1 00:00:00,372 --> 00:00:02,830 SPEAKER: The following content is provided under a Creative 2 00:00:02,830 --> 00:00:04,370 Commons license. 3 00:00:04,370 --> 00:00:06,670 Your support will help MIT OpenCourseWare 4 00:00:06,670 --> 00:00:11,060 continue to offer high quality educational resources for free. 5 00:00:11,060 --> 00:00:13,660 To make a donation or view additional materials 6 00:00:13,660 --> 00:00:17,385 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,385 --> 00:00:18,010 at ocw.mit.edu. 8 00:00:24,530 --> 00:00:28,210 JOANNE STUBBE: OK, so welcome to class today. 9 00:00:28,210 --> 00:00:30,280 Today, I'm going to be talking about one 10 00:00:30,280 --> 00:00:35,620 of my favorite topics-- enzymes and catalysis. 11 00:00:35,620 --> 00:00:38,410 And what I would like to do is give you an outline 12 00:00:38,410 --> 00:00:41,260 of where we're going today. 13 00:00:41,260 --> 00:00:46,630 First, we're going to define what a catalyst is. 14 00:00:46,630 --> 00:00:49,675 And we're going to focus on enzymes as catalysts. 15 00:00:52,400 --> 00:00:56,170 Then what we're going to do is describe 16 00:00:56,170 --> 00:01:01,780 the theory of catalysis. 17 00:01:04,989 --> 00:01:07,930 And we'll show how the theory can account 18 00:01:07,930 --> 00:01:10,960 for all experimental observations or most 19 00:01:10,960 --> 00:01:13,480 experimental observations. 20 00:01:13,480 --> 00:01:19,000 We'll then talk about the mechanisms of catalysis, 21 00:01:19,000 --> 00:01:22,030 and we'll see that there are three basic mechanisms. 22 00:01:22,030 --> 00:01:24,430 I won't write them out, but we'll come to them. 23 00:01:24,430 --> 00:01:29,800 And then if time allows, we'll talk about another property 24 00:01:29,800 --> 00:01:30,490 of enzymes. 25 00:01:30,490 --> 00:01:36,330 These are all focused with amazing rates of reaction. 26 00:01:40,030 --> 00:01:42,700 And the second property of enzymes, 27 00:01:42,700 --> 00:01:46,180 besides the fact that they can accelerate reactions 28 00:01:46,180 --> 00:01:50,860 by a million to a billion fold, is their specificity. 29 00:01:56,260 --> 00:01:58,910 So that's where we're going. 30 00:01:58,910 --> 00:02:02,530 And what I'd like to do in the very beginning 31 00:02:02,530 --> 00:02:06,430 is show you why-- spend a little time 32 00:02:06,430 --> 00:02:09,400 to show you why enzymes are important. 33 00:02:09,400 --> 00:02:11,590 Why do you care about enzymes? 34 00:02:11,590 --> 00:02:13,090 That's why you care about enzymes. 35 00:02:13,090 --> 00:02:14,620 Look at this mess. 36 00:02:14,620 --> 00:02:17,530 That's what's going on inside your body. 37 00:02:17,530 --> 00:02:21,970 There are thousands of reactions going on inside your body. 38 00:02:21,970 --> 00:02:26,980 Without enzymes, no reaction. 39 00:02:26,980 --> 00:02:29,800 So you must care about enzymes. 40 00:02:29,800 --> 00:02:33,970 So what we're going to see over the course of the semester 41 00:02:33,970 --> 00:02:36,490 is that we can break down this mess 42 00:02:36,490 --> 00:02:40,070 into a few basic reactions. 43 00:02:40,070 --> 00:02:42,670 OK, so here is Waldo. 44 00:02:42,670 --> 00:02:44,710 And over the course of this semester, 45 00:02:44,710 --> 00:02:50,350 as you've seen many times, we walk through central metabolism 46 00:02:50,350 --> 00:02:52,630 and all of the reactions. 47 00:02:52,630 --> 00:02:55,000 Now, a second thing about enzymes 48 00:02:55,000 --> 00:03:00,130 that I think will be what you guys do for a living 49 00:03:00,130 --> 00:03:03,310 if you decide to become biochemists and enzymologists 50 00:03:03,310 --> 00:03:07,660 is can we take our understanding of how these amazing catalysts 51 00:03:07,660 --> 00:03:11,620 work and design our own protein catalysts 52 00:03:11,620 --> 00:03:15,700 to do any reactions we want not involved in the 10 53 00:03:15,700 --> 00:03:19,930 or 12 basic reactions we have going on inside our body? 54 00:03:19,930 --> 00:03:22,390 And we can't do that now, but I would 55 00:03:22,390 --> 00:03:26,890 argue that understanding catalysis is a key requirement 56 00:03:26,890 --> 00:03:28,840 for getting to the point where we can actually 57 00:03:28,840 --> 00:03:30,970 do catalytic design. 58 00:03:30,970 --> 00:03:33,460 And the third thing that I think is really important 59 00:03:33,460 --> 00:03:38,560 is that 40% to 50% of all the drugs we presently 60 00:03:38,560 --> 00:03:42,280 use in treatment of antibacterial infections, 61 00:03:42,280 --> 00:03:45,130 anti-viral infections, anti-cancer infections 62 00:03:45,130 --> 00:03:49,390 are all inhibitors of enzyme-based reactions. 63 00:03:49,390 --> 00:03:52,420 And understanding catalysis helps 64 00:03:52,420 --> 00:03:54,880 us to design better inhibitors. 65 00:03:54,880 --> 00:03:59,560 So understanding catalysis is central to many things 66 00:03:59,560 --> 00:04:04,090 that are important to all of us in society. 67 00:04:04,090 --> 00:04:07,780 So let me just tell you how I got excited about enzymes. 68 00:04:07,780 --> 00:04:10,210 So I went to graduate school. 69 00:04:10,210 --> 00:04:12,190 Never had a biochemistry course. 70 00:04:12,190 --> 00:04:14,890 They didn't do anything about biochemistry 71 00:04:14,890 --> 00:04:17,350 at the molecular level. 