lecture 4 youtube: microbial metabolism part 1

So the last time we left off, we were just getting started talking or expanding a little bit more about the plasma membrane. So we've talked so much about the cell membrane, also known as a plasma membrane. We know that it is made up of phospholipids. In fact, it's a phospholipid bilayer. That means that there are two layers of phospholipids where they're hydrophilic or polar heads. are facing outwards in the aqueous environment and their hydrophobic tails or water-fearing tails are facing each other, right? The plasma membrane is so important. It functions in, basically, it is selectively permeable. It allows for things to go in and out of the cell. It senses environmental signal. And as I mentioned, It's where energy transformation in a prokaryote occurs. This is a prokaryotic class, so we're going to be focusing on that. So transport, movement of chemicals across the cell membrane. So as I mentioned, it's selectively permeable. So it allows things to go in and out of the cell. How? Through what mechanisms? Well, passive transport, meaning that Things can go in and out of the plasma membrane without requiring energy in the form of ATP. So that's passive transport. Within passive transport, there are three main types of passive transport. That would be diffusion, osmosis, which refers to the diffusion of water, and facilitated diffusion. Now, facilitated diffusion is interesting because it requires a carrier protein that's embedded within the membrane. But it's still, even though it requires a carrier, it does not require energy in the form of ATP. And the reason that it does not require energy in the form of ATP is because the diffusion or the transport of substances occurs from an area of higher concentration. to lower concentration. So it goes down the concentration gradient. So it kind of makes sense. You're going from high to low and it does not require energy. The other main type of transport is called active transport. And it's called active transport because it actively requires energy. Okay. Mostly because you can go against the concentration gradient. meaning that you can be in an area, you can go the opposite. You don't necessarily go from high to low. You can go from low concentration to high concentration. Well, to do that, it's not so natural, right? And so therefore, it requires energy. Active transport, there's different types of active transport, group translocation and bulk transport, such as we see with endocytosis, exocytosis, and penocytosis. So... We're going to go over a few of these, like simple diffusion is basically just that, going from high concentration to low. So here we see that if there is like a salt granule, if we have a salt crystal, rather a salt crystal, that crystal will begin to dissolve. And the molecules will go from this high concentration area to a lower concentration. Facilitated diffusion, as I mentioned. requires a carrier protein. Now, what is a carrier protein? I'm so confused. Look at these integral proteins that we see embedded in the membrane. It would require something like that to allow for the passage of molecules, okay? But it's going from high concentration to low concentration. I don't really get into a whole lot of the active transport methods and the sodium potassium pump. We don't really go into that in a prokaryotic class like this, okay? That's going to be more molecular or general biology or physiology. Now, the last type of movement across the cell membrane that we see is active transport. And this does require a carrier protein like facilitated diffusion. But again, now we can go against the concentration gradient. And if we're going against the concentration gradient, meaning that we're not going from high to low, we can go from low to high. You require. energy in the form of ATP. There is not an animation on Blackboard, that would be Canvas, and there isn't one. But your textbook has these animations if you wish to see them. So let's talk a little bit about simple diffusion and osmosis, okay? So when we're talking about diffusion and osmosis, we're talking about the movement of water, okay? We're talking about the movement of water inside and outside of the cell. Now, cells like to be in an isotonic environment, isotonic solution, meaning that the concentration of solute inside... and outside of the cell is the same. Now the solute would refer to the little salt crystals, if you will, okay, the molecules. It likes to have, it likes to be in an environment that is, that is in, in an isotonic type of environment and solution, okay. So when a cell is placed in an isotonic environment, meaning that the solute concentration is in the same on the inside of the cell and the outside, water can go in and out and there is no problem. If you place a cell in a hypotonic solution, now hypo means low. So that means that the beaker, if you consider this square here in the blue and the little dots, if you consider this being a beaker of solution and it has an It's a hypotonic solution. Hypo means low. So that means that there is a low concentration of solute. That means that there is less solute in this beaker than inside of the cell. So you see that the cell has a higher concentration of these molecules, right? Well, the cell doesn't like that. It wants to be in an isotonic environment. So it's going to say, you know what, I'm going to dilute myself so I can be in a sense of equilibrium, if you will. So it allows water to go inside of the cell in order to be in the same concentration as outside. The problem is that too much water would need to go into the cell so that it would be in a state of equilibrium that the cell will burst. That's cell lysis. The cell will lyse because too much water would try to rush in and dilute the inside of the cell. Now, if that cell is placed in a hypertonic solution, now this is a different beaker. Okay, let's consider this blue being the beaker, the water. In a hypertonic means that there is a high concentration of solute. Notice that there are a lot of little molecules, right, inside of this beaker. And so as a result, the cell's like, okay, no problem. I'm just going to dilute my environment so that I can be in a state of equilibrium. So the cell is going to release a lot of water to its surrounding environment to try to dilute its surrounding environment. But do you think a small little cell is going to be successful at that? No. Essentially, it's going to release all its water and it shrivels up. It shrivels up. And that's referred to as plasmolysis. These different states, you need to understand these three different states. Okay. Now in facilitated diffusion, we talked, I mentioned to you that it requires a carrier protein, but it doesn't require energy. So these proteins are embedded in the plasma membrane. And what they do is they will transport substances from an area of high concentration to low concentration. And because it's going down the concentration group, it does not require. require energy in the form of ATP. Okay, now let me go ahead and get the next one. Okay, so now we're going to start lecture four. This is a large lecture and I've divided into lecture four and five. And on Thursday, we're going to discuss cellular respiration. So you don't want to miss that. But today we're going to talk about cellular respiration. So let's get started. we're going to talk about microbial metabolism, or at least get started with the idea of microbial metabolism. And this is a great slide. And we're going to revisit this slide in a few. Okay. So let's start talking about metabolism and what is metabolism. It is the sum of chemical reactions in a cell. Okay. And oftentimes when we talk about metabolism, we talk about being metabolically active. And what does that mean? right? What does it mean to be metabolically active? It means that a cell is able to reproduce. So that is really what metabolism is all about. The ultimate function of metabolism is to reproduce the organism. So you will hear me talk about a metabolically active cell. A metabolically active cell is a cell that is alive and it's reproducing. It's going through all these different biochemical reactions. Why do we study metabolism? Well, it occurs in all living organisms. And we also use it in microbiology to identify organisms. You see, that is one of the things that you're going to be doing in lab. you're going to be performing various biochemical tests on your unknown. And based on those biochemical tests, you will eventually determine the identity of your unknown. Because not every organism is the same. And the only way you can determine if your microorganism can metabolize glucose versus fructose versus sucrose is to actually test it. So not every organism can metabolize or utilize fructose, for example, or lactose or sucrose. So that those are some biochemical tests that you will run on your organism. That's why metabolism is important. It tells us something about our microorganism. And based on the collective data, we can determine what our unknown is. OK, so it's going to be a lot of testing that you're going to do. All right. So there are two major classes of metabolic reactions, two classes of chemical reactions. They are both enzyme regulated chemical reactions. We have catabolism and we have anabolism. OK, so let's talk a little bit about these. Catabolism is the process of going from a large molecule and breaking it down to smaller molecules. or going from a large molecule and breaking it down to its monomeric units. And in the process of breaking down this large macro molecule, if you will, to smaller monomeric units, you release energy. And that energy that is released is in the form of ATP. Well, wait a minute. That kind of sounds familiar. That type of reaction to me sounds familiar. What does that remind you of? What does that kind of sound like to you? Dehydration synthesis. I don't know about dehydration synthesis. What is dehydration synthesis? Is it the breaking down of molecules? A-Lyn says no. What is it? No. It is to build or to synthesize molecules, right? But to me, catabolism does sound familiar. I think I've heard something like that before. What does it remind you of? Cellular respiration. Cellular respiration. Okay. Yeah, eventually, but we're not there yet. So let's back up. What, Heidi? Catalyst. A catalyst? Well, a catalyst speeds up reactions, and we haven't even talked about that yet either. Who goes metabolism? Well, that's