch of measurements,
29:58
you know, using different substrate concentrations and measured velocities.
30:06
I'm going to set these points here, points on this curve.
30:10
And then I can, uh I can fit the data. You know, that's what we usually do.
30:14
Once we get the data we know the equation. And then we can fit out those parameters.
30:19
But what happens if you have a time machine and you travel back 140 years ago, you know, 1913 or 1912,
30:26
uh, you got this data, how are you going to find out the Vmax and, uh, and K um, it will be a tough.
30:38
Right, because you don't have cell phone. That's no compiler death, no ChatGPT, nothing.
30:47
And you just have those data points, and you don't have Excel or other software to help you to keep the data.
30:55
That's always my people. They look at the equations and say, well, I know how to do a linear plot.
31:03
So can we turn this equation around, making it easier to, uh, to figure out the k, m and Vmax.
31:11
So it turns out that the tricks is very simple. You take the reciprocal on both sides.
31:18
So instead of we knock we have one over we not.
31:24
And if you turn this one upside down you have CM divided by Vmax uh one over the substrate concentration.
31:27
And then you have um, the one over wi max here.
31:35
So you can, you know, when you study, you can easily just take the reciprocal on both side and you will be able to come up with this equation.
31:41
Now what make this equation simpler to, uh, to interpret.
31:49
I mean, this one basically we're turning this equation into a linear equation here.
31:55
So this is y equal to m x plus a constant.
32:01
So instead of having a hyperbolic curve, if we plotted one over v not versus one of the substrate concentrations.
32:10
So this one is experimental measurement. This is the substrate concentration is how much substrate we use for the experiments.
32:18
And you can see that now we got a linear plot like this.
32:26
And the slope is cm divided by Vmax.
32:31
And then the y intercept is one over.
32:37
We max. And then the x intercept is minus one over k m.
32:40
So now by just doing this tree we can quickly estimate, uh, the parameters,
32:45
the values of the parameters that we want to find out the k m and we max just
32:52
using the y intercept or the x intercept or based on the slope of this plot.
32:57
And this one is called the line myth about, uh, plot.
33:05
Uh, by default, by two, uh, uh, different scientists.
33:09
So what you need to know is that you need to know the, uh, Mercatus Menten equation.
33:15
How does it look? And, you know, also, uh, the trick that, you know, people commonly use, you know,
33:22
turn it into a linear equation so that you can estimate the re max in the k m.
33:29
Now let's say if, um, the enzyme that we're interested in, instead of trying to enhance the function of an enzyme,
33:36
it could be this enzyme is involved in certain human disease.
33:44
And we want to inhibit this enzyme. Uh, you can see that actually a lot of drugs, they are not, you know, uh, using natural inhibitors of metabolism.
33:48
Um, well, I love drugs. They they inhibit enzyme.
33:59
Okay. So, um, there are many types of inhibitors, and some of them, um, they have reversible binding.
34:03
Uh, they, they, they bind to, uh, enzymes in a reversible way.
34:12
What that means is that instead of just, you know, bind to the enzyme and, and just stick with the enzyme, it's just like protein ligand interactions.
34:17
The inhibitor can bind it enzyme, but can also dissociate from the enzyme.
34:28
So that means the binding of the inhibitor to the enzyme is not through covalent bond.
34:33
And the inhibitor can be removed but are also enzyme.
34:39
Uh, there's also inhibitors. They are uh, the binding is irreversible.
34:44
What that means is that those uh, inhibitors, they're going to form covalent bonds with the enzyme that we,
34:49
that we're interested in, or the enzyme that we're trying to target, and it will permanently block that activity of the enzyme.
34:56
So that's two at least two types of these uh inhibitors.
35:03
So today we are going only to look at one type of inhibitors because we want to use it to, um, to illustrate the concept.
35:09
You know, how, you know, with an inhibitor, how can you change, um, some genetic parameters of an enzyme.
35:19
So we're going to look at the reversible inhibitors. That means an enzyme can bind to an inhibitor.
35:28
But also, uh, you know, over time, inhibitor can dissociate from the enzyme just like protein ligand binding.
35:35
Okay. So, um, the inhibitor that here actually, uh, compete with the substrate for binding to the enzyme to the same active site here.
35:43
So we have the substrate that can bind the enzyme and then form the enzyme substrate complex.
