1 00:00:15 --> 00:00:20 OK. So we're going to continue with the discussion about biochemistry, 2 00:00:20 --> 00:00:25 and specifically focus on enzymes today. Professor Sive introduced 3 00:00:25 --> 00:00:30 those to you briefly in her last lecture. 4 00:00:30 --> 00:00:34 I'm actually covering for her today. This is one of her lectures but she 5 00:00:34 --> 00:00:39 has given me her material, so hopefully it will go fine. 6 00:00:39 --> 00:00:44 She wanted me to remind you a little bit about energetics, 7 00:00:44 --> 00:00:49 specifically that a negative Delta G in a reaction implies that the 8 00:00:49 --> 00:00:54 reaction can occur spontaneously, that is if the products have lower 9 00:00:54 --> 00:00:59 energy than the reactants. And so given enough time this will 10 00:00:59 --> 00:01:03 happen in that direction. But importantly for many reactions 11 00:01:03 --> 00:01:07 an activation energy might be necessary to get that reaction 12 00:01:07 --> 00:01:10 started. And if the activation energy can be sufficiently high then 13 00:01:10 --> 00:01:14 in a reasonable amount of time the reaction will actually never proceed 14 00:01:14 --> 00:01:17 forward. And it's the job of enzymes actually to deal with that 15 00:01:17 --> 00:01:21 problem, as we'll discuss. So this is the third of three 16 00:01:21 --> 00:01:24 lectures in biochemistry. We're going to transition next week 17 00:01:24 --> 00:01:28 to genetics, and I'll be actually teaching that section on genetics. 18 00:01:28 --> 00:01:33 Before we get into today's lecture proper, Professor Sive wanted to 19 00:01:33 --> 00:01:38 quiz your knowledge from previous lectures. So firstly in this 20 00:01:38 --> 00:01:43 structure, this polypeptide structure, the question is was the 21 00:01:43 --> 00:01:48 valine amino acid, the Val, added last or first in this 22 00:01:48 --> 00:01:53 reaction? Specifically true or false, was it the last amino acid 23 00:01:53 --> 00:01:59 added? False. Very good. It was the first. 24 00:01:59 --> 00:02:04 Because amino acids are added in polypeptides from the amino end to 25 00:02:04 --> 00:02:09 the carboxy end. And in this case Val is next to the 26 00:02:09 --> 00:02:14 amino ends. So the second question is in this reaction shown here, 27 00:02:14 --> 00:02:19 the generation of sucrose from glucose and fructose, 28 00:02:19 --> 00:02:24 which has a positive Delta G, does this release energy or consume 29 00:02:24 --> 00:02:30 energy? Specifically true or false, does this? False. Good. 30 00:02:30 --> 00:02:34 So the Delta G is positive, which means to get this reaction to 31 00:02:34 --> 00:02:39 go you need to add energy. And there are various ways of 32 00:02:39 --> 00:02:43 adding energy. In a synthetic case a chemistry 33 00:02:43 --> 00:02:48 example you might add heat or you might link this to another reaction 34 00:02:48 --> 00:02:53 that actually produced energy. And finally is activation energy 35 00:02:53 --> 00:02:57 required only to initiate reactions with a positive Delta G? 36 00:02:57 --> 00:03:02 No. Very good. So, as we just talked about, 37 00:03:02 --> 00:03:06 activation energies can be important both for reactions that consume 38 00:03:06 --> 00:03:11 energy and give off energy. And so even in situations where 39 00:03:11 --> 00:03:15 reactions give off energy, it may be necessary for an enzyme to 40 00:03:15 --> 00:03:19 deal with this activation energy problem in order to speed up the 41 00:03:19 --> 00:03:24 rate of the reaction. OK. So Professor Sive gave you 42 00:03:24 --> 00:03:28 examples previously comparing the cell to a factory. 43 00:03:28 --> 00:03:33 And I think it's a very apt analogy. And it's particularly apt with 44 00:03:33 --> 00:03:37 respect to the subject of today's lecture which are enzymes. 45 00:03:37 --> 00:03:41 There's a great deal of interest these days in the MIT community and 46 00:03:41 --> 00:03:45 around the research community around the world in nano technology, 47 00:03:45 --> 00:03:50 building tiny little machines that can do work. Well, 48 00:03:50 --> 00:03:54 I would say that proteins are the consummate example of nano 49 00:03:54 --> 00:03:58 technology. These are small entities, five to ten nanometers in 50 00:03:58 --> 00:04:02 diameter, which carry out very specific and very powerful 51 00:04:02 --> 00:04:07 reactions. So proteins are already nano 52 00:04:07 --> 00:04:13 particles to the nth degrees. And the cell is itself remarkable 53 00:04:13 --> 00:04:18 in this respect because the cell, which is only ten to thirty microns 54 00:04:18 --> 00:04:24 in diameter not only has all these nano particles doing all this work, 55 00:04:24 --> 00:04:30 but it also contains the blueprint information for building those. 56 00:04:30 --> 00:04:32 It has the synthetic capability for making the raw materials that they 57 00:04:32 --> 00:04:35 act on. And it has the micro machining capability to build the 58 00:04:35 --> 00:04:38 nano machines. And this all takes place in a very, 59 00:04:38 --> 00:04:41 very tiny space. So the cell is an unbelievable example of engineering 60 00:04:41 --> 00:04:44 that we can only just aspire to replicate. OK. 61 00:04:44 --> 00:04:47 So, again, the subject of today's lecture is enzymes as biological 62 00:04:47 --> 00:04:52 catalysts. And I want to introduce this by 63 00:04:52 --> 00:04:59 putting some of the earlier stuff into perspective. 64 00:04:59 --> 00:05:06 So Professor Sive told you in her last lectures about 65 00:05:06 --> 00:05:17 macromolecules -- 66 00:05:17 --> 00:05:20 I'm not going to talk about all of them that she's told you about, 67 00:05:20 --> 00:05:23 but she's talked about DNA, deoxyribonucleic acid which is 68 00:05:23 --> 00:05:27 responsible for information storage. 69 00:05:27 --> 00:05:37 She also told you about RNA, 70 00:05:37 --> 00:05:43 ribonucleic acid, a related polymer, nucleic acid. RNA actually has many 71 00:05:43 --> 00:05:49 functions in the cell. Information transfer is an 72 00:05:49 --> 00:05:55 important one, but it's not limited to that. 73 00:05:55 --> 00:06:01 RNAs can also function structurally. 74 00:06:01 --> 00:06:05 When we talk about protein synthesis, for example, you'll see that RNA 75 00:06:05 --> 00:06:09 molecules surface scaffolds for the ribosome which is the machine that 76 00:06:09 --> 00:06:13 makes proteins. And they can also be catalysts. 77 00:06:13 --> 00:06:18 This was not appreciated for a long time. We now know that in biology, 78 00:06:18 --> 00:06:22 in biological systems today RNAs can function as catalysts. 79 00:06:22 --> 00:06:26 And we actually believe, in an evolutionary sense, that RNAs 80 00:06:26 --> 00:06:31 were the first catalysts. Before there were polypeptides and 81 00:06:31 --> 00:06:36 proteins, life was made possible through the catalytic properties of 82 00:06:36 --> 00:06:42 RNA molecules. And your book actually talks about 83 00:06:42 --> 00:06:47 those in the chapter that you just read. They're called ribozymes, 84 00:06:47 --> 00:06:52 for RNA ribose-based enzymes. And then there were proteins. 