72 00:04:17,350 --> 00:04:19,870 When I went to graduate school, I went to a lecture 73 00:04:19,870 --> 00:04:21,540 in the first year of graduate school 74 00:04:21,540 --> 00:04:25,240 that was given by a faculty member at Stanford. 75 00:04:25,240 --> 00:04:31,270 And he talked about an enzyme called lenosterol cyclase that 76 00:04:31,270 --> 00:04:34,270 converts a linear molecule. 77 00:04:34,270 --> 00:04:37,030 So here's the linear molecule, but I have 78 00:04:37,030 --> 00:04:41,470 it folded up into four rings. 79 00:04:41,470 --> 00:04:43,480 And these four rings provide the basis 80 00:04:43,480 --> 00:04:46,810 for all steroids like estrogen and testosterone 81 00:04:46,810 --> 00:04:47,710 and cholesterol. 82 00:04:47,710 --> 00:04:49,990 And look at what this reaction does. 83 00:04:49,990 --> 00:04:56,290 One enzyme in a single step converts this linear molecule 84 00:04:56,290 --> 00:05:00,340 through a series of cascade transformations in hydride 85 00:05:00,340 --> 00:05:03,760 and methyl shifts into this molecule, 86 00:05:03,760 --> 00:05:07,650 putting in six asymmetric centers 87 00:05:07,650 --> 00:05:14,070 in a single step in 100% yield at 37 degrees in a pH 7. 88 00:05:14,070 --> 00:05:16,980 I said, my god, why do I want to be a chemist? 89 00:05:16,980 --> 00:05:17,754 You sweat. 90 00:05:17,754 --> 00:05:19,170 There are no blocking [INAUDIBLE]. 91 00:05:19,170 --> 00:05:22,920 You to sweat to put in any kind of an asymmetric center, 92 00:05:22,920 --> 00:05:26,120 and here, this little protein has figured out 93 00:05:26,120 --> 00:05:32,050 how to do all of this under really mild conditions. 94 00:05:32,050 --> 00:05:35,310 And so this was a transformative experience for me. 95 00:05:35,310 --> 00:05:39,060 I remember the lecture clearly because I thought 96 00:05:39,060 --> 00:05:41,520 it was so amazing and I'd never seen that enzymes could 97 00:05:41,520 --> 00:05:43,890 catalyze reactions like this. 98 00:05:43,890 --> 00:05:48,210 So enzymes really are amazing. 99 00:05:48,210 --> 00:05:51,600 So what you want to do now is start 100 00:05:51,600 --> 00:05:53,790 by defining what a catalyst is. 101 00:05:59,270 --> 00:06:03,870 And a catalyst, it can be for those of you who 102 00:06:03,870 --> 00:06:05,580 have had more chemistry, it can be 103 00:06:05,580 --> 00:06:08,880 an inorganic ion for example. 104 00:06:08,880 --> 00:06:12,750 It can be a small organic molecule. 105 00:06:12,750 --> 00:06:14,430 But for us, we're going to be focused 106 00:06:14,430 --> 00:06:18,140 on large macromolecules. 107 00:06:18,140 --> 00:06:21,810 And the macromolecules, we'll see 108 00:06:21,810 --> 00:06:25,590 that we're getting focused on, could be proteins or RNA. 109 00:06:25,590 --> 00:06:28,140 But most of the reactions found in our body 110 00:06:28,140 --> 00:06:32,250 are catalyzed by protein catalysts. 111 00:06:32,250 --> 00:06:43,050 And these catalysts increase the rate of the reaction 112 00:06:43,050 --> 00:06:46,177 without themselves being changed during the reaction. 113 00:06:52,670 --> 00:06:55,580 And furthermore, while they can increase 114 00:06:55,580 --> 00:06:57,410 the rate of the reaction, they don't 115 00:06:57,410 --> 00:07:01,160 affect the overall equilibrium of the reaction. 116 00:07:01,160 --> 00:07:05,180 They just increase the rate of approach to equilibrium. 117 00:07:05,180 --> 00:07:16,530 So they have no effect on equilibrium insolution, 118 00:07:16,530 --> 00:07:22,424 but increase the rate of approach to equilibrium. 119 00:07:26,750 --> 00:07:30,500 So what I want to do now is define 120 00:07:30,500 --> 00:07:33,920 some of the basic properties of the catalysts 121 00:07:33,920 --> 00:07:38,690 that we'll be talking about for the next 15 minutes or so. 122 00:07:38,690 --> 00:07:41,330 So the first thing is that the catalysts we're going 123 00:07:41,330 --> 00:07:44,972 to be focused on are enzymes. 124 00:07:47,570 --> 00:07:50,060 Remember, we've spent the last few lectures 125 00:07:50,060 --> 00:07:51,350 talking about proteins. 126 00:07:51,350 --> 00:07:54,080 Enzymes are simply proteins, but then we 127 00:07:54,080 --> 00:07:57,140 see that they have special regions in the protein 128 00:07:57,140 --> 00:08:01,370 structure which allow them to accelerate 129 00:08:01,370 --> 00:08:04,670 rates of defined reactions. 130 00:08:04,670 --> 00:08:09,920 I also will mention that we have inside of us 131 00:08:09,920 --> 00:08:12,370 a machine called the ribosome. 132 00:08:12,370 --> 00:08:18,700 And the ribozome is the machine that makes proteins, makes 133 00:08:18,700 --> 00:08:21,020 polypeptide bonds. 134 00:08:21,020 --> 00:08:22,940 We're not going to talk about that in 507, 135 00:08:22,940 --> 00:08:25,970 but we talk about it in 508. 136 00:08:25,970 --> 00:08:29,960 And the amazing observation was made really initially 137 00:08:29,960 --> 00:08:34,350 by the seminal experiments by Harry Knoller at UC Santa Cruz 138 00:08:34,350 --> 00:08:38,240 that you don't need any proteins to make peptide bonds. 