35:53
And then the substrate will turn into product. But what happens if that's inhibitor that also can bind to the same site.
35:58
It's going to occupy the site. And the enzyme will have less chance to bind to the substrate.
36:05
But luckily, this is not, uh, this is not a permanent binding.
36:11
This is not, uh, this is reversible. That means, uh, the substrate at certain point will leave the enzyme, and, uh, sorry,
36:16
the inhibitor can leave the enzyme, and then the substrate can bind to the enzyme.
36:24
So it's like they're competing with each other for binding to the enzyme.
36:29
Now if we look at this, the curve, you know, the binding curves uh sorry the kinetics curve here.
36:34
That's the study inhibitor. That's what we saw when we first, uh, looked at the Michaelis Menten equation.
36:43
And we do see that when we increase the substrate concentrations to a very high concentration,
36:50
then we start to see, uh, the enzyme which the Vmax, the maximum rate that it can, uh, it can have.
36:56
But what happens if you add the inhibitor, you can see that it will the curve a shift to the to the right.
37:04
But when the substrate concentration is very high it will still reach the same way max.
37:12
So you can think that, you know, yes, the inhibitor can bind to the active site, can compete with the substrate binding to the active site.
37:20
But when the substrate concentration becomes so high, then the chance for the for the enzyme to find the inhibitor will be very low.
37:30
I mean, think about that. Let's say I have an enzyme and now it's surrounded by 100 substrate molecules.
37:39
But there's only one inhibitor. Most likely the enzyme goes to bind to the substrate.
37:46
And that's why when, uh, when the substrate connectivity is very high, we still got the same, uh, same Vmax.
37:52
And that's why for competitive inhibitors, it will increase the CM but do not affect the Re max because you can see that the CM now is larger.
37:59
So why does the CM become larger.
38:11
I mean, if you think about that, uh, we look at uh, um, how much protein can bind to, um, bind to the substrate.
38:15
You know, where we are saying that, you know, if, um, if the substrate has a favorable affinity with the enzyme, then a lot of, uh,
38:27
enzymes will bind to the substrate, will be in the substrate bound form because,
38:36
because k m, uh, reflects the affinity of um, the enzyme for substrate.
38:40
Now, if there are inhibitors in the solution, then enzymes will spend less time with the substrate because sometimes it will bind to the inhibitor.
38:46
So alongside the enzyme has less affinity for the substrate because it spend some time with the inhibitors.
38:57
And that's why when you plot out the curve uh, with the inhibitor the camera will become larger.
39:05
So for competitive inhibitors, it's not going to change the Vmax, but it's going to increase the cap.
39:10
Now if we if the inhibitor, you know, inhibit the enzyme through some other mechanisms, then the proof can look very different.
39:18
So here we just, you know, use the competitive inhibitors as the example to illustrate this concept.
39:26
We can also, uh, use, uh, this log metaphor plot.
39:34
Uh, turns into a linear plot so you can see that, uh, without inhibitors as what we saw,
39:39
uh, the slope is equal to cm divided by Vmax, and the y intercept is one over.
39:46
We max and the x intercept is minus one over 2 p.m.
39:51
But now with the inhibitor you can see that inhibitor doesn't change the y max.
39:55
And that's why the y intercept is still the same. But now the k m become larger.
40:02
And that's why the x intercept because it's minus one over uh k m now becomes smaller.
40:09
So the alpha basically is the factor. You know how much we increase the k m.
40:15
Remember the larger the k m that we face dividing.
40:20
So if alpha is bigger than one, that means the apparent k m is larger than the k m.
40:24
So that means with the inhibitor the k um uh will become larger.
40:31
You know, the affinity for the, for the, for the original substrate group become uh, will become smaller.
40:36
Now, uh, another concept I want to introduce.
40:46
Uh, you're going to see this one in, uh, in unity and maybe also in unity.
40:49
So this is the allosteric enzymes. Now we have an enzyme.
40:55
And I think, you know, using kind of trypsin as an example.
41:00
You can see how important is the protein structural enzyme structure.
41:04
If we change the enzyme structure what's going to happen.
41:08
I mean, especially if we change the, um, the positions of those, uh, catalytic, uh, residues,
41:12
it's not the enzyme will become malfunction of the, uh, the enzyme may not work anymore.
41:19
So it's very important to have the right structure of the enzyme.