85 00:06:52 --> 00:06:57 And we'll talk about proteins as catalysts today, 86 00:06:57 --> 00:07:03 but importantly proteins also serve structural roles. 87 00:07:03 --> 00:07:07 They compose, for example, the long cables that extend 88 00:07:07 --> 00:07:11 throughout your cells to give them the proper shape and allow them to 89 00:07:11 --> 00:07:15 move. They're important carrier proteins. For example, 90 00:07:15 --> 00:07:19 hemoglobin which carries oxygen to different parts of your body. 91 00:07:19 --> 00:07:23 There are proteins that bind to iron, for example, 92 00:07:23 --> 00:07:27 and deliver it from your diet to the relevant parts of your body that are 93 00:07:27 --> 00:07:31 protein specific to that function. And then there are catalysts. 94 00:07:31 --> 00:07:35 And it's the catalysts that we'll focus on today. 95 00:07:35 --> 00:07:39 Now, the way that proteins carry out these diverse functions is that 96 00:07:39 --> 00:07:43 by virtue of their amino acid sequence, their primary sequence of 97 00:07:43 --> 00:07:47 amino acids, they fold up into a particular shape. 98 00:07:47 --> 00:07:51 Proteins have very specific three-dimensional shapes. 99 00:07:51 --> 00:07:55 And these shapes then dictate their function. Depending on what the 100 00:07:55 --> 00:07:59 protein is supposed to do, it will have a different shape. 101 00:07:59 --> 00:08:03 So, for example, there are proteins that, 102 00:08:03 --> 00:08:07 as I mentioned, function as structural elements within the cell 103 00:08:07 --> 00:08:11 like long cables. They form polymers. 104 00:08:11 --> 00:08:15 And these proteins have shapes a little bit like Lego blocks that 105 00:08:15 --> 00:08:19 link together, one to the next, 106 00:08:19 --> 00:08:23 and as such line up into these long polymers. So some proteins have 107 00:08:23 --> 00:08:27 structures that just allow them to bind to one another and 108 00:08:27 --> 00:08:32 form long polymers. The proteins that we'll talk about 109 00:08:32 --> 00:08:36 today, catalysts, usually have, maybe always have a 110 00:08:36 --> 00:08:40 specific shape. And I'm taking a three-dimensional 111 00:08:40 --> 00:08:44 structure here and simply cutting a slice through it, 112 00:08:44 --> 00:08:48 so you're looking at sort of the middle of the protein cut in half. 113 00:08:48 --> 00:08:52 Here's the overall three-dimensional structure of the 114 00:08:52 --> 00:08:56 protein. And in this portion here, which we call the active site, is 115 00:08:56 --> 00:09:00 where the chemical reaction takes place. 116 00:09:00 --> 00:09:03 Now, we know a lot about many enzymes, as well as other proteins 117 00:09:03 --> 00:09:07 in the cell, based on the primary amino acid sequence that we can 118 00:09:07 --> 00:09:11 determine from the genes sequence. But we also have seen what these 119 00:09:11 --> 00:09:15 look like using methods in x-ray crystallography. 120 00:09:15 --> 00:09:23 We won't actually review the methods 121 00:09:23 --> 00:09:27 of x-ray crystallography with you, but basically sufficed to say that 122 00:09:27 --> 00:09:31 you can take a pure protein, allow it to form into a crystal, 123 00:09:31 --> 00:09:35 shine x-rays through it, the x-rays get diffracted based on the position 124 00:09:35 --> 00:09:38 of the atoms in the protein. And you can then interpret that 125 00:09:38 --> 00:09:42 diffraction pattern to tell you what the structure of the protein was. 126 00:09:42 --> 00:09:46 So these are not just cartoons. We actually know what these proteins 127 00:09:46 --> 00:09:50 look like in great detail. Not only do we know what the 128 00:09:50 --> 00:09:54 proteins look like in many instances, but we also often know what the 129 00:09:54 --> 00:09:58 protein looks like in complex with some reactant to which its bound. 130 00:09:58 --> 00:10:02 And these we call co-crystals. Crystals that occur between the 131 00:10:02 --> 00:10:06 protein and the substrate molecule that it's going to act upon. 132 00:10:06 --> 00:10:11 And so we have a very clear idea of what the chemistry is that's going 133 00:10:11 --> 00:10:15 on inside the enzyme's active site. And this information is not just 134 00:10:15 --> 00:10:20 useful for biological purposes, but the more we understand enzymes 135 00:10:20 --> 00:10:24 and the specific structure of enzymes the more we 136 00:10:24 --> 00:10:28 can do about it. So in disease, 137 00:10:28 --> 00:10:31 for example, where you have an enzyme that's causing some 138 00:10:31 --> 00:10:34 pathogenic problem, if you want to inhibit that enzyme, 139 00:10:34 --> 00:10:37 the more you know about what's happening here the more precise you 140 00:10:37 --> 00:10:40 can be in your design of an inhibitor. And that's now happening, 141 00:10:40 --> 00:10:44 and I'll tell you an example of that later in the lecture. 142 00:10:44 --> 00:10:48 Now, I'm just curious to know whether you have a sense for why the 143 00:10:48 --> 00:10:52 protein has its particular structure, and also what distinguishes one 144 00:10:52 --> 00:10:57 active site from another? There are thousands of enzymes 145 00:10:57 --> 00:11:01 inside this cell. Do they all look like this or does 146 00:11:01 --> 00:11:05 each one look different? Are they all the same? 147 00:11:05 --> 00:11:09 No. They're all different. They're different in shape, as I 148 00:11:09 --> 00:11:13 indicated earlier because their primary amino acid sequence causes 149 00:11:13 --> 00:11:17 them to fold up based on local interactions between amino acids 150 00:11:17 --> 00:11:20 into helices and beta pleated sheets. As I told you, 151 00:11:20 --> 00:11:24 they interact with each other. And that gives them that large sort 152 00:11:24 --> 00:11:28 of three-dimensional structure. But it's the nature of the amino 153 00:11:28 --> 00:11:32 acids in this active site which ones are there. 154 00:11:32 --> 00:11:36 Is it a glutamic acid, a valine, a methionine? 155 00:11:36 --> 00:11:40 Which specific ones are present there and how are they oriented 156 00:11:40 --> 00:11:44 which allows them to bind to particular molecules, 157 00:11:44 --> 00:11:48 substrates, and do chemistry on them? And the reason that proteins are 158 00:11:48 --> 00:11:52 much better catalysts, or much more powerful catalysts than 159 00:11:52 --> 00:11:56 RNAs, is that our RNAs are fairly boring. They only have four 160 00:11:56 --> 00:12:00 subunits. They don't have that great diversity of chemical reactive 161 00:12:00 --> 00:12:04 groups that you find in proteins. Proteins, as you heard, 162 00:12:04 --> 00:12:08 are composed of 20 distinct amino acid subunits. 