 Loot, loot, loot, loot, loot, loot, test, test. We're being invaded. All right, hello, everybody. To give us a little more time, I start 60 seconds early. All right, thanks for coming again. So we have a little bit to finish up from the last lecture. The last lecture was intense. I have office hours today after class at Sydney-Frank Hall. So please avail yourself of all the resources in the class, review the video, pause it, try to get caught up. That's going to be the most difficult lecture. Today we're going to be looking at practical examples of the theoretical framework that we experienced in the last class. But we do need to finish up by substrate reactions. So there's two different types of bisubstrate reactions. And the way you can differentiate them is by understanding the concept of a ternary complex. So a ternary complex, that's bisubstrate, right? So there's two substrates. Ternary complex is when both substrates are bound to the enzyme at the same time. Whereas in this other mechanism of a bisubstrate reaction, both substrates are not bound to the enzyme at the same time. So there's two different types of ternary complex, including mechanisms, the random order and the ordered mechanism. And so either these things come on in sequence in an ordered way, or they can come on in either order. But in both cases, you have what's called this ternary complex. And once both substrates are bound to the enzyme, then and only then can they be converted to products. The other mechanism is no ternary complex. And in this mechanism, the enzyme is oscillating between two states, schematically represented here as E and E prime. So the first substrate binds to the enzyme. That substrate, while bound to the enzyme, is converted to product. But the conversion of that substrate to product alters the enzyme in some way. So we'll see examples of this type of mechanism throughout the class. Then that first product is released. The altered enzyme, then and only then, combined the second substrate. While bound to the altered enzyme, that second substrate is converted to product. And so this is a mechanism. And associated with it is a configuration of these double reciprocal plots that we saw before. So this is similar to what we looked at with inhibition. So with inhibition, we did line weaver-berg plots at increasing inhibitor concentration. Here, we're going to do a similar thing. But instead of increasing an inhibitor, we're going to increase one of the substrates. So these are the things that differentiate these two classes by substrate reactions. And so here we have, we're increasing substrate 2. So playing the role of Mr. Inhibitor today is substrate 2. And then we're doing a titration of the amount of substrate 1. And so we're asking, how does the slope of this line change as S2 increases as a function of the first substrate concentration? And you can see that the slope of the line decreases. So remember these intercepts. It's important that you remember what those are. So this is double reciprocal plots. So this is 1 over Vmax as the y-intercept. The x-intercept is negative 1 over Km. So in this type of reaction, people figured out that this type of plot arises from this type of reaction. So what's happening to Vmax here? It's increasing. You see that? Because 1 over Vmax is decreasing. What about Km? So negative 1 over K, it's a little hard to see. Negative 1 over Km is increasing. So Km is decreasing. So people figured this out because they were able to study in-depth certain enzymes and know what kind of mechanisms they had, collect this type of kinetic data, and then they saw this type of plot. But you might wonder why does Vmax and Km go in these directions that were observed? And although we don't know for sure, this is a logical hypothesis of why this might be occurring. And so increasing the second substrate concentration increases the rate of production of products because bulk products must bind to the enzyme to have productive product formation. So that makes a bit of sense. And then what about this one? If you increase the second substrate concentration, it decreases the amount of S1 necessary to make the ternary complex, the productive ternary complex, right? Decreases the concentration of S1 necessary to achieve the half-maximal rate because the equilibrium is already favoring the formation of these two necessary steps, right? So if we decrease the concentration of the substrate necessary to fill half the binding sites, that decreases the Km. Hi, yes. It's not inhibition. That's a very good question. So this is in some sense the exact inversion of inhibition. So in a bisubstrate reaction, two substrates need to interact with each other. And the only productive outcome comes when both substrates are being transformed, or in this case, directly interacting. So it's sort of similar. And another way you can look at these plots is compare this to one of the modes of inhibition and think about why is there similarities? Okay, but I find that analysis more confusing than just providing a rational out-of-the-blue explanation. So the other one's a little bit more confusing to rationalize, but this is the pattern of data seen. So in the ping-pong mechanism, remember we're oscillating between two states of the enzyme. So as we increase the concentration of one substrate, what's happening to Vmax here? So one over Vmax is decreasing. So Vmax is increasing, just like we saw for the last one. But what about the Km? So negative one over Km is decreasing. So Km is increasing. Wouldn't that be bad? So this is the pattern you see with that type of reaction. And these are reasonable explanations. It has to do with this oscillation of the enzyme between two states. If you increase substrate two concentration, you increase the amount of enzyme that is in this not modified state. Because you drive this reaction forward. So increasing F2 increases the rate that the enzyme oscillates between its two forms. Increasing the abundance of the form of the enzyme that can convert substrate one to product. But if you increase the amount of enzyme that is in this form, well, there'll be more enzyme necessary to fill half the binding sites. Makes a certain amount of sense. Is this a law of biochemistry? No, it's rational and logical. This is the data that you see. And this perhaps is the reason why you see these patterns. So bi-substrate reactions, as mentioned, are akin to the inverse. So what is this similar to? Just in terms of pattern when compared to inhibition. So where did we see a similar pattern? You remember? That was a long lecture. No guilt if you don't remember. Yeah? Exactly. Why it has a similar pattern when you think about the mechanism I cannot come up with a reasonable explanation. But this seems reasonable. Increase one substrate, the thing is gonna be more efficiently oscillating between its forms. It's gonna be able to pump out widgets more quickly. There'll be more of the form here that combine that substrate. So if there's more of that form, you're gonna need a higher concentration of that substrate to fill half the sites because there's more there. Okay. You with me so far? That's the last painful bit of that lecture. But today we're gonna be thinking pragmatically. Okay, what does this mean for some case studies of enzymes? What