41:25
So what happens if there are small molecules of molecules that can bind to the enzyme and change the conformation,
41:30
the enzyme, it doesn't need to be just the inhibitor that can compete with the substrate for binding to active site.
41:40
The molecule can bind to some other place in the in the enzyme, but somehow the binding cause the conformation to change.
41:48
Now the enzyme may not work anymore.
41:57
So this we call this one, you know, a process by which proteins transmit the effect of binding on one side to another one.
42:00
Uh, we call this, uh, loss. You know, this is allosteric regulation.
42:07
So we can use, you know, instead of putting just an inhibitor to compete with the substrate,
42:12
we can put it in something that can bind to the enzyme, change the conformation.
42:18
Then we can also modulate the activity of the enzyme.
42:23
Some of them can make the enzyme, uh, even more efficient.
42:26
But most of the time, uh, the binding will make the enzyme, uh, less less effective in, you know, turning substrates into into product.
42:31
So make sure you, you, you know, you understand this one is the binding of, you know, there's two phenomenon.
42:42
One is finding of substrate to one active site that can influence the binding at other sites.
42:48
So that could be um, just like hemoglobin.
42:53
You know, when we look at hemoglobin, we say that binding an oxygen molecule to one subunit can change the binding affinity in and others.
42:57
So what happens if the enzymes actually have, uh, more than one active site?
43:06
It has forced, let's say four subunits come together, and then each subunit can have,
43:11
uh, have an active site that can turn that substrate into, into product.
43:18
So what happens if binding of the substrate to one active site change the confirmation
43:24
of the protein that can make other sites to be more active or less active?
43:29
Or it could be just like, uh, when DPG bind to hemoglobin, it can change the, uh, the, the confirmation of the, the enzymes.
43:35
And then that can also affect the activity of the enzyme.
43:45
I'm going to use one example to show this concept.
43:51
Um, but like I said, you're going to see more examples like this in, uh, later on in YouTube.
43:54
Uh, so here is, um, is, uh, this fossil free to, uh, kinase?
44:01
This is an enzyme you can find in, um, the, uh, I call this pathway.
44:09
So now this enzymes, um, you know, uh, accelerate the reaction in the, in step free of the guy.
44:15
Call us. And, you know, we start with who closed and end of the final product.
44:24
And this enzyme here is important for step three.
44:30
And it can turn this fructose, uh, six phosphate into a product.
44:34
And we have a kinase here. That means, uh, it's going to transfer a phosphate group from ATP to this molecule.
44:39
Now you don't need to know the the details. You know how this one work, but just, you know, feed in mind.
44:49
This is, uh, one of the reactions in the glycolysis.
44:54
Now, you know, we always think, you know, how these pathways that regulate how the cells know where to stop, when to start and where to stop.
44:58
Now with the glycolysis and you start to produce, you know, um, you know, fruit ten steps.
45:10
Uh, it's going to produce the final product. And step nine, it produced this phosphor in no pathway, this product here.
45:18
It turns out that this product here can find also to this enzyme, which is very important for step three.
45:27
And by binding the enzyme is going to uh decrease the efficiency of this enzyme to suppress this enzyme.
45:37
Now we call this one a feedback mechanism because, you know, think about that.
45:45
Uh, you know, through the guy call says you start to produce a lot of product,
45:50
but then when the concentration of the product becomes very high, it will come back to inhibit this enzyme and make it less efficient.
45:54
So, in other words, you know, once we increase, you know, once we see more products and then they will come back to shut down the pathway.
46:04
So this is a feedback mechanism. And basically it's how the cells that's enough know we have enough product.
46:13
Why don't you just slow down or just stop the reaction here.
46:20
So why is it like that? You know, how can an enzyme know when you know to stop and when to stop?
46:26
Um, now, if you look at that, enzymes just like hemoglobin, instead of having one subunits, it has four subunits here and it has full active site.
46:35
And when the inhibitor when this molecule is in high concentration, it binds to the enzyme but not to the active site.
46:47
It binds to some other parts of the of the of the enzyme.
46:59
Uh but somehow the binding change to conformations enzyme makes the active site, uh, less efficient for, for the biochemical reaction.
47:02
And what happened is that when, you know, without this, um, this inhibitor, um, we can see the binding curve is like that.