139 00:08:38,240 --> 00:08:40,669 That was heresy at the time. 140 00:08:40,669 --> 00:08:44,300 In 2001, Steitz won the Nobel Prize 141 00:08:44,300 --> 00:08:46,880 for the structure of the ribosome and Harry 142 00:08:46,880 --> 00:08:50,120 didn't get the Nobel Prize. 143 00:08:50,120 --> 00:08:50,810 Bad. 144 00:08:50,810 --> 00:08:54,170 He's the one that made the seminal discovery, 145 00:08:54,170 --> 00:08:56,030 although the structure of a ribosome, 146 00:08:56,030 --> 00:09:01,370 which is 2.3 megadaltons, is really sort of spectacular. 147 00:09:01,370 --> 00:09:04,610 I still get goosebumps when I think about that structure that 148 00:09:04,610 --> 00:09:06,470 was published in 2001. 149 00:09:06,470 --> 00:09:07,880 But Harry didn't get it. 150 00:09:07,880 --> 00:09:09,710 Anyhow, so that was a digression. 151 00:09:09,710 --> 00:09:12,760 So that took a few minutes off my 50 minutes. 152 00:09:12,760 --> 00:09:15,170 Anyhow, we're going to be focused 153 00:09:15,170 --> 00:09:17,980 on enzymes as catalysts. 154 00:09:17,980 --> 00:09:21,770 So why are enzymes important. 155 00:09:21,770 --> 00:09:24,410 They're important because as I already told you, 156 00:09:24,410 --> 00:09:31,080 they accelerate the rates of reaction 10 157 00:09:31,080 --> 00:09:38,330 to the 6-- a million-- to 10 to the 15-fold. 158 00:09:38,330 --> 00:09:39,260 Whoa. 159 00:09:39,260 --> 00:09:41,160 Can you imagine that? 160 00:09:41,160 --> 00:09:43,980 Essigmann always used to say to me, give them an example. 161 00:09:43,980 --> 00:09:45,200 That's a lot. 162 00:09:45,200 --> 00:09:49,316 It's faster than a speeding bullet. 163 00:09:49,316 --> 00:09:50,690 Do you know where that came from? 164 00:09:50,690 --> 00:09:52,990 Faster than a speeding bullet? 165 00:09:52,990 --> 00:09:57,650 See, if this is when I have a disconnection with my audience. 166 00:09:57,650 --> 00:10:02,090 It's a bird, it's a plane, able to leap tall buildings 167 00:10:02,090 --> 00:10:04,180 in a single bound. 168 00:10:04,180 --> 00:10:07,310 Superman, course five. 169 00:10:07,310 --> 00:10:10,190 That's where our course five logo came from. 170 00:10:10,190 --> 00:10:13,610 So let me just give you an example of this. 171 00:10:13,610 --> 00:10:17,780 And so this is taken from an article by Wolfenden, 172 00:10:17,780 --> 00:10:22,230 and this is the expanse of reactions 173 00:10:22,230 --> 00:10:26,630 that it rates that enzymes can catalyze that also 174 00:10:26,630 --> 00:10:28,740 can occur in solution. 175 00:10:28,740 --> 00:10:34,560 So if you take down at this end a half life of adding water 176 00:10:34,560 --> 00:10:37,760 to CO2 is five seconds. 177 00:10:37,760 --> 00:10:39,380 That's pretty fast. 178 00:10:39,380 --> 00:10:43,040 Why do you need water to hydrate CO2? 179 00:10:43,040 --> 00:10:45,410 Anybody got any ideas? 180 00:10:45,410 --> 00:10:48,760 Where have you seen that in the last few lectures? 181 00:10:48,760 --> 00:10:50,150 Hemoglobin. 182 00:10:50,150 --> 00:10:51,140 Why do you need that? 183 00:10:51,140 --> 00:10:56,420 Because in your tissues, all of the fatty acids of the glucose 184 00:10:56,420 --> 00:10:57,970 gets breakdown to CO2. 185 00:10:57,970 --> 00:11:00,720 The CO2, where does it come out? 186 00:11:00,720 --> 00:11:01,910 You exhale it. 187 00:11:01,910 --> 00:11:06,860 Somehow it has to be carried around and into your lungs 188 00:11:06,860 --> 00:11:08,500 from your tissues. 189 00:11:08,500 --> 00:11:11,970 And there's a key enzyme called carbonic anhydrase 190 00:11:11,970 --> 00:11:16,145 that accelerates even this very fast reaction by a million 191 00:11:16,145 --> 00:11:16,645 fold. 192 00:11:19,230 --> 00:11:21,680 Let's look at another one that might be familiar to you. 193 00:11:21,680 --> 00:11:24,290 Let's think about peptide bond hydrolysis. 194 00:11:24,290 --> 00:11:28,040 We just told you the ribosome makes peptides. 195 00:11:28,040 --> 00:11:30,320 What about peptide bond hydrolysis, which 196 00:11:30,320 --> 00:11:37,520 plays a major role in cell media death and blood coagulation 197 00:11:37,520 --> 00:11:41,090 and controlling the levels of proteins inside the cell? 198 00:11:41,090 --> 00:11:44,660 If you look at the half life of peptide bond 199 00:11:44,660 --> 00:11:47,900 hydrolysis, 450 years. 200 00:11:47,900 --> 00:11:50,360 That means if we needed this reaction in our lifetime, 201 00:11:50,360 --> 00:11:52,490 it wouldn't ever happen. 202 00:11:52,490 --> 00:11:55,990 So if you actually look at the rate acceleration, 203 00:11:55,990 --> 00:11:59,690 proteases, which hydrolyze peptide bonds, 204 00:11:59,690 --> 00:12:02,420 have rate constants of about 50 per second. 205 00:12:02,420 --> 00:12:05,940 This rate constant is about 10 to the minus 9 per second. 206 00:12:05,940 --> 00:12:09,110 That's the rate acceleration of 10 to the 12. 207 00:12:09,110 --> 00:12:11,510 So without these kinds of enzymes 208 00:12:11,510 --> 00:12:14,610 and many other kinds of enzymes, we would not be alive 209 00:12:14,610 --> 00:12:17,390 and we would not be able to function. 210 00:12:17,390 --> 00:12:23,150 So the description of rate accelerations 211 00:12:23,150 --> 00:12:28,370 is given by a term we're going to derive in the next lecture-- 212 00:12:28,370 --> 00:12:31,175 kcat over KM. 213 00:12:31,175 --> 00:12:33,220 A kcat is a turnover number. 