163 00:12:08 --> 00:12:11 They are all differently chemically in those R groups that Professor 164 00:12:11 --> 00:12:15 Sive told you about before, and they can do different chemistry 165 00:12:15 --> 00:12:18 in the active site. So different enzymes are different 166 00:12:18 --> 00:12:22 by virtue of their overall structure and the particulars within the 167 00:12:22 --> 00:12:26 active site that allows them to do what they do. 168 00:12:26 --> 00:12:32 I wanted to mention briefly that we often use the suffix “ase” to 169 00:12:32 --> 00:12:38 designate an enzyme, polymerase, DNA polymerase, 170 00:12:38 --> 00:12:44 sucrase. You'll see those terms all the time. Whenever you see it, 171 00:12:44 --> 00:12:50 it reflects that the protein is an enzyme, the suffix A-S-E. 172 00:12:50 --> 00:12:56 OK. Again, just for perspective, where do the proteins come from in a 173 00:12:56 --> 00:13:02 sense? How does the cell know what to make? 174 00:13:02 --> 00:13:07 We're going to get into that in later lectures, 175 00:13:07 --> 00:13:13 but just so you have a sense of it, the information to produce a 176 00:13:13 --> 00:13:18 particular protein with a particular amino acid sequence, 177 00:13:18 --> 00:13:23 and therefore shape and therefore function, is encoded in the genes, 178 00:13:23 --> 00:13:28 which are in the DNA. This information is transferred into an 179 00:13:28 --> 00:13:34 intermediary molecule, which is RNA. 180 00:13:34 --> 00:13:37 Again, you're going to learn about these details. 181 00:13:37 --> 00:13:41 You don't have to worry about them so much right now. 182 00:13:41 --> 00:13:44 You'll learn about these details later in the class. 183 00:13:44 --> 00:13:48 And the particular RNA molecule that carries the information from 184 00:13:48 --> 00:13:52 the DNA is called the mRNA, messenger RNA. And that mRNA is 185 00:13:52 --> 00:13:55 then translated into the protein itself. So the reason that we have 186 00:13:55 --> 00:13:59 proteins of particular sequence and particular shape and particular 187 00:13:59 --> 00:14:03 function is that we have different genes that carry the information to 188 00:14:03 --> 00:14:08 make those specific proteins. OK? And we'll see that again in 189 00:14:08 --> 00:14:14 detail in later lectures. OK. So very importantly we talked 190 00:14:14 --> 00:14:20 last time, you talked last time about the energetics of reactions, 191 00:14:20 --> 00:14:27 as illustrated here, that in many reactions there is the energy of the 192 00:14:27 --> 00:14:33 reactants themselves, the energy of the products, 193 00:14:33 --> 00:14:40 as well as so-called activation energy. 194 00:14:40 --> 00:14:44 That is the energy that's required to make that reaction go, 195 00:14:44 --> 00:14:49 which can be greater than the energy of the reactants. 196 00:14:49 --> 00:14:54 The important function of enzymes is to lower the activation energy to 197 00:14:54 --> 00:14:59 reduce the threshold that these reactants have to go over in order 198 00:14:59 --> 00:15:04 to carry out the reactions that lead to the products. 199 00:15:04 --> 00:15:07 The enzyme's function is to lower the activation energy. 200 00:15:07 --> 00:15:11 And the way that enzymes do that is several-fold, as we'll review. 201 00:15:11 --> 00:15:14 The most important thing I can imagine you can take away from this 202 00:15:14 --> 00:15:18 lecture is the understanding that what enzymes do as catalysts is to 203 00:15:18 --> 00:15:21 lower the activation energy. They don't change the nature of the 204 00:15:21 --> 00:15:25 reactants, they don't change the nature of the products, 205 00:15:25 --> 00:15:28 they actually don't change themselves in the course of the 206 00:15:28 --> 00:15:31 reaction, but what they do is to facilitate the reaction by lowering 207 00:15:31 --> 00:15:35 the activation energy. And that is the nature of catalysis. 208 00:15:35 --> 00:15:46 209 00:15:46 --> 00:15:55 So enzymes are biological catalysts. 210 00:15:55 --> 00:16:04 Their function is to increase the 211 00:16:04 --> 00:16:08 rate of a reaction. As I said, reactions that release 212 00:16:08 --> 00:16:13 energy will happen spontaneously, but it might take a very long time. 213 00:16:13 --> 00:16:18 Enzymes function to increase the rate at which those reactions can 214 00:16:18 --> 00:16:22 happen. And they can do so in impressive ways. 215 00:16:22 --> 00:16:27 They can increase the rate by a million-fold. So they really can 216 00:16:27 --> 00:16:32 change whether a reaction will take place in the lifetime of an 217 00:16:32 --> 00:16:37 individual compared to whether it would take place in microseconds. 218 00:16:37 --> 00:16:41 OK? And many biological processes have to happen within the course of 219 00:16:41 --> 00:16:46 microseconds or seconds. And, therefore, without enzymes 220 00:16:46 --> 00:16:51 those would not be possible. Importantly, as illustrated on this 221 00:16:51 --> 00:16:56 slide, enzymes do not change the Delta G. They don't change the 222 00:16:56 --> 00:17:01 Delta G of the reaction. Delta G is the same. 223 00:17:01 --> 00:17:07 What's being changed here, in the presence of an enzyme, 224 00:17:07 --> 00:17:13 a catalyzed reaction, is the activation state. 225 00:17:13 --> 00:17:19 The energetics of the products and the energetics of the reactants 226 00:17:19 --> 00:17:24 don't change, so the Delta G does not change. So, 227 00:17:24 --> 00:17:30 again, they do so by lowering the activation energy. 228 00:17:30 --> 00:17:36 And this is often done by combining the reactants with portions of the 229 00:17:36 --> 00:17:42 protein to create what's called a transition state complex. 230 00:17:42 --> 00:17:50 And it's the nature of that 231 00:17:50 --> 00:17:54 transition state complex, the protein bound to the substrates 232 00:17:54 --> 00:17:58 that allows the activation energy to be reduced, as you'll 233 00:17:58 --> 00:18:04 see in a moment. I want to emphasize that the enzyme 234 00:18:04 --> 00:18:12 itself is the same at the end of the reaction as at the beginning. 235 00:18:12 --> 00:18:20 The enzyme does not change. It goes through one reaction cycle. 236 00:18:20 --> 00:18:29 It's exactly as it was when it started. 237 00:18:29 --> 00:18:33 And that's important because it means that the enzyme can be reused. 238 00:18:33 --> 00:18:38 This is not a single reaction process. The enzyme can be used 239 00:18:38 --> 00:18:43 over and over and over again, which is another part of the 240 00:18:43 --> 00:18:48 definition of a catalyst. OK. When we talk about enzymes, 241 00:18:48 --> 00:18:52 reactions, reactants and products, we use slightly different 242 00:18:52 --> 00:18:57 nomenclature, and so it's important that you see that and 243 00:18:57 --> 00:19:03 get to recognize it. The enzyme, often denoted as E, 244 00:19:03 --> 00:19:10 combines with substrates, one or more substrates, 245 00:19:10 --> 00:19:18 described as S, to form a complex which is designated ES. 246 00:19:18 --> 00:19:25 That's where this transition state is taking place. 