like cellular respiration. That's what George said. Hydration synthesis reaction? Dehydration synthesis is to build something, is to make these large macromolecules. Oh my word. Hydration synthesis? Say that again? Hydrolysis? Hydration synthesis? Hydration synthesis, you mean hydrolysis. Hydrolysis. It's not synthesis. Remember what the word synthesis means? Synthesis means to make. It's hydrolysis. These are essentially hydrolytic reactions. Catabolism releases energy. These are hydrolytic reactions that use water. to break chemical bonds. And in the process of breaking down these large macromolecules into smaller monomeric molecules, you release energy in the form of ATP. This reaction is also referred to being as exergonic. It releases energy. Now that energy in the form of ATP is going to be stored. It's going to be stored so that it can be used for anabolic reactions, you see. So let's talk about anabolism. or anabolic reactions. So in an anabolic reaction, we are taking these smaller molecules or small monomeric units and we're putting them together to build or form a larger macrame. But because we're building, when we're building something, does that not require energy? Yeah. Yeah. So we need energy in the form of ATP. What does this type of reaction remind you of? Dehydration synthesis. There you go. That's your dehydration synthesis. Yeah. So what's interesting is that the catabolic reactions are going to provide us with the ATP that we need for the anabolic reactions, you see. And these anabolic reactions are endergonic. They require energy and that energy is in the form of ATP. So let's get, let's take a closer look at this relationship between catabolism and anabolism. You see, it's a beautiful story. As things are being broken down because they're not needed anymore, those monomeric units that were released. These can then be used and synthesized to form something that is needed at that time. So we can start anywhere we want. We can start here at the top. We have small, simple, monomeric units such as glucose, right? Glucose is a monosaccharide. Amino acids, glycerol, fatty acids. These are all monomeric units. And guess what? They come together in an anabolic reaction, transfer energy from ATP, release heat, and boom. We have synthesized our complex molecules, or we have synthesized our macromolecules. When the cell no longer needs these large proteins, or these lipids or starches. Why keep it? We can reuse and recycle, so to speak, right? Reuse and recycle. So through catabolic reactions, we're going to transfer that energy that was stored in these large molecules, break them down, release ATP, and now we're back to our monomeric units. Isn't that just beautiful? The way this all works. I find it fascinating. As things are broken down because they're not needed anymore, other new things are being synthesized because that's what the cell needs. It's just amazing to me. I think it's amazing. Here's another view. We have our large carbohydrates. lipids, they're going to be broken down into their basic monomeric units and to build what we need and go on and do what they do. So what we say is that catabolic reactions, catabolic reactions. Now, you know what, now that I'm thinking about it. I used to get confused, like, well, which one's anabolic? Which one's catabolic? Which one's breaking down? Which one's building? It's all so confusing. There are so many terms, right? And I think about a cat breaking things down. And so then I remember, oh, yeah, okay, catabolic. Cats break things down. The catabolic is breaking down on these large molecules. If it doesn't help you, don't use it, okay? So catabolic reactions essentially provide the... building blocks for anabolic reactions. Catabolic reactions provide the building blocks for anabolic reactions. And guess what? This is possible through ATP. It is possible through ATP. So ATP stores energy derived from catabolic reactions to later release it. to drive anabolic reactions and perform cellular work. It's just beautiful. So let's talk a little bit about ATP. Let's talk about the synthesis of ATP. Okay, so how do we make or synthesize ATP? The synthesis of ATP is a phosphorylation reaction. We see that ATP has these phosphates with these high energy bonds between them. Okay. So the synthesis of ATP, how we make this ATP is we start with ADP, adenosine diphosphate. You see a phosphate group is added to ADP. This refers to inorganic phosphate, the little I. means inorganic. So we're going to add an inorganic phosphate to ADP to yield ATP. So anabolic reactions are coupled to ATP breakdown and catabolic reactions are coupled to ATP. synthesis. So it's kind of the opposite, right? Does that make sense? Because when we break things down, we are releasing ATP. But when we are synthesizing things, we are utilizing ATP. Anabolic reactions is coupled to ATP. breakdown. Because when you use ATP, you are basically removing or breaking one of these high energy bonds to utilize it. Okay. This concept of coupled reactions, like we see here, is important. Some molecules are broken down while other molecules are synthesized. Yeah. And this balance is important because it's what maintains the cell. Very important. Maintains the cell. Okay. So we're going to be talking about enzymes next. Enzymes are our biological catalyst. We hear that a lot. They speed chemical reactions by lowering the energy of activation, how much energy is needed to get the reaction started. But what really are enzymes? Do you know what they are? What are they? What are they made out of? What are they? Amino acids, proteins. Yeah. They're essentially proteins. They're amino acids that are bonded together via dehydration synthesis. They're proteins. When enzymes are used, the enzyme is not permanently altered in the reaction. They can be reused, so to speak. Enzyme promotes a reaction by serving as a site for the substrate. And I'll talk to you a little bit more about that. So it's a little bit, it's a little bit hard to put in words. So let's see how these enzymes function. How is it that they work? Here we have two reactants. We have A and we have B, okay? We see that A in the presence of an enzyme. Hold on. Sorry, it was the opposite. We have A without an enzyme and B with an enzyme, okay? So how much energy does it take in order to get to our final product? What was that? Which thing A requires more energy than B. That is correct. A is going to require more energy. So eventually you're going to get the product, but it's going to require more energy. And sometimes that equates with time as well. All right. So in the lab, when we would do cloning and we needed to insert something in a bacteria, for example, we work with enzymes and we speed up the reactions. Using an enzyme versus not using an enzyme is a matter of... Having your reaction complete within an hour, if you incubate it at 56 degrees, let's say, versus letting the reaction run overnight at room temperature to get the same result. So using an enzyme speeds things up. But enzymes are proteins and they're finicky. They require specific temperature. They require specific pH. Being outside of the optimum is not going to make the, or allow the protein to function to the best of its potential. So using enzymes speeds up the reaction time. Hmm. I wonder if that's why it's important that we're careful or why it's so dangerous to have such a high fever. Well, what does that even mean? All right. Remember that enzymes are proteins and we know that proteins are sensitive to temperature, pH and chemicals, right? Do I have a slide on that? Let me see if I can look ahead. Oh, I think I already talked about, no, I haven't. Okay. It's up ahead. So I'll just wait on that. What I was going to say, as far as temperature is concerned, I can see in the bottom a little bit what I have. Now let's take a look at our enzyme terminology, or let's take a look at the anatomy of an enzyme and see how they work, how they function, and all the little body parts of an enzyme. First of all, a substrate is a molecule that the enzyme is going to act on. So the substrate is what the enzyme is going to work on, what's going to activate or break down, whatever it's going to do to it. So here I have an enzyme, but here I have an enzyme too. What is the difference between these two? This is called an apoenzyme. An apoenzyme is an inactive enzyme. It's inactive unless it binds a cofactor or coenzyme. Okay. So it requires a cofactor. These cofactors or coenzymes can be for inorganic ions like iron, magnesium, zinc. It can actually even be vitamins. But without them, we don't have an active enzyme. Well, why is that? Well, if we take a look at this area here that is created when this coenzyme or cofactor binds to the apal enzyme, we see that it creates an area called the active site that fits the substrate. or what it's going to act on. You see that? If we do not have this coenzyme or cofactor, will this substrate fit in here? No, it won't. No, it won't. So if this enzyme does not have that coenzyme or cofactor, it is an apoenzyme or inactive enzyme. Once it has its cofactor or coenzyme bound to this site, now we have a holoenzyme. A holoenzyme is your active enzyme. And it has the active site ready for the substrate to bind to and act on. Yeah? So binding of an apoenzyme to its cofactor. forms an active enzyme called a holoenzyme. Now, the substrate is what's going to activate this protein or this enzyme, if you will. Yeah. I threw a few terms at you, a cofactor and coenzyme. A cofactor is going to be a molecule that's needed for enzyme activity. because it helps create that active site. These are usually non-protein components. So it's not part of the enzyme that the enzyme, remember it's a protein that binds that it wraps around itself and it has this tertiary structure. Remember that? Okay. The cofactor may be organic or it may be inorganic. They can be metal ions. In fact, a coenzyme is going to be an organic molecule that acts as a carrier. of molecules or electrons such as NAD. So these are just different types of cofactors that can fit in that area. Okay. But you know what? Co-enzymes are important. And I did mention to you that some of these co-enzymes are vitamins. Yeah. So is that why it's important to take our vitamins? Is that why our parents always said, take your vitamins? Maybe. But I want to actually point a particular important vitamin to you. And that is this one, folic acid. Take a look