47:12
Just what we saw. Uh, you know, for other enzymes, it goes up, you know, the hyperbolic curve here, it goes up and they start to plateau.
47:21
And, uh, uh, substrate concentration is very high. But what happens is that with the inhibitor, you can still see, you know,
47:31
at very high substrate concentration, uh, the enzyme can reach the same Vmax.
47:40
Now, if you think about why is that the case, you know, just like, you know, um, the competitive uh, inhibitors,
47:48
when the substrate is very high in concentration, is going to push the Philippian, uh, thing about that.
47:56
Um, you know, this may be a is a way to, to, to to think about this situation.
48:02
Just like the hemoglobin, we have two stages.
48:09
We have to stay which has a lower enzyme activity.
48:13
And then we have the our state which has a higher activity.
48:17
So the low the higher activity is the one without uh, the inhibitor.
48:20
And then the one with lower activity is the one with the inhibitor.
48:26
So when the substrate concentration is very high,
48:30
it's going to push the equilibrium this way so that we still can see, uh, the enzyme reach a maximum capacity.
48:32
But when the substrate concentration is not very high and we see that, uh, it, you know, it only have a very weak, um, activity here.
48:41
And that's why we see this sigmoidal curve, just like hemoglobin.
48:53
Uh, once it's switched to our state, then the activity started going up.
48:57
So instead of going into the details here, I just want to show you, um, there are enzymes like that.
49:02
They have multiple subunits.
49:10
And the activity could be regulated by, uh, molecules that bind to other parts of the protein, not just to the active site.
49:11
Uh, very similar to hemoglobin. All right.
49:19
That's all I want to cover for, uh, for topic five.
49:25
But before we go, I just want to have a very quick recap.
49:29
Um, you know, we always have questions, especially before the midterm.
49:34
Uh, I repeated many, many times. Lecture slides, practice quizzes, also topic notes.
49:39
A lot of times, you know, people forget that. Oh, I need to read the topic because, uh, I, you know, it's always nice to know, uh,
49:47
the structures, uh, but also the one letter code, the three letter call and things like that.
49:59
So what, Paula, what you choose to represent is peptide. Football because it stands for Western.
50:05
You are not going to see this in the in the metro. All right.
50:20
Um. Thoughts? Calculations. Always. No.
50:28
Table 4.1. If you know the table 4.1, most likely we will get the right answer.
50:31
Uh, for level for protein structures, make sure you understand the definitions.
50:37
Uh, secondary structures, you know, that that stabilize by hydrogen bonding.
50:43
Uh, protein folding. Uh, understand hydrophobic effect.
50:48
What happens when a protein is denature? Uh, we run through methods that separate proteins, make sure, you know, uh,
50:54
for example, if you use a site's exclusion columns, what protein will come out first?
51:03
What protein will come out later? Uh, protein ligand binding.
51:08
Uh, knowing the equations, uh, knowing how to calculate the percent of protein in the bond form,
51:13
uh, what factors can regulate, uh, the binding of hemoglobin, for example?
51:21
Uh, chemically, the binding, uh, can regulate the binding and then collagen.
51:29
As you know, a lot of students, they got stuck with collagen.
51:36
But focus on, um, um, you know, modification of certain, uh, amino acid residues in the protein.
51:40
And here I want to emphasize that this is a revised version of this slide.
51:48
Make sure you download this version and it focus on how the guide postulates.
51:53
And the addition of, uh, sugar molecule can increase the solubility inside the cell.
51:58
Not just not just we have increased the solubility inside the cell enzymes.
52:04
We we just, uh, look at this one. Uh, the important point is that how enzyme can lower the activation energy, this is the key point.
52:11
And then even though the mechanism is very complicated, but try to focus on the importance of these three molecules, at least three residues.
52:21
What are they doing in this enzymatic reaction?
52:30
Uh, we are not going to do this to this quick, uh, question, but you can see that questions like that ask you the role of Siri in 195.
52:35
It doesn't ask you to draw the enzymatic reaction. It asks you, you know what's important.
52:43
And then, uh, you know, equations again. Uh, but you need to know what information you can get, you know, from plots like this.
52:51
And the importance of these parameters. What can means and what're max means?
53:04
That's all I want to cover. Uh, the last slide I want to share with you some insight, uh, proteins that you learned.