247 00:19:25 --> 00:19:33 And then following that the enzyme releases the products. 248 00:19:33 --> 00:19:41 And, importantly, the enzyme can then be recycled to 249 00:19:41 --> 00:19:50 do this process again on new substrates to produce new products. 250 00:19:50 --> 00:19:59 So the S in this is the reactant or substrate, and P is the product, 251 00:19:59 --> 00:20:08 and the ES is the enzyme transition state complex. 252 00:20:08 --> 00:20:21 As I said at the beginning, 253 00:20:21 --> 00:20:25 enzymes have very particular specificities. 254 00:20:25 --> 00:20:29 They are designed to do specific reactions. They don't bind to every 255 00:20:29 --> 00:20:33 old molecule in the cell. And this is determined, 256 00:20:33 --> 00:20:38 as I said, by the shape of the enzyme and its complementarity to 257 00:20:38 --> 00:20:43 the substrates to which it binds. And that's illustrated here on a 258 00:20:43 --> 00:20:48 slide from your book where you can see an enzyme with its active site. 259 00:20:48 --> 00:20:54 And here are three potential substrates. Based on the shape of 260 00:20:54 --> 00:20:59 the active site and the particular side chains on those amino acids, 261 00:20:59 --> 00:21:04 the yellow one and the red one will fit into the active site, 262 00:21:04 --> 00:21:08 but the green one will not. So specificity, 263 00:21:08 --> 00:21:12 in this case, is determined by the complementarity between the shape of 264 00:21:12 --> 00:21:16 the substrates and the shape of the active site. And then, 265 00:21:16 --> 00:21:20 by virtue of their positioning within the active site, 266 00:21:20 --> 00:21:24 the enzyme is now catalyzing the binding, covalent attachment of the 267 00:21:24 --> 00:21:28 yellow one to the red one to produce the product, as shown here. 268 00:21:28 --> 00:21:35 So specificity is achieved by the complementarity between the active 269 00:21:35 --> 00:21:42 site and the substrates. The transition state is achieved by 270 00:21:42 --> 00:21:49 a variety of conditions that the enzyme places upon the substrates. 271 00:21:49 --> 00:22:02 So the substrate fits in much the 272 00:22:02 --> 00:22:08 way a key fits into a lock into the active site. This then promotes the 273 00:22:08 --> 00:22:15 formation of this transition state. And that is done by three distinct, 274 00:22:15 --> 00:22:22 sometimes related but, distinct mechanisms. One is the fixing of 275 00:22:22 --> 00:22:29 the orientation of the two substrates to one another. 276 00:22:29 --> 00:22:33 They're not just randomly floating around in solution anymore. 277 00:22:33 --> 00:22:38 They're literally aligned next to each other in a way that will 278 00:22:38 --> 00:22:43 promote the chemical reaction. And that's one way that the 279 00:22:43 --> 00:22:48 activation state is lowered because now you don't have the problem of 280 00:22:48 --> 00:22:53 kinetic energy of the molecules floating around. 281 00:22:53 --> 00:22:58 A second is what's referred to as induced fit, and in your book 282 00:22:58 --> 00:23:03 referred to as strain. And I'll show you a slide of this in 283 00:23:03 --> 00:23:07 a second. And this is important because often times the activation 284 00:23:07 --> 00:23:12 energy is due to the fact that the molecules have to get contorted. 285 00:23:12 --> 00:23:16 It's not a native confirmation of the molecules during the chemical 286 00:23:16 --> 00:23:21 reaction. They actually have to get bent in ways that they don't like to 287 00:23:21 --> 00:23:25 be bent. The enzyme helps this by adding chemical groups around it 288 00:23:25 --> 00:23:30 which promote the bending process, promote the sort of straining of 289 00:23:30 --> 00:23:35 chemical bonds that allows additional reactions to take place. 290 00:23:35 --> 00:23:38 So the enzyme produced is this so-called induced fit. 291 00:23:38 --> 00:23:42 And, finally, the enzyme can, depending on the nature of the 292 00:23:42 --> 00:23:46 chemistry, apply charge. We know that there are charged 293 00:23:46 --> 00:23:50 amino acids, both positively and negatively charged amino acids. 294 00:23:50 --> 00:23:54 There are acid-base reactions that often take place within the enzyme's 295 00:23:54 --> 00:23:58 active site. So the presence of charges can facilitate those 296 00:23:58 --> 00:24:02 acid-base reactions. They can donate positive charge or 297 00:24:02 --> 00:24:06 donate negative charge to allow the reaction to take place more rapidly 298 00:24:06 --> 00:24:10 than it would do spontaneously. And those are illustrated in a 299 00:24:10 --> 00:24:14 following slide, actually. This just shows you an 300 00:24:14 --> 00:24:18 example, a specific example of a reaction that is catalyzed by a 301 00:24:18 --> 00:24:22 particular enzyme. Here we have the substrate. 302 00:24:22 --> 00:24:26 It happens to be a sugar, a disaccharide sugar, 303 00:24:26 --> 00:24:30 sucrose, and is made up of the subunits glucose and fructose for 304 00:24:30 --> 00:24:34 you to use sucrose, which you can eat. 305 00:24:34 --> 00:24:39 To produce energy you need to break it down into glucose and fructose. 306 00:24:39 --> 00:24:44 And this reaction is catalyzed by a particular enzyme called sucrase. 307 00:24:44 --> 00:24:49 And you can see in diagrammatic form what happens here. 308 00:24:49 --> 00:24:54 Here's sucrase. Here is its active site. You can see that its 309 00:24:54 --> 00:24:59 structure is exactly complimentary to the substrate. 310 00:24:59 --> 00:25:03 So the substrate now floats in, binds to this active site. There's 311 00:25:03 --> 00:25:07 then a chemical reaction, which is basically the addition of 312 00:25:07 --> 00:25:11 water to break this bond, which is catalyzed by the enzyme. 313 00:25:11 --> 00:25:15 And then the products are released, the enzyme remains as it was at the 314 00:25:15 --> 00:25:19 beginning of the reaction, and it can go through the reaction 315 00:25:19 --> 00:25:23 cycle one more time. This is the picture that shows you 316 00:25:23 --> 00:25:27 the various ways that the active site can promote the transition 317 00:25:27 --> 00:25:32 state complex. One, as I mentioned, 318 00:25:32 --> 00:25:36 is orientation. Again, two substrates here are positioned 319 00:25:36 --> 00:25:40 next to each other in the way that we want them to react. 