at folic acid. And what the function of folic acid, what it says here that the function of folic acid is, is that it is a coenzyme used in the synthesis of purines and pyrimidines. Whoa, wait a minute. Now we have a lot of terminology here, right? So we know that coenzymes are important because they bind to the apal enzyme to make a holo enzyme. And we know that these holo enzymes are important because they lower the activation energy and they allow reactions to take place. They speed up reactions to occur, correct? Now, it also says that this coenzyme called folic acid is used in the synthesis of purines and pyrimidines. And we know that synthesis means to make, right? Building. So it's used in the synthesis. What are purines and pyrimidines? I forgot. Where have we heard that term? DNA, pairs of DNA, nucleic acids, right? Because it's in DNA and it's in RNA too. Yeah. So those are components of nucleotides guys. Purines and pyrimidines are components of nucleus. Okay. Well, so what nucleotides are the building blocks for nucleic acids. All right. Well, so what folic acid is important. especially for pregnant women. It is so important for pregnant women to take folic acid, one milligram at minimum 800 micrograms, which you can buy over the counter. It is important for neural tube development. Very important. Folic acid supplementation can prevent neural tube defects, such as spina bifida. In fact, if you're a woman and you're even thinking about having a baby and getting pregnant, you should really start taking folic acid or prenatal vitamins, ideally six months before you even try to get pregnant. Folic acid is a B vitamin that's used in our bodies to make new cells. So you know what? These little coenzymes, there are little attachments right here. They're actually very important. So that's just to give you an idea. of the importance of these coenzymes, okay, in their function. All right, so let's talk about naming enzymes. An enzyme, you usually know that it's an enzyme because it ends in ace. Hyaluronidase, hexokinase, streptokinase. So usually it's safe to say that if the word name in ace, it probably is an enzyme, okay? Now, the names are sometimes based on the type of chemical reaction that they produce or the substrate or product. So these are some examples of different types of chemical reactions that are catalyzed. You don't need to know these. I'm not going to examine you on them. These are just some examples of different enzymes. And you'll notice that they end in A's. So generally speaking, an enzyme will end in A's. That's how you know it's an enzyme. enzyme. Okay. All right. So let's take a look at an enzyme in action. So here we assume that this is a holoenzyme. It already has its coenzyme attached. It's ready to work. And what we see is that this enzyme has its active site and the active site is shaped specifically for a substrate. Now, some people use the lock key fashion. It kind of is like that. Not everything can fit in. But it's not a perfect fit like a key and lock. The active site kind of molds into that key, if you will. But it's not promiscuous in the sense that it's going to allow anything in either. So, for example, here we have two substrates. And we can see that one of them has the shape that appears to fit in, where the other one does not, right? So, not just anything. can bind to this active site and give you product. It just doesn't happen. So we see that this green one does not fit. But the other one does. When the substrate binds the enzyme, we create something referred to as an enzyme substrate complex. Okay? An enzyme substrate complex. And then the enzyme will work on this complex and give you or release your products. Now what we see is that the active site is open. Do you see how it's open? Guess what? It can be reused. another substrate can come in, bind to this newly opened active site, and give you yet another product. So these enzymes are reusable in a sense. As soon as the active site is clear, another substrate can come in. Questions? So let's talk about the location and regularity of enzyme action. In microorganisms specifically, we talk a lot about exoenzymes and endoenzymes. Okay. And exoenzymes are going to be enzymes that are made inside of the cell and exported to the outside. Now we can test for this and we use special media in the lab to determine and detect the presence of specific exoenzymes that are released by the bacterial cell. So again, exoenzymes are made inside of the cell and released to the outside environment. Why? Well, for different purposes. Sometimes it's to break down things, maybe break down nutrients. Here we see some examples. Cellulase, amylase to break down starch. Penicillinase to break down penicillin. Is that what that means? Yes. There are some bacteria that produce an enzyme called penicillinase, also known as beta-lactamase, but you'll learn that later. And it breaks the beta-lactam ring in the penicillin. so that the penicillin or the antibiotic is no longer active. So we'll talk more about that when we talk about antibiotics. So these are enzymes made inside of the cell and released to the environment to break things down. But a bacterial cell can also produce endoenzymes. And these are enzymes that are made inside of the cell. And guess what? They're kept inside. They're kept inside for use inside of the cell. to function there, right? So two different types of enzymes that a bacterial cell will produce. Exoenzymes made inside, released to the environment, and endoenzymes made inside and kept inside for functioning within the cell. And here's a representation. An exoenzyme is made inside of the cell and then released. And it can function there. You see that it's breaking things apart. Maybe it's food products. to get nutrients in. An endoenzyme is made inside of the cell and it stays inside to function there. After all, the cell has its needs within also. Now I was alluding to this prior to this slide, and that is that there are different factors that influence enzyme activity. We know that an enzyme is a protein. And we've learned about proteins being sensitive to temperature. In fact, what happens if the temperature gets too high to a protein? They denature. They denature. That is correct. Remember that beautiful three-dimensional structure that is folded upon itself, right? If it dismembers, then it's no longer active. Enzymes are also sensitive to pH. the substrate concentration, and then we're going to talk about inhibitors, competitive versus non-competitive in a bit. So let's talk about temperature first. The example that I gave you of using an enzyme versus a non-enzyme versus, let me back up. The example that I gave you of a reaction where we use an enzyme versus a reaction where we don't use an enzyme was a difference of... running my reaction in an hour at 56 degrees, which is pretty warm. versus running my reaction overnight at room temperature. And I told you that one took one hour, the other one took overnight. Major difference to get the same product. So temperature is important. High temperature or increase in temperature does increase the rate of reaction. But that window is very narrow. Running your reaction at 37 degrees, for example, in my example with the enzyme that I use. Running the reaction at 37 degrees versus 56 degrees was a matter of 24 hour difference, essentially. Every enzyme has its optimum temperature. That means that it is the best temperature at which it will function the best. It will have its high temperature is the optimum. It will have its minimum and its maximum. But if you notice here, if we deviate just a little bit past its optimum temperature, the enzyme denatures on either side. And then the enzyme is non-functional. Because as soon as you go really past on either side, the enzyme will no longer maintain its three-dimensional shape and it unravels. Once you have a denatured protein, you don't have a functional enzyme. But it's not just affected by an increase in temperature. Just like we have an optimum temperature for an enzyme, we also have an optimum pH. Maybe the range is a little bit wider. But we see that each enzyme will function best at a specific pH. If you go beyond on either side, too low of a pH or too high of a pH, the same thing will happen. Your enzyme will denature and therefore be non-functional. Now, as far as substrate concentration goes, we have a similar thing. Under conditions. of a high substrate concentration under conditions of a high substrate concentration. Like we see here, we see that the substrate concentration has increased. That means we keep adding substrate. If you keep on adding substrate, the enzyme will be saturated. What that means is that there won't be any open active sites because you keep adding substrate. So under conditions of high substrate concentration, the enzyme is saturated and therefore its active site will always be occupied. So even if you continue to add substrate, you won't have any higher activity because it's all being used up. Every enzyme's active site is used up and that's what this represents. That's why it's flat. You see, in the lab, we always think that, or we know that if we just add substrate, we're going to get more product. And that's true. But if you continue to add more substrate, eventually all your enzymes are full. You cannot speed the reaction anymore. Okay. So if I add more substrate, when I'm already saturated, it won't affect the reaction rate. If I add more substrate, Okay. My enzymes are already full. It will not affect the rate of reaction because all the active sites are already in use. Okay. All right. So now what I want to do is I am going to pause this because I am going to, what am I going to do? I'm going to use my whiteboard. Second. Okay. Huh. I forgot what I was going to do. Okay. So we're going to talk about competitive versus non-competitive. That's what I'm going to do. Okay. So, so this is my enzyme. This is my active site. And I see that my active site has a site through which my substrate can bind, right? So when my enzyme, my enzyme is always going to look different. This is not one of my gifts. Binds my substrate. I have now formed an enzyme substrate complex, right? Yeah. And so as a result, I'm going to get my products and maybe my products look like this. It works on the substrate and it releases my products. OK, maybe it's just one product or two, whatever it may be. What if this product was dangerous? What if it was toxic