are the magic forces that enzymes are employing to provide the spectacular rate enhancements we've seen? So we'll be thinking about the transition state, making more bonds to the transition states than the ground state. We'll be coming back to this idea of cofactors. We looked at prosthetic groups and so here we're gonna be looking at coenzymes. Okay, and then we'll think in an abstract way. Okay, what are some theoretical reasons why enzymes might accelerate rates of chemical reactions? And then we're gonna go in depth, electron by electron through chymotrypsin. And you might think we could literally spend, probably many years of biochemistry lectures going through every single detail of what's known about every enzyme. But here we're just gonna get one taste of this, what's going on and you can perhaps when you encounter different enzymes be able to understand them a little deeper. So we're gonna be looking at chymotrypsin and then we're gonna look at the ways in which enzymes harness the awesome catalytic improvements that you have for chemical reactions. You do not want to unleash a grains, earth grains of sand worth of rate enhancement willy-nilly. It's an awesome power. And so there's lots of ways that have evolved to regulate enzymes. Okay, so here's Mr. Grains of Sand on Earth. So 10 of the 17-fold faster in the presence of the enzyme than in its absence. So what in the world? Surely it's not just one thing that this enzyme is doing to provide 10 of the 17-fold rate enhancement. What could be some of the things that enzymes are doing? Well, here's a picture of an enzyme. So we just talked about bisubstrate reactions. So here we have an enzyme that's catalyzing a bisubstrate reaction. And here are the two substrates in red and yellow and the enzyme's surface is highlighted in blue color. Do you see how these two substrates are held very precisely in relation to each other? They're literally being aimed at each other. In some enzymatic reactions, it's beneficial to encompass the reacting substances to prevent access from the environment. So in this case, it's a pretty deep binding group. We have these two molecules being associated. The entropy is being dramatically decreased. So out here in solution, these substrates are floating around. But here, we've limited that entropy and it made it more likely that these two molecules would react together. So besides the number of bonds that transitions to the ground state, there's the control of entropy, the beautiful control of entropy that's going on here. So here's another way to look at this. So here we have stickase, right? So stickase takes stick, this is a chemical reaction. Stick gets bent and then it reaches the breaking point. When it's really on the edge, there's an equal probability that it would go back to not broken stick compared to going to broken stick. So this stick reaction has a certain thermodynamic barrier. You can feel it, it takes muscles to bend the stick. But this is actually a metal stick. So you'd have to be Arnold Schwarzenegger to bend it. And here we have stickase enzyme that has magnets. And so this particular enzyme has high complementarity to the substrate. There's a lot of magnetic bonds between the substrate and stickase. And the result of that is something that I described in words yesterday, but I didn't show you a picture. Here's a picture. You have lots of bonds to this substrate and stickase. You have great stabilization of that molecule. Okay, and do you see how this is counterproductive? So we started from a situation where the height of the energy barrier was this high. And now it's actually even higher because we bound so tightly to Mr. Stick. So we're not, this would, if you use the Renius equation to calculate the rate, well, EA would be larger. That would not be productive. But really, what many enzymes are doing is they make some bonds. They make enough bonds to substrate to find it with specificity, but they make more bonds to the transition state, this stressed configuration of the molecule. And this lowers the free energy of the transition state more than the ground state. And so the activation energy is decreased. Delta G, double dagger. So there is no stickase, but it's a reasonable way to think about things. And here's the Renius equation. We've already covered this. It's a direct exponential relationship between the activation barrier and the rate of the reaction, right? And so as the activation barrier gets larger, the rate goes down. As it gets smaller, the rate goes up. It's inversely related. So this is important. This is the magic. We're not manipulating the equilibrium with these enzymes. The free energy of the ground state of the substrate and product is the same. The presence or absence is an enzyme. We're just decreasing the free energy of the transition state. So you might say, well, that's a nice picture of stickase. It seems like you just made that stuff up. Is there any experimental evidence for the stickase phenomena? And it turns out that there is. Creative chemists developed something called a catalytic antibody. And so there's a type of molecule called a transition state analog. Now chemists could not synthesize a transition state and have it persist, right? Because by definition, it's unstable. But a transition state analog looks geometrically and in terms of charge distribution, very similar to a transition state of a molecule, but it's more stable. And so you can use that transition state analog to immunize the animals and have them produce antibodies that bind very tightly to those transition state analogs. And then you can go and put some substrate in with that antibody and see, does the type binding of that antibody of the transition state provide a rate enhancement? Now nature is very good at this. Chemists, they're pretty good. You have to guess what the transition state is. And sometimes that's controversial, but they have been able to synthesize antibodies. They bind very tightly to the transition state and they found that those did accelerate the rate. Nowhere near a billion fold like we'll see today for chimotripsin or 10 to the 17 fold were nowhere that good. But it's a proof of principle that if you make more bonds to transition states in the ground state, you're gonna help to accelerate the rate. But is that enough to get to 10 to the nine or 10 to the 17? Just bonds to transition state? No, it's not sufficient, we need more. So here's a picture, if you don't believe me still, here's a picture of the reaction. The reactants here bound to the antibody molecule and the rate of this reaction was accelerated. Now this is important. So if you wanna develop a drug to inhibit a particular enzyme, you're gonna want that to make the structure of that drug more similar to the transition state than the ground state of the substrate of the product, right? Because you wanna make more, if you want it to be a competitive inhibitor and bind at the active site, you're gonna wanna have lots of bonds to that drug or that inhibitor. So this is important as we develop pharmaceuticals. So enzymes come in all kinds of different classes by definition and enzymes are the set of proteins that catalyze some reaction, but there's a dewey decimal system, which you might not know what dewey decimal is, that's my generation, but