320 00:25:40 --> 00:25:44 So that's helpful. A second is the straining process, 321 00:25:44 --> 00:25:48 the fact that the protein can impose, in a sense, stress on the molecules, 322 00:25:48 --> 00:25:52 the substrates, change their shape, and in that way facilitate the 323 00:25:52 --> 00:25:56 chemical reaction, and finally, as I mentioned, 324 00:25:56 --> 00:26:01 to charge. In the case of acid-base reactions 325 00:26:01 --> 00:26:06 there are positively and also negatively charged amino acid side 326 00:26:06 --> 00:26:11 chains that can contribute their positive or negative charge to 327 00:26:11 --> 00:26:16 facilitate the reaction. And this is one final example. 328 00:26:16 --> 00:26:21 Here we're talking about an enzyme that adds a phosphate group onto 329 00:26:21 --> 00:26:26 glucose in an early step in glucose metabolism. And, 330 00:26:26 --> 00:26:31 in this case, the enzyme actually changes its shape as a consequence 331 00:26:31 --> 00:26:37 of the substrate binding to it. This happens [UNINTELLIGIBLE PHRASE]. 332 00:26:37 --> 00:26:43 At least he didn't point to me. [LAUGHTER] What was that all about? 333 00:26:43 --> 00:26:50 I was actually prepared to have those sorts of interruptions on 334 00:26:50 --> 00:26:56 Monday which we always have. I don't know what that was all 335 00:26:56 --> 00:27:02 about. So what was it all about, 336 00:27:02 --> 00:27:08 pretty boy? [LAUGHTER] And who is pretty boy anyway? 337 00:27:08 --> 00:27:14 Somebody back there. OK. Well, that was fun. 338 00:27:14 --> 00:27:20 Anyway. Well, we were talking about enzymes. 339 00:27:20 --> 00:27:26 So, again, some enzymes, as indicated here in the example of 340 00:27:26 --> 00:27:32 hexokinase, will actually change their shape in response to the 341 00:27:32 --> 00:27:38 binding of the substrate, here glucose. 342 00:27:38 --> 00:27:41 And this is maybe a more interesting example of something I'm going to 343 00:27:41 --> 00:27:44 come to later, which is that proteins are not 344 00:27:44 --> 00:27:48 static in their shape. They actually do change a little 345 00:27:48 --> 00:27:51 bit, and the ability of them to change can tweak, 346 00:27:51 --> 00:27:54 can tune their activities so that positioning the exact structure of 347 00:27:54 --> 00:27:58 the active site can change based on other things that are happening 348 00:27:58 --> 00:28:02 in the protein. And that's a useful thing with 349 00:28:02 --> 00:28:06 respect to regulation. You can turn up the activity of an 350 00:28:06 --> 00:28:11 enzyme. You can turn down the activity of an enzyme based on the 351 00:28:11 --> 00:28:15 changes that the overall structure can make. OK. 352 00:28:15 --> 00:28:20 And then just to bring the subject of specificity of enzyme function 353 00:28:20 --> 00:28:24 home, here is an example from real life. This is a packet of Equal, 354 00:28:24 --> 00:28:29 which you may use. It's an artificial sweetener. 355 00:28:29 --> 00:28:33 And you may have noticed on the very back of the Equal packet it says 356 00:28:33 --> 00:28:38 phenylketonurics: contains phenylalanine. 357 00:28:38 --> 00:28:42 And you might have wondered, what the hell is that all about? 358 00:28:42 --> 00:28:47 Does anybody know? Is anybody a phenylketinuric? 359 00:28:47 --> 00:28:51 You don't actually have to say if you are or not, 360 00:28:51 --> 00:28:56 but does anybody know what this means? Yes. It's close. 361 00:28:56 --> 00:29:00 You're actually thinking of a similar disease called 362 00:29:00 --> 00:29:05 alkaptonuria. But you're on the right track. 363 00:29:05 --> 00:29:09 Right. You cannot break it down. And specifically what you cannot 364 00:29:09 --> 00:29:13 break down is phenylalanine. This is a disease that affects only 365 00:29:13 --> 00:29:17 about one in 12, 00 individuals. It's a so-called 366 00:29:17 --> 00:29:22 metabolic disease. We'll talk about metabolic diseases 367 00:29:22 --> 00:29:26 later. And the important point here is that the enzyme that's 368 00:29:26 --> 00:29:30 responsible for breaking down phenylalanine is altered in these 369 00:29:30 --> 00:29:35 individuals in one residue, one amino acid out of 451. 370 00:29:35 --> 00:29:39 Protein has 451 amino acids. One of those amino acids in the 371 00:29:39 --> 00:29:43 active site is not what it's supposed to be. 372 00:29:43 --> 00:29:47 And therefore it cannot bind properly to phenylalanine. 373 00:29:47 --> 00:29:51 And that causes a defect in the breakdown of this and the build up 374 00:29:51 --> 00:29:56 of a toxic substance, as I'll show you in a second. 375 00:29:56 --> 00:30:00 This is NutraSweet. You might not have known that it is a dipeptide 376 00:30:00 --> 00:30:04 composed of aspartic acid and a phenylalanine linked together with 377 00:30:04 --> 00:30:08 an extra group on it, probably to make it more soluble or 378 00:30:08 --> 00:30:13 ability to pass through cells more easily. 379 00:30:13 --> 00:30:17 In your body, when you take in phenylalanine from the diet like 380 00:30:17 --> 00:30:21 with respect to NutraSweet, when NutraSweet gets into your body, 381 00:30:21 --> 00:30:25 the phenylalanine and the aspartic acid get broken apart so you have 382 00:30:25 --> 00:30:29 increased phenylalanine, but however you intake phenylalanine 383 00:30:29 --> 00:30:33 from your diet it's normally converted enzymaticly by an enzyme 384 00:30:33 --> 00:30:37 called phenylalanine hydroxylase which converts it from phenylalanine 385 00:30:37 --> 00:30:41 to tyrosine. And the tyrosine is either used for 386 00:30:41 --> 00:30:45 stuff or it's broken down itself. It's actually used for making 387 00:30:45 --> 00:30:49 melanin. And so these same patients who I have just mentioned, 388 00:30:49 --> 00:30:53 these phenylketonurics also have lighter hair and lighter skin 389 00:30:53 --> 00:30:57 because they cannot make as much tyrosine, and therefore don't make 390 00:30:57 --> 00:31:00 as much melanin. But the real problem is not that. 391 00:31:00 --> 00:31:04 The real problem is that if you have too much phenylalanine in your 392 00:31:04 --> 00:31:08 blood because you cannot break it down properly. 393 00:31:08 --> 00:31:11 Then you build up phenylpyruvic acid which is a natural byproduct of 394 00:31:11 --> 00:31:15 phenylalanine. And this stuff is toxic when 395 00:31:15 --> 00:31:19 present in high levels. And so the patients, these 396 00:31:19 --> 00:31:22 phenylalanine hydroxylase mutants, which are now called 397 00:31:22 --> 00:31:26 phenylketonurics, have a defect in this enzyme, 398 00:31:26 --> 00:31:30 cannot carry out this reaction properly. And therefore this 399 00:31:30 --> 00:31:34 spontaneous reaction happens more readily. 400 00:31:34 --> 00:31:37 And therefore you have high levels of this toxic compound that causes 401 00:31:37 --> 00:31:40 mental retardation, actually. It causes some sort of 402 00:31:40 --> 00:31:44 neuro toxicity. And that's how it was originally 403 00:31:44 --> 00:31:47 defined. And the reason we're telling you about this is that this 404 00:31:47 --> 00:31:51 is an example of enzyme specificity because this enzyme is different, 405 00:31:51 --> 00:31:54 as I said, in only one of 451 amino acids. The 408 amino acid is 406 00:31:54 --> 00:31:57 supposed to be an arginine, and instead is a tryptophan. 