214 00:12:33,220 --> 00:12:35,920 It tells you how good your catalyst is in terms of per 215 00:12:35,920 --> 00:12:36,890 second. 216 00:12:36,890 --> 00:12:41,250 KM has a concentration dependence. 217 00:12:41,250 --> 00:12:46,160 So this is a second order rate constant-- concentration 218 00:12:46,160 --> 00:12:50,730 inverse, time inverse. 219 00:12:50,730 --> 00:12:54,410 And this is what we use to think about how efficient enzymes 220 00:12:54,410 --> 00:12:57,240 are, as we'll see in the next lecture. 221 00:12:57,240 --> 00:13:00,800 So what I want to show you here is another graph 222 00:13:00,800 --> 00:13:05,930 that was made by Wolfenden, who we were talking about data 223 00:13:05,930 --> 00:13:07,670 in the previous slide. 224 00:13:07,670 --> 00:13:10,760 And what I want to do is show you his comparison 225 00:13:10,760 --> 00:13:13,620 of enzyme catalyzed reactions and 226 00:13:13,620 --> 00:13:15,510 non-enzyme catalyzed reactions. 227 00:13:15,510 --> 00:13:20,060 And we just heard with peptide bond hydrolysis, 450-fold rate 228 00:13:20,060 --> 00:13:20,880 acceleration. 229 00:13:20,880 --> 00:13:22,040 That's a lot. 230 00:13:22,040 --> 00:13:23,750 What do you notice immediately about 231 00:13:23,750 --> 00:13:26,300 enzyme catalyzed reactions? 232 00:13:26,300 --> 00:13:37,570 The kcat over KM is on the order of 10 to the 6 to 10 to the 8 233 00:13:37,570 --> 00:13:40,640 per molar per second. 234 00:13:40,640 --> 00:13:43,310 Does that ring a bell with anybody? 235 00:13:43,310 --> 00:13:46,490 Where have you seen a number like 10 to the 6 to 10 to the 8 236 00:13:46,490 --> 00:13:48,240 per molar per second? 237 00:13:48,240 --> 00:13:53,480 What that is is a diffusion constant of any two molecules 238 00:13:53,480 --> 00:13:55,370 finding each other in solution. 239 00:13:55,370 --> 00:13:57,500 So what is that telling us? 240 00:13:57,500 --> 00:14:03,210 That's telling us that inside the cell, 241 00:14:03,210 --> 00:14:07,280 enzymes have evolved to be so efficient 242 00:14:07,280 --> 00:14:09,170 that the rate-limiting steps are going to be 243 00:14:09,170 --> 00:14:10,520 finding each other in solution. 244 00:14:10,520 --> 00:14:11,230 It's physical. 245 00:14:11,230 --> 00:14:13,500 It has nothing to do with the chemistry. 246 00:14:13,500 --> 00:14:15,800 So you've had billions of years to figure out 247 00:14:15,800 --> 00:14:18,832 all this chemistry, and what limits everything-- 248 00:14:18,832 --> 00:14:20,540 and we'll come back to this in a minute-- 249 00:14:20,540 --> 00:14:25,410 is the enzyme and the small molecule finding themselves. 250 00:14:25,410 --> 00:14:28,020 And so that's where this number-- of 10 to the 6 or 10 251 00:14:28,020 --> 00:14:30,410 to the 8 per molar per second comes from. 252 00:14:30,410 --> 00:14:33,530 If you look at the non-enzymatic reactions, 253 00:14:33,530 --> 00:14:35,850 we just talked about hydration of CO2 254 00:14:35,850 --> 00:14:38,270 versus enzyme catalyzed reaction, 255 00:14:38,270 --> 00:14:42,020 what you see is that they're all over the place. 256 00:14:42,020 --> 00:14:46,010 So the staggering rate accelerations 257 00:14:46,010 --> 00:14:49,710 of 10 to the 6 to 10 to the 15 that you see 258 00:14:49,710 --> 00:14:54,980 are really based on the rates of the non-enzymatic reactions. 259 00:14:54,980 --> 00:14:57,140 And the enzymes have evolved-- most of them 260 00:14:57,140 --> 00:14:59,730 have evolved over billions of years 261 00:14:59,730 --> 00:15:03,360 to be incredibly effective at what they do. 262 00:15:09,330 --> 00:15:11,970 So the other thing that I wanted to say about enzymes 263 00:15:11,970 --> 00:15:16,520 at this stage is that enzymes are usually 264 00:15:16,520 --> 00:15:19,490 in addition to being great catalysts, 265 00:15:19,490 --> 00:15:24,050 they're also-- you learn, I think, if you've seen enzymes 266 00:15:24,050 --> 00:15:34,370 before that they are very specific for the substrates, 267 00:15:34,370 --> 00:15:38,040 which I'll call S and we'll come back to this in a minute. 268 00:15:38,040 --> 00:15:41,220 So they only react-- you have hundreds of metabolites 269 00:15:41,220 --> 00:15:42,270 inside of our body. 270 00:15:42,270 --> 00:15:46,790 That only will pick up and react with one of those metabolites. 271 00:15:46,790 --> 00:15:50,540 But in reality, I think what we found over the last 15 years 272 00:15:50,540 --> 00:15:53,960 or so is enzymes are not all that specific. 273 00:15:53,960 --> 00:15:58,130 They are specific for what they encounter inside us. 274 00:15:58,130 --> 00:16:00,140 So if you take them out as a biochemist 275 00:16:00,140 --> 00:16:01,950 and start messing around with them, 276 00:16:01,950 --> 00:16:03,800 they aren't anywhere near as specific. 277 00:16:03,800 --> 00:16:06,960 They don't have to be that specific because they never 278 00:16:06,960 --> 00:16:10,470 encounter these molecules inside the cell. 279 00:16:10,470 --> 00:16:16,250 So they are very specific for substrates in vivo. 280 00:16:16,250 --> 00:16:19,980 And in fact, many of them are promiscuous in vitro. 281 00:16:19,980 --> 00:16:22,740 And I think that's something that's been under-appreciated. 