to the cell, to the body? What if we did not want this product? What do we do? Well, we can try to prevent the substrate from binding. If we try to prevent from this happening, Do you agree that if the substrate doesn't bind to the enzyme, that we will not have this product? Do you agree with that? Okay. So that is called competitive inhibition. That is the basis for some drug development. And that's what I did before. I designed drugs. And what I did, not the recreational type back now, not the recreational type, but what I did is I tried to design a drug that would compete with the substrate in an effort of not having the substrate bind to the active site and give me this dangerous, toxic byproduct. How? Well, this is what I did. And this is for tuberculosis. So here's my active site. Here's my enzyme. I know that I have my substrate. So what I did is I designed a drug. It's too pink. I designed a drug that looked, that looked like the substrate. And the hopes is that my drug will bind. will bind the active site before the substrate binds and therefore no product will occur. That's pretty cool, huh? This is referred to as competitive inhibition or what I created was a competitive inhibitor. The goal is that I would design a drug that would get to the active site first. But you know what? That's very difficult to do. Do you know why? First of all, I need to design something that's going to have a very similar structure so that it will bind. OK, well, that's that's OK. I can do that. Looking at the atomic structure of the protein, I can design a drug that will interfere, inhibit or activate the active site. I have no problem with that. I know my amino acids. I know how they arrange. I can design that. There's a few problems. The active site sometimes is not in the location. an easy location to access. The biggest problem is that the affinity of my new product that I make, that I design, it has to want to bind the active site at a higher affinity than the native substrate. You see the substrate has a strong bond to the active site. It has a high affinity. I have to design a drug that's going to want to bind the active site with a stronger intensity, if you will, than the native natural substrate for it. That's hard because oftentimes what happens that, yeah, you know what? My competitive inhibitor will bind here if it doesn't have competition, but usually who wins is the substrate. Most of the time, the substrate will bind before my little competitive inhibitor even had a chance. You see, that's tough. That is competitive inhibition. Using a competitive inhibitor to intersect, right? That's what we're doing. We're intercepting the binding of the substrate, of the natural substrate to the active site. In an effort to stop. products from being produced. Did you guys notice that in my drawings I drew this site? Do you wonder why did she do that? What is that? Is that another active site? Sort of, but we don't call it an active site. It's the non-competitive co-transporter. It is referred to as the allosteric site. Okay. So sometimes the active side, you just cannot compete with the substrate. So I'm like, you know what? I'm not even going to try to compete with the active site, but there is a special site here called an allosteric site. And I think I can do something with that. So this is the way this works. Here is my enzyme with its active site that gets really weird every time I make it. OK, so instead of trying to compete with the active site. What if I design a drug that can bind to the allosteric site? So this is referred to as an allosteric inhibitor or a non-competitive inhibitor. Gotta know how to spell. So what? Upon binding of the allosteric inhibitor to the allosteric site, that will change the confirmation. changes the conformation of the active site. So that, guess what? Whoa, what happened? Oh, darn it. My daughter connected her AirPods. Oh, sorry. Doesn't she know I'm using it? No, she doesn't. Hold on a second, guys. Done. How do I do this? Let's see if I can still connect it. Can you see it? All right. So what I was saying is that upon binding of the allosteric inhibitor or the non-competitive inhibitor to the allosteric site, that changes the confirmation of the active site so that my substrate can no longer bind because it doesn't fit. You see that? So would this be used when we can't compete with the affinity of the native substrate? Exactly. So see what happens, Adrienne? It's like, you know what? I know that I cannot compete with this substrate. I know that I cannot compete with it. So you know what? I'm not even going to try. Instead, I'm going to design something to bind to the allosteric site. So therefore, changing the conformational shape of the active site so that the native substrate cannot. find. Do all enzymes then have like allosteric sites that you can do this? Nope, not all enzymes have allosteric sites. No, not all enzymes have allosteric sites. But this is really interesting because this is how I used it in drug research because that's what I did prior to going into academia. But the cell uses this to stop reactions from continuing to go on and on and on. When you really think about it, how do you stop product from forming? This is one way. Well, it's usually a series of steps, and we're going to discuss that in just a second. But it's pretty cool, isn't it? Pretty darn cool. Okay, so let's continue with the lecture. I like to draw it first before I actually show you the slide on it. because I can kind of do it, discuss it as I draw it step by step. And so that's what this slide is. Let me get you guys back. I can't see it. It always does this. I'm sharing it. Do you see my PowerPoint? Yes. Yeah. Thank you. Because I don't see you. I don't see anything. All right, there we go. All right, that's it. Okay, so I have a question. Yes. Over the allosteric inhibitor that we just went over. Does that one also not give you a product at the end? Just like you kind of went over when you mentioned a competitive inhibitor? Yeah, that is exactly the point of it. If you change the conformational shape of the active site, then the substrate cannot bind. Therefore, no product. That is the whole goal. So these are two different types of enzyme inhibitors. Competitive inhibitor. and a non-competitive inhibitor. The competitive inhibitor, like I mentioned, is going to compete for the active site. You see that? But I already talked to you about some of the limitations and some of the challenges of designing or having a competitive inhibitor is that the affinity for one, right? It needs to have to bind first. When that is really hard to compete with, you may have an option, another option. And that is a non-competitive inhibitor. The non-competitive inhibitor is going to bind to a site other than the active site known as this right here, the allosteric site. The non-competitive inhibitor, also known as allosteric inhibitor, is going to bind the allosteric site. And in doing so, it changes the conformational shape of the active site. This is what the native active site looked like. This is what the active site looks after the non-competitive inhibitor binds the allosteric site. It changes the conformational shape so that the substrate, the native substrate, can no longer bind the active site. And as a result, you get no product. Oh, I'm running out of time, huh? Wow. Okay, I better hurry. I don't know how many slides I have left. This is the last slide actually. Okay. So someone asked, you know, do all the cells have this? And no, but let me show you why this is important. I talked to you about my drug research and what I did in designing these drugs, but the cell does this, the cell utilizes this as such. Here we have enzyme number one. Let's analyze enzyme number one. We see that enzyme number one has an active site and it has an allosteric site. Here is the native substrate for enzyme number one. The substrate is going to bind enzyme number one to give you an enzyme substrate complex, correct? Yes. Yes. Yes. This enzyme substrate complex is going to give you a product. It's going to be releasing this and give you a product called intermediate A. Notice that intermediate A is now going to be the substrate for enzyme number two. Interesting how the product of one enzyme substrate reaction is going to be the substrate for the next reaction. So intermediate A is a substrate for enzyme number two. We have yet another enzyme substrate complex for me. And the reaction or the product rather, the product of this enzyme substrate complex is going to be intermediate B. Intermediate B is now going to be the substrate for enzyme number three. Until we finally get our end product. This is what we wanted all along, but we had to go through all these enzyme substrate reactions to get to our final end product. And the cell continues to deliver this and continues to deliver this. And at some point, the cell says, you know what? I think I'm done. I don't think I need this end product anymore. How do we shut this system down? Interestingly enough, this end product can serve its purpose, but it also serves as an allosteric inhibitor. So you see that this final end product can then go up to the first enzyme, enzyme number one that we saw had an allosteric site. bind to the allosteric site, therefore or thereby causing a conformational change in the active site so that the substrate can no longer bind. And as a result, this whole system shuts down. This is referred to as feedback inhibition. It's fascinating. Review it before you forget it. Review competitive inhibition, review non-competitive inhibition, review just enzyme substrate reactions, because right now it makes sense, but if you don't review it, you will forget it. So would this pathway be more, would this be, would this pathway be found more in like hormonal feedback, positive and negative feedback? So we don't talk about positive feedback so much. And remember, this is a prokaryotic class. You know, in physiology would probably, or even maybe, I don't even know if anatomy would discuss this, but probably more physiology positive feedbacks where like, let's say we're talking about blood clotting, right? Like clotting or causing a scar formation, clotting, that's positive feedback where you're trying to clot something. This is more for stopping enzyme reactions in bacterial cells. Got it. Okay. Thank you. You're welcome. Any other questions? If you have no questions, this concludes this lecture.