407 00:31:57 --> 00:32:01 And as a tryptophan it cannot properly bind to or carry out the 408 00:32:01 --> 00:32:05 chemical reaction. And therefore the enzyme fails, 409 00:32:05 --> 00:32:10 levels build up, and the individuals have a very, very severe phenotype. 410 00:32:10 --> 00:32:15 A very, very severe disease presentation, I should say. 411 00:32:15 --> 00:32:20 Now, I indicated a little bit ago that enzymes can be tweaked in their 412 00:32:20 --> 00:32:25 function. Enzymes are not just static in their ability to interact 413 00:32:25 --> 00:32:30 with substrates and catalyze reactions. 414 00:32:30 --> 00:32:35 In fact, they are highly regulated. And they're regulated by a number 415 00:32:35 --> 00:32:41 of different both external and internal processes. 416 00:32:41 --> 00:32:47 So regulation of enzyme function is critical with respect to producing 417 00:32:47 --> 00:32:53 the right sort of products at the right sort of times and rates within 418 00:32:53 --> 00:32:59 your cells. And one key factor that determines the regulation of a given 419 00:32:59 --> 00:33:05 enzyme is the pH, the pH of the solution that the 420 00:33:05 --> 00:33:11 enzyme finds itself. Now, why would that be? 421 00:33:11 --> 00:33:18 Does anybody have a sense for why that would be? 422 00:33:18 --> 00:33:26 Why does the pH of the cell's environment determine the enzyme's 423 00:33:26 --> 00:33:31 function? Yeah. Say it again. Right. So proteins can denature at, 424 00:33:31 --> 00:33:35 extreme pHs in both directions, actually. But more importantly 425 00:33:35 --> 00:33:39 they're optimized based on the side chains that are present within the 426 00:33:39 --> 00:33:43 active sites. So, as I said, there are charged amino 427 00:33:43 --> 00:33:46 acids which have different pKas. And depending on the pH of the 428 00:33:46 --> 00:33:50 solution, they'll either be protonated or not protonated. 429 00:33:50 --> 00:33:54 And their state of protonation, whether or not that they have a 430 00:33:54 --> 00:33:58 proton bound to them, will affect their ability to carry 431 00:33:58 --> 00:34:02 out the chemistry. So enzymes are perfected to function 432 00:34:02 --> 00:34:07 in the pH conditions they find themselves. So, 433 00:34:07 --> 00:34:12 for example, salivary amylase, which breaks down carbohydrates in 434 00:34:12 --> 00:34:16 your saliva, has a pH of around seven because your saliva is around 435 00:34:16 --> 00:34:21 pH seven. So it's been evolved to function best at that pH. 436 00:34:21 --> 00:34:26 If you increase the pH or decrease the pH it doesn't work so well, 437 00:34:26 --> 00:34:31 as indicated by this reduced reaction rate at higher 438 00:34:31 --> 00:34:35 and lower pHs. In contrast pepsin, 439 00:34:35 --> 00:34:39 which is an enzyme that is present in your stomach acid and in your 440 00:34:39 --> 00:34:43 small intestine where the pH is very, very low, works best at low pH. 441 00:34:43 --> 00:34:47 If you raise this to increase pH, it doesn't work at all well because 442 00:34:47 --> 00:34:51 presumably at the increased pH things that should be protonated are 443 00:34:51 --> 00:34:55 not protonated. And now those reactions that should 444 00:34:55 --> 00:34:59 be taking place in the active site don't. 445 00:34:59 --> 00:35:02 This is also another interesting example of regulation in the sense 446 00:35:02 --> 00:35:06 that pepsin breaks down proteins. And you actually don't want rampant 447 00:35:06 --> 00:35:10 protein breaking down enzymes floating around in your body. 448 00:35:10 --> 00:35:14 Particularly, you don't want them in the cells that make pepsin. 449 00:35:14 --> 00:35:18 You could imagine that the cells that make pepsin run the risk that 450 00:35:18 --> 00:35:22 pepsin is going to eat all the proteins in those cells. 451 00:35:22 --> 00:35:26 It doesn't happen because the pH of those cells is around seven, 452 00:35:26 --> 00:35:30 and therefore the pepsin isn't active. 453 00:35:30 --> 00:35:34 It only becomes active when it gets dumped into the acid environment of 454 00:35:34 --> 00:35:39 the stomach and the small intestine. So that's another justification for 455 00:35:39 --> 00:35:44 tweaking the activity of enzymes. Another example is temperature. 456 00:35:44 --> 00:35:52 Again, based on the structure of the 457 00:35:52 --> 00:35:56 protein, which is strongly influenced by the temperature, 458 00:35:56 --> 00:36:00 proteins have optima. Most of our proteins function best 459 00:36:00 --> 00:36:04 at what temperature? 37 degrees Centigrade. 460 00:36:04 --> 00:36:08 98.6, or whatever, Fahrenheit. But that's not true of all 461 00:36:08 --> 00:36:12 organisms. As I mentioned in the first lecture, 462 00:36:12 --> 00:36:17 there are organisms that live in thermal vents where the regular 463 00:36:17 --> 00:36:21 temperature is 80 degrees centigrade or higher. Their enzymes actually 464 00:36:21 --> 00:36:26 work like crap at 37 degrees, but they work great at 72 or 75 465 00:36:26 --> 00:36:30 degrees. They've been optimized, based on their structure, to 466 00:36:30 --> 00:36:35 function best at the temperature in which they find themselves. 467 00:36:35 --> 00:36:41 And finally, or almost finally, covalent modification. 468 00:36:41 --> 00:36:48 And there are different ways that 469 00:36:48 --> 00:36:52 proteins can be modified after they've been made in the translation 470 00:36:52 --> 00:36:56 process. Other stuff can get added to them covalently. 471 00:36:56 --> 00:37:00 And I'll give you an example of phosphorylation -- 472 00:37:00 --> 00:37:04 -- because it's going to come up in later lectures, 473 00:37:04 --> 00:37:09 too. Phosphorylation which means that the protein gets an extra 474 00:37:09 --> 00:37:14 phosphate group added to it. And if you imagine, for example, 475 00:37:14 --> 00:37:19 an enzyme, it has an active site here -- 476 00:37:19 --> 00:37:29 -- which is blocked at its front 477 00:37:29 --> 00:37:33 door. Stuff cannot get into it because this little arm is kind of 478 00:37:33 --> 00:37:38 hanging over the front of the active site. What can happen is that a 479 00:37:38 --> 00:37:43 reaction, another chemical reaction that adds a phosphate group -- 480 00:37:43 --> 00:38:00 Phosphorylation, 481 00:38:00 --> 00:38:06 which is another enzymatic reaction, can cause the enzyme to open up and 482 00:38:06 --> 00:38:12 allow substrates to come through. And this is a reversible process. 483 00:38:12 --> 00:38:18 Other enzymes called phosphatases can come along, 484 00:38:18 --> 00:38:24 clip the phosphate off and return the enzyme to its inactive state. 