this is a way to organize books, right? If you go to the library and they just drew the books all over the library, you wouldn't be able to find anything. Well, so the same kind of thing with enzymes. There's tens of thousands of different enzymes in a human cell. How are we gonna organize those? How are we gonna order them from a company? Well, we need to have a dewey decimal system for enzymes. And this system is based on the type of chemistry each enzyme catalyzes. You have broad groups, and then you have subgroups and you have subgroups for the subgroups. So the broad groups describe very general chemical phenomena. So things that push electrons around between molecules, those are aceto reductases, transfer of electrons, transfer races, so the names make sense. Aceto reductase is gonna change oxidation states and reduction states. And transfer races, well, those are gonna transfer molecules or parts of molecules between each other. Hydrolases, you think of hydro water. So water is involved in these reactions, hydrolysis reactions, hydrolyzing various chemical bonds. Y-aces are not necessarily the most obvious name for this class of enzymes. You might've called them eliminases or additionases, right? So these are enzymes that catalyze elimination and addition reactions. See, those are called lyeases. They're somerases or are somerasing substrates. Lyegases are making new bonds to carbon. So these are the broad families of enzymes. Within each family, we can have sub-family. So for example, transfer racing. What are you transfer racing, right? You have 2.1. Well, that's transferacing one carbon group. 2.2, transferacing aldehydes, they're ketone groups. So we can organize all these enzymes based on their chemistry that they're catalyzing. So let's come back to modes of catalysis. So binding energy is important. So formation of hydrogen bonds, electrostatic bonds, hydrophobic interactions is important in sometimes making more bonds to the transition state than the ground state. De-solvation, controlling the entropy of the substrates as well as the water in the reaction. You might think hydrolysis, well, we could push those reactions forward if those active sites were more exposed because you'd have a large concentration of water available. But other types of reactions, you might want to have a closed active site where you envelop the substrates. And so, but you also have to always consider when you're thinking about entropy, the increase in entropy of the water molecules as they don't have to encase the substrates anymore, they can make interactions with other water molecules. We can precisely position, control the entropy of the substrates by aiming the reactive groups at each other. We can transfer protons to and from substrates. Acid-based catalysis. We can make new covalent bonds between the enzyme and the substrates. That's called covalent catalysis. We'll see an example of that today. Metal ions, sometimes amino acids are not sufficient to provide catalysis for all types of chemical reactions. So for example, oxidation reduction reaction. The propensity of metal ions to change their oxidation state can be leveraged by enzymes. So enzymes can bind with their amino acids, and certain metal ions, and those metal ions can change oxidation state and change the oxidation state of substrate molecules. So all kinds of different modes of catalysis. So remember when we did myoglobin and hemoglobin? So those were prosthetic groups, right? So the heme group was a prosthetic group of the myoglobin and hemoglobin protein. But you can also have something called a coenzyme. And these, unfortunately, I cannot give you a precise definition that differentiates a coenzyme from a prosthetic group. A prosthetic group binds more tightly to the enzyme than a coenzyme. But both of them are functionally the same. They're providing some lacking chemical capability that's not provided by the amino acids in the enzyme. So when we have a polypeptide, that's called the APO enzyme, and if the polypeptide needs one of these helper groups, when you add the helper group, either a prosthetic group or a coenzyme to the amino acid-only polypeptide, that's now called the holoenzyme, or the whole enzyme is the way to remember this. It's not a whole enzyme without those extra groups. Okay, so this is just semantic definition so far. We need to talk about this. So Art, one question about binding in the transition state. Does the enzyme form bonds to the intermediate state after orienting the substrate toward each other? Right, so intermediate state is a bit of a vague term, so that's more thought of as a stable intermediate. Yes, the enzyme provides ideas that you wanna provide more bonds to the transition state than the substrate in its ground state, product in its ground state, or for more complex reactions, intermediate state. So, okay, any other questions? Okay, it gets better. We'll get out of definition land in a moment. So, you might have heard of vitamins. Vitamins are typically things that we cannot produce that we get from our environment. Evolution has said, why do you have to make it? So you just eat some plants, you know, or some Cheerios. Right, so here vitamins are coenzymes in many cases. So vitamins get converted in your body to form the molecules that are useful to help in catalysis. So for example, biotin, it can be used to transfer groups, CO2 groups between molecules. Coenzyme A is good for transferring A-cell groups and so forth. So these things are non-amino acid molecules that are providing useful capabilities to the enzyme, and we call them prosthetic groups for this group. So these are the metal ions, the ones with the green dot can change their oxidation state. So it could be used in oxidation reduction reaction. Metal ions also can be used to hold a substrate precisely. So magnesium, for example, has a propensity to bind an important molecule called ATP we'll learn in this class. And it holds that ATP molecule so that it's optimally positioned to transfer phosphate to a substrate, for example. Metal ions can also be involved in acid-based catalysis. So the amino acids alone made of the organic elements are insufficient for this chemistry. To carry out all types of chemical reactions, we need the transition state elements. We need metal ions because those provide efficient ways to catalyze those reactions. So let's think about, in a very abstract way, how controlling the entropy of a reaction can accelerate rates. So here we have two simple molecules an ester and an acid. And those can come together to make an N-hydride. So that reaction requires those two reactants to find each other, orient each other in relation to, they have to have a certain orientation so that this reaction can occur. So the oxygen attacks, carbonyl, electrons that go up and back down. And kick out the alcohol, or the, yeah, the alcohol. And so that has a certain rate associated with it. But it makes sense entropically if they don't have to go through this struggle of finding each other. In other words, if the two reacting groups are tethered together on the same molecule, well, that's gonna accelerate the rate, right? There might be still a lot of flexibility in the case of this contrived example. There's single bonds, there's degrees of rotation. So they're