In fact, it's a phospholipid bilayer. That means that there are two layers of phospholipids where they're hydrophilic or polar heads. are facing outwards in the aqueous environment and their hydrophobic tails or water-fearing tails are facing each other, right?

The plasma membrane is so important. It functions in, basically, it is selectively permeable. It allows for things to go in and out of the cell. It senses environmental signal. And as I mentioned, It's where energy transformation in a prokaryote occurs.

This is a prokaryotic class, so we're going to be focusing on that. So transport, movement of chemicals across the cell membrane. So as I mentioned, it's selectively permeable. So it allows things to go in and out of the cell. How?

Through what mechanisms? Well, passive transport, meaning that Things can go in and out of the plasma membrane without requiring energy in the form of ATP. So that's passive transport.

Within passive transport, there are three main types of passive transport. That would be diffusion, osmosis, which refers to the diffusion of water, and facilitated diffusion. Now, facilitated diffusion is interesting because it requires a carrier protein that's embedded within the membrane.

But it's still, even though it requires a carrier, it does not require energy in the form of ATP. And the reason that it does not require energy in the form of ATP is because the diffusion or the transport of substances occurs from an area of higher concentration. to lower concentration. So it goes down the concentration gradient. So it kind of makes sense.

You're going from high to low and it does not require energy. The other main type of transport is called active transport. And it's called active transport because it actively requires energy. Okay.

Mostly because you can go against the concentration gradient. meaning that you can be in an area, you can go the opposite. You don't necessarily go from high to low.

You can go from low concentration to high concentration. Well, to do that, it's not so natural, right? And so therefore, it requires energy. Active transport, there's different types of active transport, group translocation and bulk transport, such as we see with endocytosis, exocytosis, and penocytosis.

So... We're going to go over a few of these, like simple diffusion is basically just that, going from high concentration to low. So here we see that if there is like a salt granule, if we have a salt crystal, rather a salt crystal, that crystal will begin to dissolve.

And the molecules will go from this high concentration area to a lower concentration. Facilitated diffusion, as I mentioned. requires a carrier protein.

Now, what is a carrier protein? I'm so confused. Look at these integral proteins that we see embedded in the membrane. It would require something like that to allow for the passage of molecules, okay?

But it's going from high concentration to low concentration. I don't really get into a whole lot of the active transport methods and the sodium potassium pump. We don't really go into that in a prokaryotic class like this, okay?

That's going to be more molecular or general biology or physiology. Now, the last type of movement across the cell membrane that we see is active transport. And this does require a carrier protein like facilitated diffusion. But again, now we can go against the concentration gradient. And if we're going against the concentration gradient, meaning that we're not going from high to low, we can go from low to high.

You require. energy in the form of ATP. There is not an animation on Blackboard, that would be Canvas, and there isn't one.

But your textbook has these animations if you wish to see them. So let's talk a little bit about simple diffusion and osmosis, okay? So when we're talking about diffusion and osmosis, we're talking about the movement of water, okay? We're talking about the movement of water inside and outside of the cell.

Now, cells like to be in an isotonic environment, isotonic solution, meaning that the concentration of solute inside... and outside of the cell is the same. Now the solute would refer to the little salt crystals, if you will, okay, the molecules.

It likes to have, it likes to be in an environment that is, that is in, in an isotonic type of environment and solution, okay. So when a cell is placed in an isotonic environment, meaning that the solute concentration is in the same on the inside of the cell and the outside, water can go in and out and there is no problem. If you place a cell in a hypotonic solution, now hypo means low.

So that means that the beaker, if you consider this square here in the blue and the little dots, if you consider this being a beaker of solution and it has an It's a hypotonic solution. Hypo means low. So that means that there is a low concentration of solute. That means that there is less solute in this beaker than inside of the cell. So you see that the cell has a higher concentration of these molecules, right?

Well, the cell doesn't like that. It wants to be in an isotonic environment. So it's going to say, you know what, I'm going to dilute myself so I can be in a sense of equilibrium, if you will.

So it allows water to go inside of the cell in order to be in the same concentration as outside. The problem is that too much water would need to go into the cell so that it would be in a state of equilibrium that the cell will burst. That's cell lysis.

The cell will lyse because too much water would try to rush in and dilute the inside of the cell. Now, if that cell is placed in a hypertonic solution, now this is a different beaker. Okay, let's consider this blue being the beaker, the water.

In a hypertonic means that there is a high concentration of solute. Notice that there are a lot of little molecules, right, inside of this beaker. And so as a result, the cell's like, okay, no problem.

I'm just going to dilute my environment so that I can be in a state of equilibrium. So the cell is going to release a lot of water to its surrounding environment to try to dilute its surrounding environment. But do you think a small little cell is going to be successful at that? No. Essentially, it's going to release all its water and it shrivels up.

It shrivels up. And that's referred to as plasmolysis. These different states, you need to understand these three different states.

Okay. Now in facilitated diffusion, we talked, I mentioned to you that it requires a carrier protein, but it doesn't require energy. So these proteins are embedded in the plasma membrane. And what they do is they will transport substances from an area of high concentration to low concentration. And because it's going down the concentration group, it does not require.

require energy in the form of ATP. Okay, now let me go ahead and get the next one. Okay, so now we're going to start lecture four. This is a large lecture and I've divided into lecture four and five.

And on Thursday, we're going to discuss cellular respiration. So you don't want to miss that. But today we're going to talk about cellular respiration.

So let's get started. we're going to talk about microbial metabolism, or at least get started with the idea of microbial metabolism. And this is a great slide. And we're going to revisit this slide in a few.

Okay. So let's start talking about metabolism and what is metabolism. It is the sum of chemical reactions in a cell.

Okay. And oftentimes when we talk about metabolism, we talk about being metabolically active. And what does that mean? right? What does it mean to be metabolically active?

It means that a cell is able to reproduce. So that is really what metabolism is all about. The ultimate function of metabolism is to reproduce the organism. So you will hear me talk about a metabolically active cell. A metabolically active cell is a cell that is alive and it's reproducing.

It's going through all these different biochemical reactions. Why do we study metabolism? Well, it occurs in all living organisms. And we also use it in microbiology to identify organisms. You see, that is one of the things that you're going to be doing in lab.

you're going to be performing various biochemical tests on your unknown. And based on those biochemical tests, you will eventually determine the identity of your unknown. Because not every organism is the same. And the only way you can determine if your microorganism can metabolize glucose versus fructose versus sucrose is to actually test it. So not every organism can metabolize or utilize fructose, for example, or lactose or sucrose.

So that those are some biochemical tests that you will run on your organism. That's why metabolism is important. It tells us something about our microorganism.

And based on the collective data, we can determine what our unknown is. OK, so it's going to be a lot of testing that you're going to do. All right. So there are two major classes of metabolic reactions, two classes of chemical reactions. They are both enzyme regulated chemical reactions.

We have catabolism and we have anabolism. OK, so let's talk a little bit about these. Catabolism is the process of going from a large molecule and breaking it down to smaller molecules. or going from a large molecule and breaking it down to its monomeric units. And in the process of breaking down this large macro molecule, if you will, to smaller monomeric units, you release energy.

And that energy that is released is in the form of ATP. Well, wait a minute. That kind of sounds familiar.

That type of reaction to me sounds familiar. What does that remind you of? What does that kind of sound like to you?

Dehydration synthesis. I don't know about dehydration synthesis. What is dehydration synthesis? Is it the breaking down of molecules?

A-Lyn says no. What is it? No. It is to build or to synthesize molecules, right? But to me, catabolism does sound familiar.

I think I've heard something like that before. What does it remind you of? Cellular respiration. Cellular respiration.

Okay. Yeah, eventually, but we're not there yet. So let's back up. What, Heidi?

Catalyst. A catalyst? Well, a catalyst speeds up reactions, and we haven't even talked about that yet either. Who goes metabolism?

Well, that's like cellular respiration. That's what George said. Hydration synthesis reaction?

Dehydration synthesis is to build something, is to make these large macromolecules. Oh my word. Hydration synthesis?

Say that again? Hydrolysis? Hydration synthesis?

Hydration synthesis, you mean hydrolysis. Hydrolysis. It's not synthesis.

Remember what the word synthesis means? Synthesis means to make. It's hydrolysis. These are essentially hydrolytic reactions.

Catabolism releases energy. These are hydrolytic reactions that use water. to break chemical bonds. And in the process of breaking down these large macromolecules into smaller monomeric molecules, you release energy in the form of ATP. This reaction is also referred to being as exergonic.