485 00:38:24 --> 00:38:32 And then, finally, 486 00:38:32 --> 00:38:36 there are partners, other molecules that the enzyme 487 00:38:36 --> 00:38:41 binds that help the enzyme do its thing. And these are summarized on 488 00:38:41 --> 00:38:45 this slide, which comes from your book. There are three groups that 489 00:38:45 --> 00:38:49 are shown here. You should probably be familiar 490 00:38:49 --> 00:38:54 with what these groups are and examples from within them. 491 00:38:54 --> 00:38:58 For example, cofactors. These are usually small metal 492 00:38:58 --> 00:39:02 molecules, atoms. Iron, copper and zinc are three that 493 00:39:02 --> 00:39:06 are shown here. These participate in the chemistry. 494 00:39:06 --> 00:39:10 They actually participate in the catalysis for enzymes that require 495 00:39:10 --> 00:39:13 them. And, actually, many enzymes in your bodies do 496 00:39:13 --> 00:39:17 require such cofactors. And that's one of the reasons it's 497 00:39:17 --> 00:39:20 encouraged, you're encouraged to eat zinc and stuff like that, 498 00:39:20 --> 00:39:24 because many of your enzymes need it. Another class called coenzymes, 499 00:39:24 --> 00:39:28 this happens to be a horrible name in my opinion, a really, 500 00:39:28 --> 00:39:32 really bad name. It's one of these historical names 501 00:39:32 --> 00:39:36 that we're stuck with. But these are, again, partner 502 00:39:36 --> 00:39:40 molecules. They're really substrates. They're really 503 00:39:40 --> 00:39:44 substrates in the reaction, but they're called coenzymes because 504 00:39:44 --> 00:39:49 they're used by many different enzymes in coupled reactions. 505 00:39:49 --> 00:39:53 And an example here is NAD which is involved in a hydrogen donating and 506 00:39:53 --> 00:39:57 receiving. And this I mention specifically because it's the 507 00:39:57 --> 00:40:01 product, or it's the byproduct of one of the vitamins you 508 00:40:01 --> 00:40:05 eat, vitamin B. And many of these coenzymes are the 509 00:40:05 --> 00:40:09 products of vitamins. And so that's one of the reasons 510 00:40:09 --> 00:40:12 it's important to eat your vitamins. And finally what are called 511 00:40:12 --> 00:40:16 prosthetic groups, the same word as artificial arms and 512 00:40:16 --> 00:40:19 legs. Prosthetic groups like heme, which is present in hemoglobin, 513 00:40:19 --> 00:40:23 flavins, other things, which are involved, again, 514 00:40:23 --> 00:40:26 in helping the enzyme or the protein do its thing. And the distinction 515 00:40:26 --> 00:40:30 between these and these is that they are larger. 516 00:40:30 --> 00:40:34 They're actually synthesized by the body, as opposed to these which are 517 00:40:34 --> 00:40:39 just taken up in the diet. OK. This is an example, and I'll 518 00:40:39 --> 00:40:44 go through it quickly for lack of time. This is an example of one 519 00:40:44 --> 00:40:48 chemical reaction. What we're talking about here is an 520 00:40:48 --> 00:40:53 enzyme called dihydrofolate reductase. This is an important 521 00:40:53 --> 00:40:58 enzyme in producing molecules that are required to build nucleotides in 522 00:40:58 --> 00:41:03 your body, both DNA and RNA precursors. 523 00:41:03 --> 00:41:06 Without this enzyme you cannot make those things, you would be dead. 524 00:41:06 --> 00:41:10 It's a very critical enzyme both in biology and in medicine. 525 00:41:10 --> 00:41:14 Dihydrofolate reductase happens to be one of the targets of an 526 00:41:14 --> 00:41:18 important chemotherapeutic agent called Methotrexate. 527 00:41:18 --> 00:41:22 It's used because cancer cells grow a lot. You want to inhibit their 528 00:41:22 --> 00:41:26 ability to grow, so you inhibit their ability to 529 00:41:26 --> 00:41:30 carry out this reaction to produce this relevant product. 530 00:41:30 --> 00:41:35 And also in bacteria it's the target for a particular antibiotic called 531 00:41:35 --> 00:41:40 Trimethoprim. Folic acid you know that you take in from your diet. 532 00:41:40 --> 00:41:45 And it's then broken down in a way, or not broken down. It's modified 533 00:41:45 --> 00:41:51 in a way that it becomes useful for these synthetic reactions. 534 00:41:51 --> 00:41:56 And that's the job of dihydrofolate reductase. Dihydrofolate has two 535 00:41:56 --> 00:42:02 hydrogens positioned at the seven and eight positions. 536 00:42:02 --> 00:42:06 And tetrahydrofolate has four hydrogens at these four positions. 537 00:42:06 --> 00:42:11 And it's the tetrahydrofolate that you want to use, 538 00:42:11 --> 00:42:16 that you need to use for these subsequent reactions. 539 00:42:16 --> 00:42:21 So the enzyme then, dihydrofolate reductase adds hydrogens. 540 00:42:21 --> 00:42:26 It reduces dihydrofolate to tetrahydrofolate. 541 00:42:26 --> 00:42:31 And it utilizes a cofactor NADP shown in green here to do that. 542 00:42:31 --> 00:42:35 It's the NADP, NADPH which transfers the hydrogens 543 00:42:35 --> 00:42:39 to the dihydrofolate. This is a little bit hard to see 544 00:42:39 --> 00:42:43 because it goes pretty quickly. You might look at it on the Web or 545 00:42:43 --> 00:42:47 at home. This is the dihydrofolate coming in. You can see in white the 546 00:42:47 --> 00:42:51 enzyme actually moving. It moves in order to carry out the 547 00:42:51 --> 00:42:55 chemical reaction. And you can see in green the NADP, 548 00:42:55 --> 00:42:59 initially NADPH coming in, interacting with the dihydrofolate, 549 00:42:59 --> 00:43:03 transferring the hydrogens and allowing it to become 550 00:43:03 --> 00:43:07 tetrahydrofolate. OK? So that's one example of an 551 00:43:07 --> 00:43:13 enzymatic reaction. OK. In the final eight minutes, 552 00:43:13 --> 00:43:19 and we do have a lot to get through, so if you could just hang in there 553 00:43:19 --> 00:43:25 until five of that would be good, I want to talk about another form of 554 00:43:25 --> 00:43:31 regulation, and that is specifically the existence of inhibitors 555 00:43:31 --> 00:43:36 and activators. So, again, the regulation of enzyme 556 00:43:36 --> 00:43:40 function is extremely important. You want to make sure that the 557 00:43:40 --> 00:43:44 enzyme is working at optimal rates, higher or lower, depending on the 558 00:43:44 --> 00:43:48 circumstances. And this can be adjusted naturally 559 00:43:48 --> 00:43:53 inside the cell by other molecules that can function to inhibit the 560 00:43:53 --> 00:43:57 enzyme or to activate the enzyme. And, as I said, we can also do that 561 00:43:57 --> 00:44:01 medically by making inhibitors or activators that change the activity 562 00:44:01 --> 00:44:07 of enzymes inside our cells. Terminology. These inhibitors and 563 00:44:07 --> 00:44:13 activators can be reversible or irreversible. They can either bind 564 00:44:13 --> 00:44:20 and come off and bind and never come off. This takes the enzyme out of 565 00:44:20 --> 00:44:26 play. It goes to the bench. It cannot work anymore. This one 566 00:44:26 --> 00:44:33 can come back off and the enzyme can continue to function. 