282 00:16:28,740 --> 00:16:30,480 So this is number three. 283 00:16:30,480 --> 00:16:32,730 I wanted to talk about specificity. 284 00:16:32,730 --> 00:16:37,310 Number four, enzymes in general, if you 285 00:16:37,310 --> 00:16:41,480 look at that metabolic chart, almost all those reactions 286 00:16:41,480 --> 00:16:45,020 can be subdivided into 10 to 12 reactions. 287 00:16:45,020 --> 00:16:47,120 And those 10 to 12 reactions, even 288 00:16:47,120 --> 00:16:49,670 though it looks like a jungle and a mess, 289 00:16:49,670 --> 00:16:52,950 are found in the lexicon that you have been 290 00:16:52,950 --> 00:16:55,230 given in the first lecture. 291 00:16:55,230 --> 00:16:59,890 So that lexicon provides a framework to think about all 292 00:16:59,890 --> 00:17:01,710 of primary metabolism. 293 00:17:01,710 --> 00:17:04,980 Now, in reality, there are many other kinds of reactions. 294 00:17:04,980 --> 00:17:09,359 But the ones that you're going to see in 507 295 00:17:09,359 --> 00:17:12,660 can be limited to 10 to 12 reactions. 296 00:17:12,660 --> 00:17:29,270 So enzymes have a limited repertoire of reactions 297 00:17:29,270 --> 00:17:30,415 in primary metabolism. 298 00:17:34,290 --> 00:17:38,740 And so in this case, let me give a plug for the chemists. 299 00:17:38,740 --> 00:17:40,690 Chemists have the whole periodic table. 300 00:17:40,690 --> 00:17:42,590 Do we have a periodic table here? 301 00:17:42,590 --> 00:17:43,180 No. 302 00:17:43,180 --> 00:17:44,430 We're in the wrong department. 303 00:17:44,430 --> 00:17:45,760 We're in the wrong building. 304 00:17:45,760 --> 00:17:48,390 Anyhow, we have hundreds-- not hundreds-- 305 00:17:48,390 --> 00:17:52,800 we have 50 elements where we can use to catalyze reactions. 306 00:17:52,800 --> 00:17:56,440 We can do all kinds of reactions catalytically, 307 00:17:56,440 --> 00:17:58,560 and we can do it with something small, 308 00:17:58,560 --> 00:18:01,240 like a proton, or something small, 309 00:18:01,240 --> 00:18:05,440 like a metal with a little organic spinach hanging off 310 00:18:05,440 --> 00:18:06,220 of it. 311 00:18:06,220 --> 00:18:07,690 But what are we doing with enzymes? 312 00:18:07,690 --> 00:18:10,580 We have these big huge molecules. 313 00:18:10,580 --> 00:18:11,640 So there's a playoff. 314 00:18:11,640 --> 00:18:15,220 Enzymes have a very limited repertoire 315 00:18:15,220 --> 00:18:19,990 of reactions they catalyze, while chemists actually 316 00:18:19,990 --> 00:18:24,300 are limited by their imagination to catalyze these reactions. 317 00:18:24,300 --> 00:18:28,350 However, as the world becomes more and more green, 318 00:18:28,350 --> 00:18:31,480 chemists are no longer allowed to use metals. 319 00:18:31,480 --> 00:18:34,510 For example, they can be toxic to people. 320 00:18:34,510 --> 00:18:37,800 And so people are rethinking and refocusing 321 00:18:37,800 --> 00:18:41,580 on developing green catalysts. 322 00:18:41,580 --> 00:18:43,550 So the question that you can ask yourself, 323 00:18:43,550 --> 00:18:47,020 is there any way that enzymes can 324 00:18:47,020 --> 00:18:49,660 enhance their repertoire of reactions 325 00:18:49,660 --> 00:18:51,360 that they can catalyze? 326 00:18:51,360 --> 00:18:53,306 And they can. 327 00:18:53,306 --> 00:18:57,460 They do that by using the vitamins on the vitamin bottle. 328 00:18:57,460 --> 00:18:59,850 So enzymes have a limited repertoire, 329 00:18:59,850 --> 00:19:12,700 but they increase this repertoire using vitamins. 330 00:19:12,700 --> 00:19:15,550 This is what we eat out of our vitamin bottle that 331 00:19:15,550 --> 00:19:18,660 are converted into co-factors. 332 00:19:18,660 --> 00:19:23,210 So the vitamins we eat have to be subtly modified 333 00:19:23,210 --> 00:19:26,280 and then get incorporated into the protein catalysts 334 00:19:26,280 --> 00:19:29,490 and greatly expand the repertoire. 335 00:19:29,490 --> 00:19:36,070 So many of you probably-- how many of you take vitamins? 336 00:19:36,070 --> 00:19:38,415 Everybody should be taking vitamins. 337 00:19:38,415 --> 00:19:39,880 Why don't you take vitamins? 338 00:19:39,880 --> 00:19:46,570 Anyhow, so you can see vitamin B6, vitamin B2, vitamin B1. 339 00:19:46,570 --> 00:19:50,070 And over the next three weeks or so, 340 00:19:50,070 --> 00:19:54,430 we talk about the chemistry of how these vitamins interact 341 00:19:54,430 --> 00:19:56,950 with the protein catalyst to increase 342 00:19:56,950 --> 00:20:03,580 the repertoire of reactions to 10 that actual enzymes can 343 00:20:03,580 --> 00:20:04,930 catalyze. 344 00:20:04,930 --> 00:20:06,580 But in addition to the vitamins, I 345 00:20:06,580 --> 00:20:09,700 want to make mention of another type of catalyst. 346 00:20:09,700 --> 00:20:13,740 So most of the vitamins are organic molecules. 347 00:20:13,740 --> 00:20:17,130 One also needs to think about inorganic molecules. 348 00:20:23,340 --> 00:20:28,050 Inorganic molecules-- copper, zinc, iron, 349 00:20:28,050 --> 00:20:30,150 all those if you look at your vitamin bottle 350 00:20:30,150 --> 00:20:34,200 are at the bottom and they're labeled inorganic. 