It releases energy. Now that energy in the form of ATP is going to be stored. It's going to be stored so that it can be used for anabolic reactions, you see.

So let's talk about anabolism. or anabolic reactions. So in an anabolic reaction, we are taking these smaller molecules or small monomeric units and we're putting them together to build or form a larger macrame. But because we're building, when we're building something, does that not require energy?

Yeah. Yeah. So we need energy in the form of ATP. What does this type of reaction remind you of? Dehydration synthesis.

There you go. That's your dehydration synthesis. Yeah.

So what's interesting is that the catabolic reactions are going to provide us with the ATP that we need for the anabolic reactions, you see. And these anabolic reactions are endergonic. They require energy and that energy is in the form of ATP.

So let's get, let's take a closer look at this relationship between catabolism and anabolism. You see, it's a beautiful story. As things are being broken down because they're not needed anymore, those monomeric units that were released.

These can then be used and synthesized to form something that is needed at that time. So we can start anywhere we want. We can start here at the top. We have small, simple, monomeric units such as glucose, right?

Glucose is a monosaccharide. Amino acids, glycerol, fatty acids. These are all monomeric units. And guess what?

They come together in an anabolic reaction, transfer energy from ATP, release heat, and boom. We have synthesized our complex molecules, or we have synthesized our macromolecules. When the cell no longer needs these large proteins, or these lipids or starches.

Why keep it? We can reuse and recycle, so to speak, right? Reuse and recycle. So through catabolic reactions, we're going to transfer that energy that was stored in these large molecules, break them down, release ATP, and now we're back to our monomeric units.

Isn't that just beautiful? The way this all works. I find it fascinating. As things are broken down because they're not needed anymore, other new things are being synthesized because that's what the cell needs.

It's just amazing to me. I think it's amazing. Here's another view. We have our large carbohydrates.

lipids, they're going to be broken down into their basic monomeric units and to build what we need and go on and do what they do. So what we say is that catabolic reactions, catabolic reactions. Now, you know what, now that I'm thinking about it.

I used to get confused, like, well, which one's anabolic? Which one's catabolic? Which one's breaking down?

Which one's building? It's all so confusing. There are so many terms, right?

And I think about a cat breaking things down. And so then I remember, oh, yeah, okay, catabolic. Cats break things down. The catabolic is breaking down on these large molecules.

If it doesn't help you, don't use it, okay? So catabolic reactions essentially provide the... building blocks for anabolic reactions. Catabolic reactions provide the building blocks for anabolic reactions. And guess what?

This is possible through ATP. It is possible through ATP. So ATP stores energy derived from catabolic reactions to later release it. to drive anabolic reactions and perform cellular work.

It's just beautiful. So let's talk a little bit about ATP. Let's talk about the synthesis of ATP.

Okay, so how do we make or synthesize ATP? The synthesis of ATP is a phosphorylation reaction. We see that ATP has these phosphates with these high energy bonds between them.

Okay. So the synthesis of ATP, how we make this ATP is we start with ADP, adenosine diphosphate. You see a phosphate group is added to ADP. This refers to inorganic phosphate, the little I.

means inorganic. So we're going to add an inorganic phosphate to ADP to yield ATP. So anabolic reactions are coupled to ATP breakdown and catabolic reactions are coupled to ATP.

synthesis. So it's kind of the opposite, right? Does that make sense? Because when we break things down, we are releasing ATP.

But when we are synthesizing things, we are utilizing ATP. Anabolic reactions is coupled to ATP. breakdown.

Because when you use ATP, you are basically removing or breaking one of these high energy bonds to utilize it. Okay. This concept of coupled reactions, like we see here, is important. Some molecules are broken down while other molecules are synthesized. Yeah.

And this balance is important because it's what maintains the cell. Very important. Maintains the cell. Okay. So we're going to be talking about enzymes next.

Enzymes are our biological catalyst. We hear that a lot. They speed chemical reactions by lowering the energy of activation, how much energy is needed to get the reaction started. But what really are enzymes?

Do you know what they are? What are they? What are they made out of?

What are they? Amino acids, proteins. Yeah.

They're essentially proteins. They're amino acids that are bonded together via dehydration synthesis. They're proteins. When enzymes are used, the enzyme is not permanently altered in the reaction. They can be reused, so to speak.

Enzyme promotes a reaction by serving as a site for the substrate. And I'll talk to you a little bit more about that. So it's a little bit, it's a little bit hard to put in words. So let's see how these enzymes function.

How is it that they work? Here we have two reactants. We have A and we have B, okay? We see that A in the presence of an enzyme.

Hold on. Sorry, it was the opposite. We have A without an enzyme and B with an enzyme, okay? So how much energy does it take in order to get to our final product?

What was that? Which thing A requires more energy than B. That is correct. A is going to require more energy.

So eventually you're going to get the product, but it's going to require more energy. And sometimes that equates with time as well. All right. So in the lab, when we would do cloning and we needed to insert something in a bacteria, for example, we work with enzymes and we speed up the reactions.

Using an enzyme versus not using an enzyme is a matter of... Having your reaction complete within an hour, if you incubate it at 56 degrees, let's say, versus letting the reaction run overnight at room temperature to get the same result. So using an enzyme speeds things up.

But enzymes are proteins and they're finicky. They require specific temperature. They require specific pH. Being outside of the optimum is not going to make the, or allow the protein to function to the best of its potential. So using enzymes speeds up the reaction time.

Hmm. I wonder if that's why it's important that we're careful or why it's so dangerous to have such a high fever. Well, what does that even mean? All right. Remember that enzymes are proteins and we know that proteins are sensitive to temperature, pH and chemicals, right?

Do I have a slide on that? Let me see if I can look ahead. Oh, I think I already talked about, no, I haven't. Okay.

It's up ahead. So I'll just wait on that. What I was going to say, as far as temperature is concerned, I can see in the bottom a little bit what I have. Now let's take a look at our enzyme terminology, or let's take a look at the anatomy of an enzyme and see how they work, how they function, and all the little body parts of an enzyme.

First of all, a substrate is a molecule that the enzyme is going to act on. So the substrate is what the enzyme is going to work on, what's going to activate or break down, whatever it's going to do to it. So here I have an enzyme, but here I have an enzyme too. What is the difference between these two? This is called an apoenzyme.

An apoenzyme is an inactive enzyme. It's inactive unless it binds a cofactor or coenzyme. Okay. So it requires a cofactor.

These cofactors or coenzymes can be for inorganic ions like iron, magnesium, zinc. It can actually even be vitamins. But without them, we don't have an active enzyme.

Well, why is that? Well, if we take a look at this area here that is created when this coenzyme or cofactor binds to the apal enzyme, we see that it creates an area called the active site that fits the substrate. or what it's going to act on. You see that? If we do not have this coenzyme or cofactor, will this substrate fit in here?

No, it won't. No, it won't. So if this enzyme does not have that coenzyme or cofactor, it is an apoenzyme or inactive enzyme. Once it has its cofactor or coenzyme bound to this site, now we have a holoenzyme.

A holoenzyme is your active enzyme. And it has the active site ready for the substrate to bind to and act on. Yeah?

So binding of an apoenzyme to its cofactor. forms an active enzyme called a holoenzyme. Now, the substrate is what's going to activate this protein or this enzyme, if you will. Yeah. I threw a few terms at you, a cofactor and coenzyme.

A cofactor is going to be a molecule that's needed for enzyme activity. because it helps create that active site. These are usually non-protein components.

So it's not part of the enzyme that the enzyme, remember it's a protein that binds that it wraps around itself and it has this tertiary structure. Remember that? Okay.

The cofactor may be organic or it may be inorganic. They can be metal ions. In fact, a coenzyme is going to be an organic molecule that acts as a carrier. of molecules or electrons such as NAD.

So these are just different types of cofactors that can fit in that area. Okay. But you know what? Co-enzymes are important.

And I did mention to you that some of these co-enzymes are vitamins. Yeah. So is that why it's important to take our vitamins?

Is that why our parents always said, take your vitamins? Maybe. But I want to actually point a particular important vitamin to you. And that is this one, folic acid.

Take a look at folic acid. And what the function of folic acid, what it says here that the function of folic acid is, is that it is a coenzyme used in the synthesis of purines and pyrimidines. Whoa, wait a minute. Now we have a lot of terminology here, right?