567 00:44:33 --> 00:44:37 They can be competitive. And I'll show you what this means 568 00:44:37 --> 00:44:48 in a second. Competitive -- 569 00:44:48 --> 00:44:52 -- versus noncompetitive. And, again, I'll tell you what that 570 00:44:52 --> 00:45:01 means in a second. 571 00:45:01 --> 00:45:06 Competitive is illustrated here. If this is the active site and this 572 00:45:06 --> 00:45:11 is the substrate which would normally fit into that active site, 573 00:45:11 --> 00:45:16 a competitive inhibitor, like the name suggests, 574 00:45:16 --> 00:45:21 competes with the active site. It gets in there and prevents the 575 00:45:21 --> 00:45:26 substrate from binding. Pretty simple concept, right? 576 00:45:26 --> 00:45:31 A noncompetitive inhibitor functions by binding somewhere else 577 00:45:31 --> 00:45:36 on the protein and changing the structure of the active site. 578 00:45:36 --> 00:45:40 So here again is the substrate. It could bind to this active site, 579 00:45:40 --> 00:45:44 but when the noncompetitive inhibitor binds over here it changes 580 00:45:44 --> 00:45:49 the active site. And now the substrate cannot bind. 581 00:45:49 --> 00:45:53 So it's not competing with the substrate directly, 582 00:45:53 --> 00:45:57 but it's affecting the ability of the substrate to bind 583 00:45:57 --> 00:46:03 to the active site. Now, these noncompetitive inhibitors, 584 00:46:03 --> 00:46:09 and you can also have molecules that activate the enzyme at these other 585 00:46:09 --> 00:46:16 sites, are binding to portions of the protein that we call allosteric 586 00:46:16 --> 00:46:22 sites. A term you should be familiar with, 587 00:46:22 --> 00:46:29 allostery, allosteric regulation. 588 00:46:29 --> 00:46:33 And what this is, again, are molecules. 589 00:46:33 --> 00:46:37 Sometimes they're products of the reactions. Sometimes they are other 590 00:46:37 --> 00:46:41 things that bind somewhere on the protein, not at the active site, 591 00:46:41 --> 00:46:46 which change the nature of the active site. So in this example we 592 00:46:46 --> 00:46:50 see an allosteric site next to an active site. The binding of an 593 00:46:50 --> 00:46:54 activator can lock this enzyme, it happens to have four subunits, 594 00:46:54 --> 00:46:59 into a confirmation where the active site is open. 595 00:46:59 --> 00:47:03 Or another small molecule, an inhibitor can bind to the 596 00:47:03 --> 00:47:07 allosteric site and lock the protein into an inactive confirmation. 597 00:47:07 --> 00:47:11 OK? So allosteric regulation, sensing some other molecule and 598 00:47:11 --> 00:47:15 changing the activity of the enzyme. Why would you want to do that? 599 00:47:15 --> 00:47:19 Before I get to that, let me give you one real-life 600 00:47:19 --> 00:47:23 example of inhibition. It comes from my world. 601 00:47:23 --> 00:47:27 Cancer drugs increasingly are becoming more specific, 602 00:47:27 --> 00:47:31 and this is the best example. I don't have time to go into it in 603 00:47:31 --> 00:47:35 detail now for lack of time, but this is a drug made by a local 604 00:47:35 --> 00:47:39 pharmaceutical company called Novartis which binds to one of these 605 00:47:39 --> 00:47:43 kinases, these phosphorylating enzymes, important in a particular 606 00:47:43 --> 00:47:46 type of cancer. It is a competitive inhibitor of 607 00:47:46 --> 00:47:50 ATP. ATP needs to bind to an active site for this protein to function. 608 00:47:50 --> 00:47:54 The drug called Gleevec is a competitive inhibitor of ATP, 609 00:47:54 --> 00:47:58 therefore ATP cannot bind, therefore the enzyme cannot function, 610 00:47:58 --> 00:48:02 therefore the cancer cells cannot stay alive, therefore the cancer 611 00:48:02 --> 00:48:06 patient is cured. Great example. 612 00:48:06 --> 00:48:11 It happens to be true. So this is not just theory, 613 00:48:11 --> 00:48:15 not just bench biology. This is real-life in pharmacy in this 614 00:48:15 --> 00:48:20 example. And this is a three-dimensional picture of Gleevec 615 00:48:20 --> 00:48:25 in green bound to the active site of the kinase called able shown in the 616 00:48:25 --> 00:48:30 red and blue ribbons. OK. So, again, feedback regulation. 617 00:48:30 --> 00:48:34 The reason that we have allostery and changes in regulation relate to 618 00:48:34 --> 00:48:38 a diagram like this. And the important points here are 619 00:48:38 --> 00:48:43 that these enzymes that we've been talking about almost never function 620 00:48:43 --> 00:48:47 in isolation. They're almost always in pathways. Something produces 621 00:48:47 --> 00:48:51 product one, the next enzyme works on product one to make product two, 622 00:48:51 --> 00:48:56 and so on and so forth. And you actually, the cell wants to 623 00:48:56 --> 00:49:00 coordinate the activity of these enzymes so that the right amount of 624 00:49:00 --> 00:49:05 product is made at the bottom of the process. 625 00:49:05 --> 00:49:08 It's like the regulation of an assembly line in a factory. 626 00:49:08 --> 00:49:12 You don't want to make too many tires if you don't have enough cars. 627 00:49:12 --> 00:49:16 So when you have enough tires you feedback the tire generation and you 628 00:49:16 --> 00:49:20 bump up the car generation. The same thing happens in biology. 629 00:49:20 --> 00:49:24 This pathway A goes to B goes to C. C splits to D and F goes to G goes 630 00:49:24 --> 00:49:28 to E. Depending on how much G you have, you might feedback on this 631 00:49:28 --> 00:49:32 enzyme to make less F. And you might feed forward, 632 00:49:32 --> 00:49:37 you might have positive feedback on this enzyme to make more E. 633 00:49:37 --> 00:49:41 And this regulation is done by this sort of allosteric process whereby 634 00:49:41 --> 00:49:46 the G product to an allosteric reaction might inhibit the enzyme 635 00:49:46 --> 00:49:51 producing F and might activate the enzyme producing E. 636 00:49:51 --> 00:49:55 And this example shown here from an actual metabolic process, 637 00:49:55 --> 00:50:00 the generation of isoleucine is a specific example whereby when you 638 00:50:00 --> 00:50:05 make enough isoleucine the isoleucine will bind back to the 639 00:50:05 --> 00:50:10 enzyme way up here in the pathway to shut it down. 640 00:50:10 --> 00:50:14 Once I have enough isoleucine, isoleucine binds to this allosteric 641 00:50:14 --> 00:50:19 site on this enzyme, which now slows down the production 642 00:50:19 --> 00:50:23 of these intermediates, and therefore results in the 643 00:50:23 --> 00:50:28 production of less isoleucine. I left off two slides on energetics 644 00:50:28 --> 00:50:31 and ATP, but I'll mention those next time.