53:10
Uh, in this part of the course. Uh, we learned, you know, about called chaperons.
53:18
Uh chaperons. How they how other proteins to fold.
53:23
We learn about Miles global and how they find these oxygens.
53:26
And we also learn about collagen. But what happens if we take the amino acid sequences of all these proteins?
53:29
Map it on the keyboard of a piano. What type of music are we going to get?
53:36
So let's first listen to HSP 70, a chapter on protein.
53:42
Now, this protein sounds very helpful because they have other protein to follow.
54:04
So here we have the option binding protein. And let's see how it's done.
54:09
Now let's listen to collagen. Remember, this is a rare protein.
54:33
I mean, at least in terms of the protein sequence. There's a lot of glycine, a lot of proline.
54:38
It's just repeating, you know, the glycine proline, hydroxyl protein.
54:44
So how would this sound? No on that.
54:48
It just sounds like that's because it's glycine proline and hydroxyl protein.
55:09
So hopefully that helps you to remember the sequence of college.
55:14
And that's all I want to say. Thank you very much.
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Notes
Enzymes are biological catalysts, mostly proteins, that accelerate biochemical reactions without being consumed in the process. They achieve reaction rates billions to trillions of times faster than uncatalyzed reactions and are characterized by high specificity, accuracy, and the ability to function under mild conditions (e.g., physiological temperature and pressure).
General Characteristics of Enzymes
Catalytic Acceleration: Enzymes drastically increase reaction rates.
Unchanged by Reaction: Enzymes are regenerated after catalysis and can be reused multiple times.
Specificity: They recognize and act on specific molecules (substrates) out of many available in a cell.
Accuracy: They rarely make mistakes, converting substrates into desired products.
Mild Conditions: Operate efficiently at physiological temperatures and pH.
Terminology
Substrate: The molecule an enzyme acts upon, binding to its active site and being converted into a product.
Active Site: A small, specific region on the enzyme where catalysis occurs. Its precise structure is crucial for function.
Product: The molecule(s) formed after the enzyme catalyzes the reaction of the substrate.
Enzyme-Substrate Complex (ES Complex): A transient intermediate formed when the enzyme binds to its substrate.
Enzyme Function and Energy Landscape
Enzymes accelerate reactions by lowering the free energy of activation () of the reaction. They do not change the overall free energy difference () between reactants and products, meaning they do not affect the equilibrium of the reaction—they only make it happen faster.
Transition State
The transition state is a highly unstable, high-energy intermediate form between reactants and products. It is the highest point on the reaction coordinate diagram.
The free energy of activation () is the energy difference between the reactants and the transition state. Enzymes lower this barrier.
Mechanisms of Lowering Activation Energy
Proximity and Orientation: Enzymes bind multiple reactants, holding them close together and in the correct orientation for efficient reaction.
Changing Charge Distribution: Enzymes can rearrange electrons in the substrate, altering its charge distribution to favor the reaction.
Stabilizing the Transition State: Enzymes preferentially bind to and stabilize the transition state, effectively lowering its energy. This means the enzyme has a higher affinity for the transition state than for the substrate or product.
Cofactors
Some enzymes require non-protein partners, known as cofactors, to function. These can be:
Metal Ions: Inorganic ions (e.g., , ) that bind to the enzyme.
Organic Molecules (Coenzymes): Organic molecules that assist in catalysis.
Co-substrates: Bind transiently to the active site and are modified during the reaction (e.g., ).
Prosthetic Groups: Bind tightly, often covalently, to the enzyme and remain associated (e.g., heme group in hemoglobin).
Chymotrypsin: A Serine Protease Example
Chymotrypsin is a member of the serine protease family, enzymes that hydrolyze peptide bonds. It is synthesized in the pancreas as an inactive precursor called chymotrypsinogen and activated by cleavage in the intestine to prevent premature protein digestion.
Catalytic Triad
Chymotrypsin's active site contains a catalytic triad of three critical amino acid residues, brought close together by protein folding, even though they are far apart in the primary sequence:
Aspartic Acid 102 (Asp102): Stabilizes the positive charge of His57 through hydrogen bonding.
Histidine 57 (His57): Acts as a general acid-base catalyst, accepting and donating protons.
Serine 195 (Ser195): Provides the nucleophilic oxygen that attacks the peptide bond.