So we know that coenzymes are important because they bind to the apal enzyme to make a holo enzyme. And we know that these holo enzymes are important because they lower the activation energy and they allow reactions to take place. They speed up reactions to occur, correct? Now, it also says that this coenzyme called folic acid is used in the synthesis of purines and pyrimidines.

And we know that synthesis means to make, right? Building. So it's used in the synthesis. What are purines and pyrimidines?

I forgot. Where have we heard that term? DNA, pairs of DNA, nucleic acids, right?

Because it's in DNA and it's in RNA too. Yeah. So those are components of nucleotides guys. Purines and pyrimidines are components of nucleus.

Okay. Well, so what nucleotides are the building blocks for nucleic acids. All right. Well, so what folic acid is important. especially for pregnant women.

It is so important for pregnant women to take folic acid, one milligram at minimum 800 micrograms, which you can buy over the counter. It is important for neural tube development. Very important.

Folic acid supplementation can prevent neural tube defects, such as spina bifida. In fact, if you're a woman and you're even thinking about having a baby and getting pregnant, you should really start taking folic acid or prenatal vitamins, ideally six months before you even try to get pregnant. Folic acid is a B vitamin that's used in our bodies to make new cells.

So you know what? These little coenzymes, there are little attachments right here. They're actually very important.

So that's just to give you an idea. of the importance of these coenzymes, okay, in their function. All right, so let's talk about naming enzymes.

An enzyme, you usually know that it's an enzyme because it ends in ace. Hyaluronidase, hexokinase, streptokinase. So usually it's safe to say that if the word name in ace, it probably is an enzyme, okay? Now, the names are sometimes based on the type of chemical reaction that they produce or the substrate or product. So these are some examples of different types of chemical reactions that are catalyzed.

You don't need to know these. I'm not going to examine you on them. These are just some examples of different enzymes. And you'll notice that they end in A's.

So generally speaking, an enzyme will end in A's. That's how you know it's an enzyme. enzyme.

Okay. All right. So let's take a look at an enzyme in action. So here we assume that this is a holoenzyme.

It already has its coenzyme attached. It's ready to work. And what we see is that this enzyme has its active site and the active site is shaped specifically for a substrate.

Now, some people use the lock key fashion. It kind of is like that. Not everything can fit in.

But it's not a perfect fit like a key and lock. The active site kind of molds into that key, if you will. But it's not promiscuous in the sense that it's going to allow anything in either. So, for example, here we have two substrates. And we can see that one of them has the shape that appears to fit in, where the other one does not, right?

So, not just anything. can bind to this active site and give you product. It just doesn't happen. So we see that this green one does not fit. But the other one does.

When the substrate binds the enzyme, we create something referred to as an enzyme substrate complex. Okay? An enzyme substrate complex.

And then the enzyme will work on this complex and give you or release your products. Now what we see is that the active site is open. Do you see how it's open? Guess what?

It can be reused. another substrate can come in, bind to this newly opened active site, and give you yet another product. So these enzymes are reusable in a sense. As soon as the active site is clear, another substrate can come in.

Questions? So let's talk about the location and regularity of enzyme action. In microorganisms specifically, we talk a lot about exoenzymes and endoenzymes. Okay. And exoenzymes are going to be enzymes that are made inside of the cell and exported to the outside.

Now we can test for this and we use special media in the lab to determine and detect the presence of specific exoenzymes that are released by the bacterial cell. So again, exoenzymes are made inside of the cell and released to the outside environment. Why? Well, for different purposes.

Sometimes it's to break down things, maybe break down nutrients. Here we see some examples. Cellulase, amylase to break down starch. Penicillinase to break down penicillin. Is that what that means?

Yes. There are some bacteria that produce an enzyme called penicillinase, also known as beta-lactamase, but you'll learn that later. And it breaks the beta-lactam ring in the penicillin. so that the penicillin or the antibiotic is no longer active. So we'll talk more about that when we talk about antibiotics.

So these are enzymes made inside of the cell and released to the environment to break things down. But a bacterial cell can also produce endoenzymes. And these are enzymes that are made inside of the cell.

And guess what? They're kept inside. They're kept inside for use inside of the cell.

to function there, right? So two different types of enzymes that a bacterial cell will produce. Exoenzymes made inside, released to the environment, and endoenzymes made inside and kept inside for functioning within the cell. And here's a representation. An exoenzyme is made inside of the cell and then released.

And it can function there. You see that it's breaking things apart. Maybe it's food products. to get nutrients in.

An endoenzyme is made inside of the cell and it stays inside to function there. After all, the cell has its needs within also. Now I was alluding to this prior to this slide, and that is that there are different factors that influence enzyme activity. We know that an enzyme is a protein. And we've learned about proteins being sensitive to temperature.

In fact, what happens if the temperature gets too high to a protein? They denature. They denature.

That is correct. Remember that beautiful three-dimensional structure that is folded upon itself, right? If it dismembers, then it's no longer active. Enzymes are also sensitive to pH. the substrate concentration, and then we're going to talk about inhibitors, competitive versus non-competitive in a bit. So let's talk about temperature first.

The example that I gave you of using an enzyme versus a non-enzyme versus, let me back up. The example that I gave you of a reaction where we use an enzyme versus a reaction where we don't use an enzyme was a difference of... running my reaction in an hour at 56 degrees, which is pretty warm. versus running my reaction overnight at room temperature.

And I told you that one took one hour, the other one took overnight. Major difference to get the same product. So temperature is important.

High temperature or increase in temperature does increase the rate of reaction. But that window is very narrow. Running your reaction at 37 degrees, for example, in my example with the enzyme that I use. Running the reaction at 37 degrees versus 56 degrees was a matter of 24 hour difference, essentially.

Every enzyme has its optimum temperature. That means that it is the best temperature at which it will function the best. It will have its high temperature is the optimum. It will have its minimum and its maximum.

But if you notice here, if we deviate just a little bit past its optimum temperature, the enzyme denatures on either side. And then the enzyme is non-functional. Because as soon as you go really past on either side, the enzyme will no longer maintain its three-dimensional shape and it unravels. Once you have a denatured protein, you don't have a functional enzyme.

But it's not just affected by an increase in temperature. Just like we have an optimum temperature for an enzyme, we also have an optimum pH. Maybe the range is a little bit wider. But we see that each enzyme will function best at a specific pH. If you go beyond on either side, too low of a pH or too high of a pH, the same thing will happen. Your enzyme will denature and therefore be non-functional.

Now, as far as substrate concentration goes, we have a similar thing. Under conditions. of a high substrate concentration under conditions of a high substrate concentration.

Like we see here, we see that the substrate concentration has increased. That means we keep adding substrate. If you keep on adding substrate, the enzyme will be saturated. What that means is that there won't be any open active sites because you keep adding substrate.

So under conditions of high substrate concentration, the enzyme is saturated and therefore its active site will always be occupied. So even if you continue to add substrate, you won't have any higher activity because it's all being used up. Every enzyme's active site is used up and that's what this represents.

That's why it's flat. You see, in the lab, we always think that, or we know that if we just add substrate, we're going to get more product. And that's true.

But if you continue to add more substrate, eventually all your enzymes are full. You cannot speed the reaction anymore. Okay. So if I add more substrate, when I'm already saturated, it won't affect the reaction rate. If I add more substrate, Okay.

My enzymes are already full. It will not affect the rate of reaction because all the active sites are already in use. Okay. All right.

So now what I want to do is I am going to pause this because I am going to, what am I going to do? I'm going to use my whiteboard. Second. Okay. Huh.

I forgot what I was going to do. Okay. So we're going to talk about competitive versus non-competitive.

That's what I'm going to do. Okay. So, so this is my enzyme. This is my active site.

And I see that my active site has a site through which my substrate can bind, right? So when my enzyme, my enzyme is always going to look different. This is not one of my gifts.

Binds my substrate. I have now formed an enzyme substrate complex, right? Yeah.

And so as a result, I'm going to get my products and maybe my products look like this. It works on the substrate and it releases my products. OK, maybe it's just one product or two, whatever it may be. What if this product was dangerous?

What if it was toxic to the cell, to the body? What if we did not want this product? What do we do? Well, we can try to prevent the substrate from binding.