351 00:20:34,200 --> 00:20:41,280 And they almost always in introductory biochemistry 352 00:20:41,280 --> 00:20:43,662 courses get swept under the table. 353 00:20:43,662 --> 00:20:45,870 And in fact, many biologists don't think about metals 354 00:20:45,870 --> 00:20:46,710 at all. 355 00:20:46,710 --> 00:20:50,790 But 30% to 35% of all the enzymes 356 00:20:50,790 --> 00:20:52,750 have metals incorporated. 357 00:20:52,750 --> 00:20:56,910 And these metals are essential for the repertoire of reactions 358 00:20:56,910 --> 00:20:58,350 that enzymes can catalyze. 359 00:20:58,350 --> 00:21:00,990 So without going into any details, 360 00:21:00,990 --> 00:21:03,570 I just want to whet your appetite. 361 00:21:03,570 --> 00:21:05,250 Look at this guy. 362 00:21:05,250 --> 00:21:07,905 Well, what are we looking at here? 363 00:21:07,905 --> 00:21:10,650 These yellow things are sulfurs. 364 00:21:10,650 --> 00:21:14,720 The purple thing is molybdenum, and the green things are iron. 365 00:21:14,720 --> 00:21:17,490 And in the middle of all these irons 366 00:21:17,490 --> 00:21:23,790 is this silver thing, which is a carbon bonded to four irons. 367 00:21:23,790 --> 00:21:25,950 Most of you probably aren't sophisticated 368 00:21:25,950 --> 00:21:29,400 enough yet to think that's amazing, 369 00:21:29,400 --> 00:21:33,600 but it was only two years ago that the x-ray crystallography 370 00:21:33,600 --> 00:21:35,670 where we can look at things at atomic resolution 371 00:21:35,670 --> 00:21:38,290 was good enough so we can see that guy. 372 00:21:38,290 --> 00:21:39,540 So what does this guy do? 373 00:21:39,540 --> 00:21:40,920 What's its function? 374 00:21:40,920 --> 00:21:42,720 Pretty damn important. 375 00:21:42,720 --> 00:21:46,000 It converts nitrogen into ammonia. 376 00:21:46,000 --> 00:21:49,020 So it turns out to be an eight electron reduction 377 00:21:49,020 --> 00:21:51,660 because not only do you produce ammonia-- 378 00:21:51,660 --> 00:21:53,880 two molecules of ammonia-- but you also 379 00:21:53,880 --> 00:21:56,920 have to produce a molecule of hydrogen during that reaction. 380 00:21:57,725 --> 00:21:59,820 So this is the basic way we control 381 00:21:59,820 --> 00:22:03,000 nitrogen-- one of the basic ways we control 382 00:22:03,000 --> 00:22:04,770 nitrogen in the environment. 383 00:22:04,770 --> 00:22:07,980 So chemists would love to understand 384 00:22:07,980 --> 00:22:11,310 how this spectacular inorganic molecule can 385 00:22:11,310 --> 00:22:15,300 mediate what turns out to be a six electron reduction. 386 00:22:15,300 --> 00:22:18,730 Another molecule-- co-factor molecule 387 00:22:18,730 --> 00:22:21,780 that's all metal-based that I think is equally amazing 388 00:22:21,780 --> 00:22:23,670 is this one. 389 00:22:23,670 --> 00:22:26,840 We recently got an atomic resolution structure down 390 00:22:26,840 --> 00:22:28,950 to 1.5 angstroms. 391 00:22:28,950 --> 00:22:31,540 It has four manganese and a calcium. 392 00:22:31,540 --> 00:22:34,470 Anybody have any idea what this does? 393 00:22:34,470 --> 00:22:39,720 This is the co-factor that takes water 394 00:22:39,720 --> 00:22:42,060 in the presence of light-- sunlight-- 395 00:22:42,060 --> 00:22:43,950 and converts it to oxygen gas. 396 00:22:43,950 --> 00:22:45,750 Why is that important? 397 00:22:45,750 --> 00:22:49,110 Because we need oxygen gas to breathe. 398 00:22:49,110 --> 00:22:53,850 So anyhow, on this one co-factor mediates that transformation. 399 00:22:53,850 --> 00:22:55,500 Pretty amazing. 400 00:22:55,500 --> 00:22:57,450 And that's a major focus of people 401 00:22:57,450 --> 00:23:01,560 who want to think about how these catalysts actually work, 402 00:23:01,560 --> 00:23:04,830 but we won't be discussing that further. 403 00:23:04,830 --> 00:23:08,190 We won't be discussing that further in 507. 404 00:23:08,190 --> 00:23:15,720 So I just wanted to point out here that, again, enzymes 405 00:23:15,720 --> 00:23:18,980 have a limited repertoire. 406 00:23:18,980 --> 00:23:22,560 Their repertoire is much less than what chemists can do, 407 00:23:22,560 --> 00:23:26,050 but they're amazingly efficient at what they do. 408 00:23:26,050 --> 00:23:28,500 So I would argue if we really could understand 409 00:23:28,500 --> 00:23:31,830 the basis of catalysis and how these things evolve 410 00:23:31,830 --> 00:23:35,370 to be able to do these amazing transformations, 411 00:23:35,370 --> 00:23:38,700 we might, if I was able to come back 50 years from now, 412 00:23:38,700 --> 00:23:42,240 see that we had designer proteins all over the place 413 00:23:42,240 --> 00:23:45,930 that could catalyze the specific reactions that we're 414 00:23:45,930 --> 00:23:51,570 interested in, not the ones that are found in our bodies. 415 00:23:51,570 --> 00:23:56,340 OK, so the next thing I want to briefly mention 416 00:23:56,340 --> 00:23:58,505 is that enzymes, so if you look at an enzyme, 417 00:23:58,505 --> 00:24:01,020 it's a big macro molecule. 418 00:24:01,020 --> 00:24:04,560 We've looked at these in the last few lectures. 419 00:24:04,560 --> 00:24:08,370 The region where the chemistry or catalysis occurs 420 00:24:08,370 --> 00:24:10,440 is called the active site. 421 00:24:10,440 --> 00:24:14,700 And we've seen this before in the TIN barrel 422 00:24:14,700 --> 00:24:17,860 superfamily of proteins. 