not gonna be reacting if the two groups are distant from each other, but they don't have to get, they're already pretty close to each other. And that provides 100,000 fold rate enhancement just by holding the two things together. So for an enzyme catalyzing a by substrate reaction, just by holding those together, you're providing a huge rate enhancement. But you can do better than that. Beyond holding them together on the same molecule, you can aim the two reacting groups at each other. So here we have a confirmationally restricted molecule where these two reacting groups are literally aimed at each other. And you get another 1,000 fold rate enhancement by the aiming of the substrates at each other. So this is one of the ways enzymes are providing the magic by controlling the entropy. We think of enzymes as endergonics, hexagonics, not endothermic, exothermic, because control of entropy is important for these to control the reaction rate. What is the concentration of water in water? Water. Undefined? I think I heard it, 55 molar. So if you wanna push a reaction, you might say okay, how do you measure that? I don't know. 55 molar. If you wanna push a reaction forward and water happens to be a substrate involved in a reaction, that's pretty handy. You might wanna leverage the fact that there's a very high concentration of water molecules in aqueous solutions, right? And so it depends what direction you wanna go here. For example, if you wanna condense, could do a condensation reaction where water is a product, what might you wanna do with your substrates? If water is a product and you wanna have this reaction to occur quickly, do you wanna have your substrates that expose the environment or shield it? Shield it, right? So we'll be learn about the enzymes as it turns out RNA ribozyme that catalyzes reaction. They literally curl up and it's this den of condensation where these reactions occur, where the access to water is precisely controlled. Now the inverse direction, if we're hydrolyzing a bond, we're gonna wanna have an exposed active site where that 55 molar concentration of water can push the equilibrium forward by mass action. So here's a variety of hydrolysis reactions. We'll see all kinds throughout the semester so we can hydrolyze fossil and hydride bond, phosphate ester bonds, carboxylate esters, acyl phosphate bonds. And so these hydrolysis reactions, you might wanna have, you can imagine for the enzymes that catalyze these reactions, those are gonna tend to have an exposed active site where water molecule, one of the reactants in the reaction, can be at high abundance, push the reaction forward. Okay, so we have condensation reactions and hydrolysis reactions. The other way that enzymes are accelerating rate is by acid-based catalysis. What's the difference between specific and general acid-based catalysis? Do you remember, yes, hi. Yep. So specific involves you're passing protons to or from a water molecule and for a general acid-based catalysis, you're passing protons to or from a not water molecule, some other molecule. What types of amino acids might be involved in general acid-based catalysis? The acidic and basic ones, the ones on the edge, histidine, alcohol-containing ones, you believe it? PTAs. You'll see in a moment. So we can have general-based catalysis, but look at why is it helping to accelerate the rate? So look at this reaction. You have the reaction of a amide bond with an alcohol-containing molecule and that's going to make this configuration. What do we call this? If my name were Michaelis or Menton, what might I call this? Is this highly energetic? Is this stable? Transition state? So this is a transition state. How happy is an oxygen atom with a positive charge? Not happy, it's electronegative. Okay, and so here we have a positively charged, unstable intermediate with this hydrogen atom here. And so what the enzyme is doing is it's picking up that hydrogen and said, wait a minute, why don't I just move you? I'm gonna pick it up here and I'm gonna put it somewhere else. Chemically, why is this helpful? So see where it's moved it? So it's picked up this hydrogen and moved it down here. What have you done in terms of organic chemistry here? You've increased the leaving groupability of this, right? So if you wanna break this bond, if your protonated nitrogen wanna be positively charged, no, that makes it unhappy. So one of the possible outcomes of that is to break that bond. And that's by moving the proton away from here, that stabilizes that bond. So you're pushing the reaction in the way that you want it to go by having amino acids on the enzyme take a proton off and have other amino acids or the same amino acid put a proton on. But also, we'll see examples today where water molecules can be escorted into an enzyme active site where they can take protons on or off a substrate as well. So water molecules can use, or enzymes can use water molecules. So specific and general acid-based catalysis. You have to be passing a proton somewhere for this acid-based catalysis. Okay, so we're able to infer, it's thinking about this logically. Well, the types of amino acids involved in acid-based catalysis are the acidic and basic ones. But you might not be aware that alcohol containing amino acids can be involved in this. You might say, wow, that's counterintuitive considering they're pKa. But pKa is of amino acid side chains and a protein can be manipulated compared to free amino acids in solution. So we can actually drop, pull this proton off this alcohol, we'll see an example of that today. So acid-based catalysis is important here. So now we're gonna dive in a little bit deeper and do a case study. How do we bring all these modes of catalysis together in one enzyme? In this case, we're gonna be looking at chymotrypsin. Chymotrypsin accelerates the reaction rate one billion fold. So how does it do this? Chymotrypsin is a peptidase, or if you're younger, you say protease. It's something that hydrolyzes an amide bond. There's different types of peptidases. They're all different proteins and they're defined by the specificity of their cleavage. So proteases, or peptidases in general, cleave at certain amino acids, okay? And so, for example, chymotrypsin cleaves C-terminally of phenylalanine, tryptophen, and tyrosine. So this C abbreviation applies to all these amino acids. So what does that mean? Does it cut the bond at the alpha carbon? Now, these are hydrolyzing amide bonds, but for every alpha carbon, there's gonna be an amide bond to the right and the left. One is going towards the C-terminus, the other is going towards the N-terminus. Chymotrypsin cuts to the right, cuts C-terminal amide bond next to an alpha carbon holding a phenylalanine, tryptophen, or tyrosine side change. Got it? Another common protease used is trypsin. That cleaves C-terminally of basic amino acids, lysine, and arginine. So there's two things that this enzyme has to do. It has to provide one billion-fold rate enhancement because amide bonds are stable. They have a large activation barrier to their hydrolysis, but it also has to provide specificity. So we're gonna see both, and these are separate things. Okay, so here's the primary structure of chymotrypsin. It has 245 amino acids. Do you see how there's these nicks here? So the single