If we try to prevent from this happening, Do you agree that if the substrate doesn't bind to the enzyme, that we will not have this product? Do you agree with that? Okay.

So that is called competitive inhibition. That is the basis for some drug development. And that's what I did before.

I designed drugs. And what I did, not the recreational type back now, not the recreational type, but what I did is I tried to design a drug that would compete with the substrate in an effort of not having the substrate bind to the active site and give me this dangerous, toxic byproduct. How?

Well, this is what I did. And this is for tuberculosis. So here's my active site. Here's my enzyme.

I know that I have my substrate. So what I did is I designed a drug. It's too pink. I designed a drug that looked, that looked like the substrate.

And the hopes is that my drug will bind. will bind the active site before the substrate binds and therefore no product will occur. That's pretty cool, huh?

This is referred to as competitive inhibition or what I created was a competitive inhibitor. The goal is that I would design a drug that would get to the active site first. But you know what?

That's very difficult to do. Do you know why? First of all, I need to design something that's going to have a very similar structure so that it will bind.

OK, well, that's that's OK. I can do that. Looking at the atomic structure of the protein, I can design a drug that will interfere, inhibit or activate the active site. I have no problem with that. I know my amino acids.

I know how they arrange. I can design that. There's a few problems. The active site sometimes is not in the location.

an easy location to access. The biggest problem is that the affinity of my new product that I make, that I design, it has to want to bind the active site at a higher affinity than the native substrate. You see the substrate has a strong bond to the active site.

It has a high affinity. I have to design a drug that's going to want to bind the active site with a stronger intensity, if you will, than the native natural substrate for it. That's hard because oftentimes what happens that, yeah, you know what?

My competitive inhibitor will bind here if it doesn't have competition, but usually who wins is the substrate. Most of the time, the substrate will bind before my little competitive inhibitor even had a chance. You see, that's tough. That is competitive inhibition.

Using a competitive inhibitor to intersect, right? That's what we're doing. We're intercepting the binding of the substrate, of the natural substrate to the active site.

In an effort to stop. products from being produced. Did you guys notice that in my drawings I drew this site?

Do you wonder why did she do that? What is that? Is that another active site?

Sort of, but we don't call it an active site. It's the non-competitive co-transporter. It is referred to as the allosteric site.

Okay. So sometimes the active side, you just cannot compete with the substrate. So I'm like, you know what?

I'm not even going to try to compete with the active site, but there is a special site here called an allosteric site. And I think I can do something with that. So this is the way this works.

Here is my enzyme with its active site that gets really weird every time I make it. OK, so instead of trying to compete with the active site. What if I design a drug that can bind to the allosteric site? So this is referred to as an allosteric inhibitor or a non-competitive inhibitor.

Gotta know how to spell. So what? Upon binding of the allosteric inhibitor to the allosteric site, that will change the confirmation.

changes the conformation of the active site. So that, guess what? Whoa, what happened? Oh, darn it.

My daughter connected her AirPods. Oh, sorry. Doesn't she know I'm using it?

No, she doesn't. Hold on a second, guys. Done. How do I do this? Let's see if I can still connect it.

Can you see it? All right. So what I was saying is that upon binding of the allosteric inhibitor or the non-competitive inhibitor to the allosteric site, that changes the confirmation of the active site so that my substrate can no longer bind because it doesn't fit. You see that? So would this be used when we can't compete with the affinity of the native substrate?

Exactly. So see what happens, Adrienne? It's like, you know what?

I know that I cannot compete with this substrate. I know that I cannot compete with it. So you know what? I'm not even going to try.

Instead, I'm going to design something to bind to the allosteric site. So therefore, changing the conformational shape of the active site so that the native substrate cannot. find. Do all enzymes then have like allosteric sites that you can do this? Nope, not all enzymes have allosteric sites.

No, not all enzymes have allosteric sites. But this is really interesting because this is how I used it in drug research because that's what I did prior to going into academia. But the cell uses this to stop reactions from continuing to go on and on and on.

When you really think about it, how do you stop product from forming? This is one way. Well, it's usually a series of steps, and we're going to discuss that in just a second.

But it's pretty cool, isn't it? Pretty darn cool. Okay, so let's continue with the lecture. I like to draw it first before I actually show you the slide on it.

because I can kind of do it, discuss it as I draw it step by step. And so that's what this slide is. Let me get you guys back.

I can't see it. It always does this. I'm sharing it.

Do you see my PowerPoint? Yes. Yeah. Thank you.

Because I don't see you. I don't see anything. All right, there we go.

All right, that's it. Okay, so I have a question. Yes.

Over the allosteric inhibitor that we just went over. Does that one also not give you a product at the end? Just like you kind of went over when you mentioned a competitive inhibitor? Yeah, that is exactly the point of it. If you change the conformational shape of the active site, then the substrate cannot bind.

Therefore, no product. That is the whole goal. So these are two different types of enzyme inhibitors.

Competitive inhibitor. and a non-competitive inhibitor. The competitive inhibitor, like I mentioned, is going to compete for the active site. You see that? But I already talked to you about some of the limitations and some of the challenges of designing or having a competitive inhibitor is that the affinity for one, right?

It needs to have to bind first. When that is really hard to compete with, you may have an option, another option. And that is a non-competitive inhibitor.

The non-competitive inhibitor is going to bind to a site other than the active site known as this right here, the allosteric site. The non-competitive inhibitor, also known as allosteric inhibitor, is going to bind the allosteric site. And in doing so, it changes the conformational shape of the active site. This is what the native active site looked like.

This is what the active site looks after the non-competitive inhibitor binds the allosteric site. It changes the conformational shape so that the substrate, the native substrate, can no longer bind the active site. And as a result, you get no product. Oh, I'm running out of time, huh?

Wow. Okay, I better hurry. I don't know how many slides I have left.

This is the last slide actually. Okay. So someone asked, you know, do all the cells have this?

And no, but let me show you why this is important. I talked to you about my drug research and what I did in designing these drugs, but the cell does this, the cell utilizes this as such. Here we have enzyme number one. Let's analyze enzyme number one.

We see that enzyme number one has an active site and it has an allosteric site. Here is the native substrate for enzyme number one. The substrate is going to bind enzyme number one to give you an enzyme substrate complex, correct? Yes. Yes.

Yes. This enzyme substrate complex is going to give you a product. It's going to be releasing this and give you a product called intermediate A.

Notice that intermediate A is now going to be the substrate for enzyme number two. Interesting how the product of one enzyme substrate reaction is going to be the substrate for the next reaction. So intermediate A is a substrate for enzyme number two.

We have yet another enzyme substrate complex for me. And the reaction or the product rather, the product of this enzyme substrate complex is going to be intermediate B. Intermediate B is now going to be the substrate for enzyme number three. Until we finally get our end product. This is what we wanted all along, but we had to go through all these enzyme substrate reactions to get to our final end product.

And the cell continues to deliver this and continues to deliver this. And at some point, the cell says, you know what? I think I'm done. I don't think I need this end product anymore.

How do we shut this system down? Interestingly enough, this end product can serve its purpose, but it also serves as an allosteric inhibitor. So you see that this final end product can then go up to the first enzyme, enzyme number one that we saw had an allosteric site. bind to the allosteric site, therefore or thereby causing a conformational change in the active site so that the substrate can no longer bind. And as a result, this whole system shuts down.

This is referred to as feedback inhibition. It's fascinating. Review it before you forget it.

Review competitive inhibition, review non-competitive inhibition, review just enzyme substrate reactions, because right now it makes sense, but if you don't review it, you will forget it. So would this pathway be more, would this be, would this pathway be found more in like hormonal feedback, positive and negative feedback? So we don't talk about positive feedback so much.

And remember, this is a prokaryotic class. You know, in physiology would probably, or even maybe, I don't even know if anatomy would discuss this, but probably more physiology positive feedbacks where like, let's say we're talking about blood clotting, right? Like clotting or causing a scar formation, clotting, that's positive feedback where you're trying to clot something. This is more for stopping enzyme reactions in bacterial cells.

Got it. Okay. Thank you. You're welcome.

Any other questions? If you have no questions, this concludes this lecture.

Transcript for:lecture 4 youtube: microbial metabolism part 1

Transcript for:
lecture 4 youtube: microbial metabolism part 1