423 00:24:17,860 --> 00:24:22,410 And so there's a region of about 10 angstroms. 424 00:24:22,410 --> 00:24:24,234 We have your amino acid side chains 425 00:24:24,234 --> 00:24:26,400 that I asked you to try to remember and think about. 426 00:24:26,400 --> 00:24:28,710 We'll see those are key to making these rate 427 00:24:28,710 --> 00:24:32,340 accelerations so fantastic. 428 00:24:32,340 --> 00:24:34,620 This is where the chemistry happens. 429 00:24:34,620 --> 00:24:36,930 But I think it's now clear from studies 430 00:24:36,930 --> 00:24:39,210 that have been done in the last 15 years 431 00:24:39,210 --> 00:24:41,580 or so this is not true. 432 00:24:41,580 --> 00:24:45,420 One can make changes out here or here. 433 00:24:45,420 --> 00:24:50,850 One can change the amino acids and totally turn off the enzyme 434 00:24:50,850 --> 00:24:52,800 or turn on the enzyme. 435 00:24:52,800 --> 00:24:56,340 So chemists use these small little molecules, 436 00:24:56,340 --> 00:24:58,860 biology uses big huge molecules. 437 00:24:58,860 --> 00:25:02,880 Everybody initially focused on this one little region 438 00:25:02,880 --> 00:25:06,120 where you can see the chemical transformation occurring. 439 00:25:06,120 --> 00:25:08,350 But what about the rest of the molecule? 440 00:25:08,350 --> 00:25:11,580 The rest of the molecule is also important. 441 00:25:11,580 --> 00:25:16,950 You cannot remove, in general, all of this spinach and come up 442 00:25:16,950 --> 00:25:21,420 with a catalyst that has these amazing rate accelerations. 443 00:25:21,420 --> 00:25:23,850 So the active site is very important. 444 00:25:28,930 --> 00:25:39,341 But so are specific amino acids outside of the active site. 445 00:25:43,720 --> 00:25:46,750 And people have studied this because of technology 446 00:25:46,750 --> 00:25:48,970 of sight directed mutagenesis, which many of you 447 00:25:48,970 --> 00:25:55,180 have probably done in either 702 or in 335. 448 00:25:55,180 --> 00:25:57,280 So what implications does that have? 449 00:25:57,280 --> 00:25:59,750 And I just want to mention one more thing. 450 00:25:59,750 --> 00:26:01,600 I don't want to spend a lot of time on this, 451 00:26:01,600 --> 00:26:07,390 but our thinking about catalysis is changing dramatically 452 00:26:07,390 --> 00:26:09,430 and has changed and continues to change. 453 00:26:09,430 --> 00:26:14,200 I continue to study this, even to teach 507. 454 00:26:14,200 --> 00:26:18,730 Because it turns out, how does change out here 455 00:26:18,730 --> 00:26:21,280 govern what's going on in this region 456 00:26:21,280 --> 00:26:23,860 where you think the chemistry happens? 457 00:26:23,860 --> 00:26:27,040 And it governs that chemistry because 458 00:26:27,040 --> 00:26:30,460 of conformational changes and movements. 459 00:26:30,460 --> 00:26:33,040 So another thing about enzymes that we 460 00:26:33,040 --> 00:26:34,930 need to do more thinking about-- and this 461 00:26:34,930 --> 00:26:40,330 is a major focus of what people are thinking about now-- 462 00:26:40,330 --> 00:26:47,110 is dynamics in enzyme catalyzed reactions. 463 00:26:47,110 --> 00:26:50,230 And so if you look at the time scale-- and I made 464 00:26:50,230 --> 00:26:53,590 you think about size scale in the first few lectures. 465 00:26:53,590 --> 00:26:58,210 Like how long is a hydrogen bond? 466 00:26:58,210 --> 00:27:00,450 How long is a carbon nitrogen bond? 467 00:27:00,450 --> 00:27:01,720 A carbon oxygen bond? 468 00:27:01,720 --> 00:27:05,060 You also need to think about time scales. 469 00:27:05,060 --> 00:27:08,110 And this is particularly true in the case of catalysis. 470 00:27:08,110 --> 00:27:12,070 What happens on the fantasecond time scale? 471 00:27:12,070 --> 00:27:13,540 That's pretty fast. 472 00:27:13,540 --> 00:27:16,270 That's a vibration of the bond. 473 00:27:16,270 --> 00:27:19,540 But what are you doing during an enzyme catalyzed reaction? 474 00:27:19,540 --> 00:27:22,030 You're breaking the bond and you're making the bond. 475 00:27:22,030 --> 00:27:24,790 So we'll see that the transition state of the reaction 476 00:27:24,790 --> 00:27:27,480 happens on the fantasecond time scale. 477 00:27:27,480 --> 00:27:31,210 Yet, if you look at the criteria kcat, which 478 00:27:31,210 --> 00:27:33,100 is a turnover number, the enzyme, which 479 00:27:33,100 --> 00:27:35,500 is given in time inverse, they're 480 00:27:35,500 --> 00:27:41,850 usually on 10 per second to 1,000 per second. 481 00:27:41,850 --> 00:27:44,480 So they're on the millisecond to second time scale. 482 00:27:44,480 --> 00:27:48,130 So catalysis is happening way up here. 483 00:27:48,130 --> 00:27:49,960 Now, I've just told you that mutations 484 00:27:49,960 --> 00:27:53,080 outside the active site can affect catalysis, 485 00:27:53,080 --> 00:27:57,010 and so one also needs to think about the time scales 486 00:27:57,010 --> 00:27:59,530 in between these two extremes. 