Catalytic Mechanism (Hydrolysis of Peptide Bonds)
Substrate Binding: The substrate binds to the active site, positioning the scissile (to be cut) peptide bond near Ser195.
Nucleophilic Attack (Step 1): His57 deprotonates Ser195, making Ser195's oxygen a strong nucleophile. This oxygen attacks the carbonyl carbon of the peptide bond, forming a tetrahedral intermediate (Transition State 1). Asp102 stabilizes the positive charge on His57 during this process.
Acyl-Enzyme Intermediate Formation: The nitrogen of the peptide bond gains a proton from His57, breaking the peptide bond and releasing the C-terminal part of the substrate. The N-terminal part remains covalently attached to Ser195, forming an acyl-enzyme intermediate (a relatively stable covalent intermediate).
Water Entry and Nucleophilic Attack (Step 2): A water molecule enters the active site. His57 deprotonates the water, making its oxygen a nucleophile. This oxygen attacks the carbonyl carbon of the acyl-enzyme intermediate, forming a second tetrahedral intermediate (Transition State 2).
Enzyme Regeneration: His57 donates a proton back to Ser195, breaking the covalent bond between the enzyme and the N-terminal product, which is then released. The enzyme is regenerated and ready for another catalytic cycle.
Oxyanion Hole
Another key feature is the oxyanion hole, a region in the active site that stabilizes the negatively charged oxygen atoms of the tetrahedral intermediates. It forms hydrogen bonds with the backbone N-H groups of Gly193 and Ser195, preferentially stabilizing the transition states and further lowering activation energy.
Substrate Specificity
Serine proteases achieve specificity through a specificity pocket adjacent to the active site. Variations in this pocket dictate which amino acid side chains the enzyme prefers:
Chymotrypsin: Has a deep, wide, hydrophobic pocket (due to glycine and serine residues) that accommodates large, bulky hydrophobic side chains (e.g., Phenylalanine, Tryptophan) for cleavage.
Trypsin: Has a negatively charged aspartic acid (Asp189) at the bottom of its pocket, preferring positively charged side chains (e.g., Lysine, Arginine).
Elastase: Possesses a shallow pocket with bulky valine and threonine residues, favoring small, nonpolar side chains (e.g., Alanine, Glycine).
Enzyme Kinetics
Enzyme kinetics studies the rates of enzyme-catalyzed reactions.
Michaelis-Menten Kinetics
This model describes how reaction velocity () changes with substrate concentration ().
Hyperbolic Curve: At low , increases linearly with . At high , the enzyme becomes saturated, and approaches a maximum velocity, forming a hyperbolic curve.
Michaelis-Menten Equation:
(derived by Maud Menten and Leonor Michaelis)(Maximum Velocity): The highest reaction rate achieved when the enzyme is saturated with substrate.
(Michaelis Constant): The substrate concentration at which the reaction velocity is half of (). It reflects the apparent affinity of the enzyme for its substrate; a lower indicates higher affinity. is independent of enzyme concentration.
Lineweaver-Burk Plot (Double Reciprocal Plot)
To simplify the determination of and , the Michaelis-Menten equation can be linearized by taking the reciprocal of both sides:
This equation forms a straight line when is plotted against ().
Y-intercept:
X-intercept:
Slope:
Enzyme Inhibitors
Enzyme inhibitors are molecules that decrease enzyme activity.
Reversible Inhibitors: Bind non-covalently to the enzyme and can dissociate.
Irreversible Inhibitors: Form covalent bonds with the enzyme, permanently blocking its activity.
Competitive Inhibition
Mechanism: A competitive inhibitor directly competes with the substrate for binding to the enzyme's active site.
Effect on Kinetics: Increases the apparent (lower apparent affinity), but remains unchanged (at very high substrate concentrations, the inhibitor can be outcompeted).
Lineweaver-Burk Plot: The y-intercept remains the same (), but the slope and x-intercept ( ) change.
Allosteric Enzymes
Allosteric regulation involves the binding of a molecule (an allosteric effector) to a site on the enzyme other than the active site, causing a conformational change that modulates the enzyme's activity. This can increase or decrease activity.
Characteristics: Often multi-subunit enzymes, exhibit sigmoidal kinetics (similar to hemoglobin's oxygen binding curve), and allow for complex regulation, such as feedback inhibition (e.g., phosphofructokinase in glycolysis).