polypeptide is synthesized initially, but that polypeptide is cut. But the pieces are held together by disulfide bonds, these cysteines, right? And so these disulfide bonds, you can see them here, they're yellow. They hold this together. So would this structure be called the quaternary or the tertiary structure? Quaternary structure is multiple polypeptides, right? It is synthesized. Initially it's one polypeptide. Whatever structure that single polypeptide adopts would be the tertiary structure. But as soon as you get in here and start cleaving these amide bonds, well, that's now quaternary structure. And look at this active site. It's a hydrolysis reaction. Do you see how it's just sort of sitting on the surface of the enzyme? But there's this one pocket layer of doom. What might go in there? This pocket has to do with the specificity of the enzyme. These peptidates are pretty amazing. They have high fidelity. They only cut where they're supposed to cut, in this case aromatic amino acids. Some of the amino acids that we'll be talking about today in this enzyme are 1557, ispartic acid 102, and serine 195. These guys are gonna be teaming up on the substrate. So this is the structure of the molecule. And here you can see a picture of what might be sitting in the layer of doom providing specificity. So here is a general schematic of the reaction. Wakey, wakey. Okay, let me come out. Come back in. So here is the reaction. So here we have serine 195. The enzyme is abstracted to be a blue amoeba, right? And so it's holding B, so it's some basic amino acid, and there's a serine 195, and that's exposed in the enzyme active site. And here is the reaction, it's catalyzed. It's another instance of chibis being lazy. What are we missing? There's two things missing from this picture. What are they? What is left out? Critically efficient. Lone pairs is one, that is correct. Lone pairs are not shown here. What else is left out in terms of electron pushing? Is this a correct way to push these electrons? What would you get on an ORGO exam if you drew this picture? Zero, baby. That's right. You would, we're not that mean, okay. So the oxygen, you're actually gonna have an alkoxide ion, whoa. And that's gonna nucleophilically attack this carbonyl carbon. The electrons kick up, go from a trigonal configuration to a tetrahedral configuration. We're gonna use the fact that the mechanism of this reaction needs to have this geometric change to push the reaction rate forward. And then we have collapse of the tetrahedral intermediate and eventually the cleavage of this amide bond, okay. But this is gonna first occur by making a covalent bond of half of the polypeptide molecule to the enzyme. What is the substrate of a peptidase? Is it a small molecule? It's another protein. It could be tightened. It could have millions of amino acids, right. And so R2 and R1 are large things. Another good reason why this reaction occurs on the surface, right. Because you can accommodate, you just have this massive protein, kind of trips and docks with that. It scans along the polypeptide sequence until it finds a aromatic amino acid. It stops and then it cuts, okay. So this is this reaction. Okay, this is the enzyme active site. Some features of this active site. See how these three amino acids are collaborating together an evil plot to catalyze this reaction. So here you have something called a hydrophobic pocket. That solely provides selectivity to this reaction, does not provide rate enhancement. And then we have something called an oxide anion hole. It's like, hmm. So we call this a catalytic triad. They're teeming up and beating up this substrate. Right, and so we have an aspartic acid tugging on a proton on histidine. Now this particular form of histidine in this enzyme has just one proton, right. So aspartic acid's pulling on this proton. Histidine is becoming very upset. Because it just has one, it does not want to share. And so then it's so upset that this histidine is gonna yank the proton off of the serine alcohol. PKA, it looks like six. How does that work? Isn't that weird? Is PKA a constant? Do you see how it's being manipulated here? You're pulling this proton off of this histidine. It's like, dude, I need a proton man. And it gets, so that's, you're literally, it's like a rheostat. The fact that aspartic acid is tugging on this, it's positioning the histidine precisely, pointing it at this alcohol group on the serine, and it's dialing up, right, the PKA here. This is more basic. It's basic enough to pull the proton off the alcohol. Okay, and then we also have another important thing that's providing catalysis. We have, wait, serine NH? What's the side chain of a serine amino acid? OH. Did they make a mistake here, or what are they trying to show? So, might this not be the side chain? Might this be the amino group that's attached to every amino acid? Here we have glycine. What's the side chain of glycine? Is it NH? No. This is the amino group next to the glycine. These amino groups are pointing at this anion hole, waiting for something meaningful to happen, okay. Here's the whole picture. Of course, it's almost impossible to see, and likely it's difficult to see up there. But the one feature you can see is there's two steps. We bind the substrate, release one product, and then after a while, we release the second product, but the two-step process, okay. And here is another molecule that comes in. A water molecule is important in this process. We're gonna go through this in gory detail, step by step, to help you to appreciate this rate enhancement. So, the first step, well, here's the substrate, phenylalanine, tryptophan, tyrosine, whatever, some bulky hydrophobic amino acid nestles down into that pocket, and then it can't move. It's anchored by this hydrophobic pocket, and the substrate comes in and binds in this active site. So, this is an abstraction of the active site. It actually looks something like this, okay. And if you're really clever, you'll see that this is inverted from our normal orientation, right. C-terminus, N-terminus here, but here we've flipped it around. You can look at it later to see that. It's not important to what I'm saying. Why did you mention it? Observation, man. Okay, so now we formed a complex between an enzyme and a substrate. What do we call that? If you're a McKillis-Minton? The enzyme substrate complex. That's what it looks like. Before, it was just this abstract arrow going to ES. It doesn't really have any meaning. There it is. That's an enzyme substrate complex. Things, so this, the substrate here is a very large molecule. It's another protein. It's being stabilized. It's interaction between the substrate and the enzyme is stabilized by the placement of this large bulky ring in this position. But there's also other positions that are making contact and holding onto this substrate in addition to that. So initially we find, and now we're gonna start catalyzing some reactions. So we have PKA-adjusted histidine about to pull a proton off the serine, making an alkoxide ion. And when that happens, we're gonna have nucleophilic attack of that serine alkoxide ion and the carbonyl carbon forming a tetrahedral intermediate. Do you see the change in geometry, trigonal planar tetrahedral, that oxygen has shifted? Oxygen ion hole initially, when the substrate binds, is not precisely aligned with this carbonyl oxygen. But when we go through the transition state, the oxygen shifts into this hole. So only when this oxygen is in this hole can it have these stronger hydrogen bonds when the polypeptide backbone next to glycine and serine, okay? And so why is this accelerating rate? It's one of the key concepts. Why is it accelerating rate? Can you think of it? Sometimes, yes, that's right. And so how many bonds are you making to the substrate here? Less than the number of bonds to the substrate here. This is the enzyme providing this rate enhancement by making more bonds to the transition state than the ground state configuration. What about this pocket over here? Is that providing rate enhancement? The hydrophobic pocket. Why or why not in terms of number of bonds? Same number of bonds. It's not accelerating anything. It is providing substrate specificity, but it's not helping to push the reaction forward. If you just, yeah, go ahead. It's precisely held in place, right? And so you're not, what would you do? If you had to pull this hydrogen off, what would you make? That would be pretty weird, right? Partial double bonded nitrogen losing a proton. So there's a bit of a chemical barrier to that. So this is in the polypeptide, the representation is confusing. This is the polypeptide backbone. The nitrogen groups in the polypeptide backbone have a partial double bond to their neighbor. It's first because it's not close enough. These are hydrogen bonds, yes. Do you think it would be? So that's a very good point. So free amino acid is different than amino group in an amide bond. That physiological pH would not be protonated. In the gas phase it would, and that's why we can do high throughput sequencing by mass spec. But in solution, this is not protonated there. It doesn't have two protons. Very good point. It's confusing because there's things abstracted here. This is the polypeptide backbone. There isn't a positive charge. Now if it were the interminous, yeah, it would have an extra proton, right? Very, very good question. Thank you for asking. So there are also two questions about specificity. Is chymotrypsin specific to phenylalanine, tryptophan, and tyrosine because they're chemically similar and do most protease work this way? Yes, because they fit in the pocket. If you don't have something fitting in that pocket, you can imagine what types of amino acids the chymotrypsin protein might surround that pocket, right? Hydrophobic amino acid, your hydrophobic interaction. And then a follow-up to that. How can peptidases be specific about which residue to cleave at? Because if you don't drop an anchor, you float away. Ooh. It's so I'm a sailor. So it literally is the anchor, right? So there will be contact. What these enzymes are doing is they're scanning along polypeptide sequences. And when a bulky aromatic amino acid ends up in that binding pocket, it stops. Then it might just miss. Maybe it's sort of off to the side. But the only way it's gonna stop, it has to stop for the reaction to be catalyzed. You gotta hold things, right? And so the specificity is actually pretty spectacular. If you look at it by a math spec. Good question. Okay. All right, so now we're gonna catalyze this reaction. We're gonna make the, let's see, tetrahedral intermediates. So here we have the alkoxide ion attacking the carbonyl carbon. Go to transition state where this negatively charged oxygen is now has more bonds to it because it sits in the oxyanine hole. It's a little harder to see it here. Here you see one set of dashes. And here you see two. But really, if you look at the actual structure, it's a big shift and the oxygen makes, really only makes hydrogen, strong hydrogen bonds when it's in this tetrahedral configuration. So this is the transition state intermediate. We're forming more bonds to that than the ground state. Next, we're going to drop off half this molecule. So the transition state's gonna collapse. Remember, equal probability would go in either direction. But if it goes forward, it's gonna be irreversible because we're going to break a bond. So now we have an ester linkage of half our polypeptide substrate attached to the enzyme. Okay, what do we call this? What type of catalysis is this? Drop it. Covalent catalysis, right? We've made a covalent bond here. It didn't have to do this. There's some like HIV protease doesn't make a covalent bond to its substrate. It just positions water very precisely. But here, because we have this bond to the enzyme, we can stabilize it by this change in geometry because we necessarily go through this intermediate. So then we have ester-linked half of polypeptide sitting on the enzyme. Obviously, if this were the end of the story, you would have to make one chymotrypsin enzyme for every peptide bond broken. So we need to break this ester linkage. And so water is introduced here and it's guided into place very precisely by the histidine amino acid, right? And so this histidine amino acid holds the water molecule precisely positioning it so that it can attack here. So is this, what kind of reaction is this? Is this a monosubstrator bisubstrate reaction? What form, ternary or ping-pong? Oh, come on, ping-pong sort of ternary, half ternary, half the substrate one's attached. But if you look at the kinetics of this, as it turns out, it is a ping-pong bisubstrate reaction because the enzyme has been altered. You're looking at E prime. E prime is half of substrate attached to enzyme. That is not the same as E because it now has an ester linkage to a polypeptide. So the water molecules guided in place, right? Actually, I failed to highlight the fact that we have a little bit of acid-based catalysis going on here. So we pulled that proton off the serine and it was sitting on histidine and then we can donate that back to help increase the leaving groupability of this amino group, right? Help push that reaction forward by acid-based catalysis. Would that be general or specific? Full of questions. General because it's a base that is not water, right? Or it's an acid that's not water. And here we have the water molecule guided into place. It's gonna attack and go through a second transition state stabilized by a change in geometry. Again, tetrahedral intermediate. Amide bonds are broken in the same way that ester linkages are. And so you have a tetrahedral intermediate, electrons go up, oxygen shifts over. You make more bonds to that than the previous state of things. But now we wanna help catalyze breaking a bond, right? And so we're going to donate. We're gonna protonate this, increase its nucleofugacity, increase its leaving groupability. That also dates me, okay? And so here we have cut the ester linkage, right? Half the water molecule ended up here. The other half ended up here. And so now we have a EP, right? It's an enzyme product complex. And the only last step is release of the product. Regenerating the enzyme. I'm allowing it to be processive, okay? So this is chymotrypsin. This is a mechanism of action. Let's recap. A lot of things happened while we were looking. So we saw the acylation phase. You had this cleavage of the peptide bond where you made an ester linkage, a stable intermediate, ester-linked serine attached to half the substrate molecule. The oxyanion hole was providing rate enhancement by making more bonds to the transition state of two different reactions here than the ground states. And then we hydrolysis of the ester linkage. We saw some examples of general acid-base catalysis by other groups in the active site. So all of these factors are culminating in a billion fold rate enhancement. Thank goodness that AMI bonds are stable. All our proteins would immediately disintegrate if they weren't. But sometimes it's necessary to turn proteins over. And so we need to be able to lower this barrier and increase the rate. And that's what this enzyme is doing. So this is recapping the structure. The triad, the gang of amino acids, are culminating to catalyze this reaction. Any question about chymotrypsin? No? Okay, so there was a wonderful question in last lecture about, well, wait a minute. How does pH affect Kcat and KM? And I did not provide a suitable example at that time, but actually chymotrypsin has this type of behavior. So here's chymotrypsin. As you change the pH, the Kcat is increasing, right? Something magical is happening right around pH seven. You might guess what that would be. And then at even higher pHs, KM goes from being small, inversely related to the parent binding affinity. So that's strong binding to a weaker binder. So in general, KM, the effect of pH on KM, what involves ionization of the substrate or enzyme, might affecting the tightness of binding to the substrate. So KM has this relationship to binding affinity. And when you ionize either the enzyme or the substrate, you could affect that. Kcat, you're talking about the maximal processivity of the enzyme, the chemical transformation. So ionization of things can affect how efficiently the enzyme machine is operating, right? So ionization of key residues could affect the ability of the enzyme to be in its active configuration. So can you think of what might be causing this? Kcat, say again? What's the y-axis? The y-axis depends on whether you're talking about Kcat. So this is Kcat, kilojoules, or I don't know what the units are. And this is KM units. So Kcat is increasing. What's causing Kcat to go up as the pH goes up? What happens right around here? pH seven. So it has to do with the chemistry. So there's something going on here. What's happening at pH seven there? You guys asked the question, so do you see it? So this histidine is gonna begin to become protonated. If that histidine is protonated, how good is it gonna be to pull off this hydrogen atom from the alcohol? That destroys the ability to do covalent catalysis. Okay, so protonation, changing the pH state, affects the ionization state of the histidine, making, removing one of the forms of... And now, yeah dude, you can turn the volume back up. Back up, up, up, up, up. I'm gonna bring it up. Okay, got it, got it. Thank you, thank you. Don't say anything. So there's experiments underlying the mechanism that I've described to you. There's never the two different types of inhibition. Irreversible inhibitors, these make dead and covalent bonds with the enzyme and prevent the enzyme from ever catalysing another reaction. Then we also have reversible inhibitors. So what we're gonna do here is we're gonna use the irreversible inhibitor of growth to extend them to the alien world and we have to determine what the mechanism of action is. We're also gonna get chromogenic substrate. So we're gonna land the probe and then we have glycine, alien, and hydrogen. We're gonna cover something up. How about growth? We're taking into this enzyme-acid site to determine the mechanism. This sort of looks like the Mars and all the rice. That's too much. We have a really wonderful leaving group, this oral group. And so this inactivator is smelling, is this a file? Is this a file? Very much a file, what's going on here? It's looking for a nucleosilic group. When you send a probe to the alien world, you don't know what you're gonna see when you do the experiment. So you might be thinking, why did they do this? Why should it work? They didn't know it was gonna work when they sent the probe in. But what they found is that this molecule made a covalent bond that's adhering 195. They could determine that it was a new bond that's adhering 195 by sequencing the polycuton. And they said, whoa, there's our probe sitting on one and only one amino acid. So if they're sitting in one probe, they're sitting in the second probe. They say, that was useful, only one amino acid found. So this has been probe number two. It looks like there's not so much in the alien chloromethylcetamine. This is also looking for a nucleosilic group. And what they found was that this TPCK probe was a covalently modified histidine. So adhering seems to be important here. Histidine seems to be important here. Somehow, this guy is not normal. It's altered in some way. So they began to come up with this hypothesis of a triad, right? So they say, if it's an amino acid, that seems to be unusually reactive to some of these theories, right? So this is some of the evidence that you're making the covalent bond to substrate. So also, they did this thing. They sent in a probe that was a chromogenic substrate. So paramexocinol acidase is not colorless, it's colorless. But when this ester makes it in its place, it gets paramexocinol. And see how this is widening from this probe in, not an amycon, why isn't it an amycon? It still cleans it, right? And so what they found was when they measured the rate of formation of this colored product, they saw an initial fast burst of production of this product, and then a separate lower stage of production of the product. So when you think about it, this is the thing of making, if it is making a covalent bond, we're swapping an ester in your substrate with an ester for the theory, right? So the thing that generates color is measuring longest to detect the rate that this product consists. So there's something that happens quickly, but then something happens more slowly. So that's how they're probably two steps here. The covalent bond formation was your substrate and the subsequent hydrolysis of the ester between the theory and the happy ester. And so they said, okay, well that must mean that the hydrolysis of the ester is very limited. It's lower. So up to a certain point, you saw all the exercises in the substrate, and they're all found in the substrate, but then it takes some time to clear that absolute length between the theory and the happy ester. So this is direct evidence that supports the hypothesis that there is a covalent intermediate between the end bond and the substrate. So this is where we need to go. Okay, any questions on chemistry? I'm going to move on. Yeah. So chemistry is another example about permeating the ester. We need to recall chemistry is a steering permeate. When you say steering permeate, you're not talking about the specificity of the permeate. You're talking about it's catalytic mechanism. The steering permeates all have triads, right? And they all have a form of a covalent bond with steering amino acids and the substrate. The steering permeate is a sample-based permeate in coca, which is important in hypothesis and subject. Well, those forms covalent bond with the steering and the substrate have steering permeates in the covalent. So all of these are named according to the type of chemistry that a lot of Indians un-reacted by, not by the substrate, when it comes to the general family. They all, all these steering permeates is the same mechanism as the maximum triad. They just said it worked before, but really didn't, no reason to make it change. Okay. So this is an amazing rate, and it has a 10 in the nine fold, one billion fold faster. And so surely once a permeate is made in a ribosome, you don't want it to start cutting up proteins. It'll start to chop away the proteins on the ribosome, and it'll inappropriately just cut everything up. You want that thing turned on, only when it's appropriate. And so we'll see how chemistry can be regulated by starting a new treatment. It could be referred to as a gynecologist. You can also have reversible covalent modification that's, yeah, the Alaskan Modulation. So reversible covalent modification is also a form of Alaskan Modulation. We learned about Alaskan Modulation when we looked at humans. It wasn't in the case of a person in the body who was alive. Let's consider this. Well, here's a time shift, then. In this case, when synthesized by the ribosome, the single poly-cut side, it's not able to achieve the pre-menstrual structure necessary to align the catalytic triad and to catalyze the reaction that it needs to catalyze. In order to first achieve that orientation, you must break these bonds. You fold it up, make some vegetable fun lengths, and then make two lengths, shift the shape of the parachute and turning it on. This is a one-way, it's not Alaskan, Alaskan is reversible. This is a one-way activation. The only way you're going to turn this off is actually to degrade the person. And that's what it is about. Unimproviation is also the use of strategies of activation by flea goods. It's irreversible. When we have the one type of way in which we can activate or turn off the protein, it's by making a modification to that protein. And these are enzymes. Each enzyme in this cascade is processive. So this one enzyme turns over more than one substrate. Some enzymes have an enzymatic activity that is to make a chemical multiplication in another protein, causing it to turn on or turn off. So here we have an enzyme, let's say the kinase, the type of enzyme that has a top state, one of these that's marked. Then what's easy to find are modified by a population. There's a enzyme, and each one of those is processive. So from a tiny starting point, you have a massive amplification, a larger response. So when you turn on this signal, you just turn it up into an environmental skew, turn it on, and then you'll have a very rapid and robust response. Here's one example of this, blood is a clotting cascade. This is a curvilidation, you're all dying again. So if you want blood clotting, you go slowly. No, you will die if you go slowly. You want this to go like this. But you don't want, I mean, this cascade involves clean as a protein. You don't want this to happen at the wrong time. You would be clotting your blood. You would also be in bad shape. So when you get the right signal, and there's a cut, the blood clotting is initiated, and this is a hydrogen cascade, one enzyme cleaning the neck, cleaning the neck, leading to a rapid and robust response to the initial input phase. So we have blood clotting, and we can also, the resolution of the clot, the removal of the clot, involves a smaller cascade of this percent. And so it's less urgent to clean your blood, because you're not leading to that, right? So that can be a lack of ample thought. When we talked about outstretched modulus, you define those as 6 to 5 in the site, other than the active site, changing the shape of the enzyme, you're determining it off their eyes. So here, for example, we have an R-Ris C sub-unit of the protein, R for regulatory C for catalytic. The regulatory sub-unit binds a regulatory form of the allosteric regulator, and that binding event changes the shape, it shifts to the right. And the changes of shape allow the acceptability of the active site to the substrate, where it allows the chemistry to happen, it changes the optimal orientation. We have some definitions. It's the allosteric regulator binding the site other than the active site, it's a chain, it's a substrate that's called homotropic allosteric regulation. It's a regular, significant model of the substrate, it's heterotropic. So remember when we looked at hemoglobulin, we saw both homotropic and heterotropic regulation as homotropic, and the rest of those guys are heterotropic regulations. If the visiting's gonna come throughout the plan by watching, there's a non-hyperbolic relationship between the reaction rate and substrate concentration for allosterically regulated processes. So when we design a KM for an allosterically regulated enjoyment, would the line we've reversed possibly linear? No, it would not. It would be closed, but it would not be linear. So instead of saying KM, we say K.5, and that's defined as the substrate concentration necessary to fill out the binding site. So it's not exactly equal to the KM. We cannot get a traditional McHale-Smith-Smith-Smith-Smith to calculate it. We have to use a more advanced type of calculation. So we can have heterotropic or homotropic allosteric regulation that is positive or negative. In other words, the binding of the regular can cause a more optimal configuration on your enzyme to catalyze reactions from less optimal considerations. We can do the subset by changing either the V-map or the KM. So if it's a positive heterotropic regulator, it shifts the curve to the left. So that decreases the number of substrate models necessary to achieve half-maximal rate. That positive, that negative, it shifts the curve to the right. Well, it takes a higher concentration of substrate models to the end after it's achieved its half-maximal rate. We can also modulate the V-map. And so if you shift up, well, it's more perceptive. If you shift down, it's less perceptive. This is all by controlling shape transitions in animal. I think I'm going to come back to this, because today's clicker will be the hardest of the semester. Sorry. I used to have it on an exam. You guys, do you want more time? Any more time? Can you fill the remaining minutes with explanations or discussion amongst yourself, either of which are good? Give it 10 more seconds. For those that haven't voted, you either need to turn in or vote within the next not 10 seconds. You need a piece of paper. Great. We have a banner ID and a vote. All right. I think we're going to stop the polling, Alex. OK, let's go through each of these answers separately. What about acid-base catalysis? Why not? What's left? Right? The things that are doing acid-base catalysis are mutated in this example. What about transition state stabilization? I phone likes that answer. OK, maybe covalent catalysis. That amino acid's gone. Hydrophobic effect. Are you sure? Why not? But the substrate couldn't bind without the hydrophobic pocket. Is that the question I'm asking? No. Come on. No. Tell them. So how would a hydrophobic pocket provide rate enhancement? Only if it made more bonds to the substrate gone states than the transition states. Hardest one of the semester. You made it through. You got to get really well, actually.