487 00:27:59,530 --> 00:28:02,650 I've also told you that finding an enzyme, finding 488 00:28:02,650 --> 00:28:08,000 its substrate in solution, can often be the slow step. 489 00:28:08,000 --> 00:28:11,737 So here you have nanosecond, microsecond time scales, 490 00:28:11,737 --> 00:28:13,570 and I'm not going to spend any time on this, 491 00:28:13,570 --> 00:28:16,000 but you come back and look at this 492 00:28:16,000 --> 00:28:17,770 and think about you've got all these side 493 00:28:17,770 --> 00:28:19,190 chains of your amino acids. 494 00:28:19,190 --> 00:28:21,670 You might have loops that are moving in and out 495 00:28:21,670 --> 00:28:24,160 and covering the active site. 496 00:28:24,160 --> 00:28:28,660 All of this dynamic interaction plays a key role in catalysis, 497 00:28:28,660 --> 00:28:33,610 making the enzyme as a whole important 498 00:28:33,610 --> 00:28:37,090 in the overall catalytic process. 499 00:28:37,090 --> 00:28:42,400 So that's my introduction to you for what an enzyme catalyst is. 500 00:28:42,400 --> 00:28:49,120 And so now what I want to do is look at the second bullet 501 00:28:49,120 --> 00:28:52,290 we were going to talk about, which I've already lost. 502 00:28:58,120 --> 00:28:59,770 How do we describe catalysis? 503 00:28:59,770 --> 00:29:04,870 How do we try to conceptualize in a theoretical framework 504 00:29:04,870 --> 00:29:07,180 all of the experimental observations that 505 00:29:07,180 --> 00:29:09,310 have been made for decades? 506 00:29:09,310 --> 00:29:12,400 And there are many things that are wrong with this theory, 507 00:29:12,400 --> 00:29:16,180 but this theory has stood the test of time, 508 00:29:16,180 --> 00:29:20,830 not only for biochemists, but for also chemists. 509 00:29:20,830 --> 00:29:24,820 And I think it helps us to think about how enzymes 510 00:29:24,820 --> 00:29:28,900 are able with just the amino acid side chains for protein 511 00:29:28,900 --> 00:29:33,460 to give us these amazing rate accelerations and specificity 512 00:29:33,460 --> 00:29:35,300 that we actually observe. 513 00:29:35,300 --> 00:29:46,020 So what we want is a theory to conceptualize catalysis. 514 00:29:48,700 --> 00:29:50,680 And this is transition state theory. 515 00:29:58,480 --> 00:30:02,710 And this is-- many of you have seen this in some form 516 00:30:02,710 --> 00:30:05,140 before, either in freshman chemistry 517 00:30:05,140 --> 00:30:07,570 or maybe if you've had 560. 518 00:30:07,570 --> 00:30:10,870 People go through and derive all of the rate equations. 519 00:30:10,870 --> 00:30:14,500 What I'm going to do is just show you 520 00:30:14,500 --> 00:30:17,230 a picture of how this theory helps 521 00:30:17,230 --> 00:30:20,170 us think about these catalytic transformations 522 00:30:20,170 --> 00:30:23,920 and then how this picture helps us think more specifically 523 00:30:23,920 --> 00:30:26,320 about these amazing rate accelerations 524 00:30:26,320 --> 00:30:28,850 that we actually observe. 525 00:30:28,850 --> 00:30:33,430 OK, so I can't remember what's on the next slide, 526 00:30:33,430 --> 00:30:36,280 but this is a picture you often see when you're 527 00:30:36,280 --> 00:30:40,240 thinking about catalysis. 528 00:30:40,240 --> 00:30:45,310 So this is chemical catalysis, but again, chemical catalysis, 529 00:30:45,310 --> 00:30:49,060 biological catalysis, really the same basic principles 530 00:30:49,060 --> 00:30:59,190 hold that we have some substrates A 531 00:30:59,190 --> 00:31:05,090 and B going to products and what's required. 532 00:31:05,090 --> 00:31:07,370 So I think all of this is intuitive, 533 00:31:07,370 --> 00:31:10,880 but if you have two things coming together, 534 00:31:10,880 --> 00:31:13,340 they have to come together in exactly the right way 535 00:31:13,340 --> 00:31:15,890 to be able to make a bond. 536 00:31:15,890 --> 00:31:20,810 They have to remove all the solvent from outside them. 537 00:31:20,810 --> 00:31:23,420 They have to come together with enough force 538 00:31:23,420 --> 00:31:27,050 to be able to get over the barrier, whatever it is, 539 00:31:27,050 --> 00:31:30,770 to break one bond and to form a new bond. 540 00:31:30,770 --> 00:31:35,630 So that's true of all reactions and everybody faces 541 00:31:35,630 --> 00:31:40,250 the same issues in terms of conversion of substrate 542 00:31:40,250 --> 00:31:41,600 into products. 543 00:31:41,600 --> 00:31:44,030 And the highest point along the reaction 544 00:31:44,030 --> 00:31:46,700 coordinate-- so this is what we call 545 00:31:46,700 --> 00:31:48,370 a reaction coordinate diagram. 546 00:31:55,120 --> 00:31:57,130 And this is energy. 547 00:31:57,130 --> 00:32:00,940 So the highest point along the reaction coordinate diagram 548 00:32:00,940 --> 00:32:04,930 is called the transition state-- TS. 549 00:32:04,930 --> 00:32:08,060 This is transition state theory. 550 00:32:08,060 --> 00:32:10,390 OK, TS theory. 551 00:32:10,390 --> 00:32:13,180 And this is where-- this is the point where we can ever 552 00:32:13,180 --> 00:32:15,879 isolate it because this is a point where 553 00:32:15,879 --> 00:32:17,170 all the chemistry is happening. 554 00:32:17,170 --> 00:32:19,210 The bonds are being made and broken. 555 00:32:19,210 --> 00:32:21,690 And the lifetime I just showed you on the previous slide 556 00:32:21,690 --> 00:32:23,530 is fast-- fentaseconds. 557 00:32:23,530 --> 00:32:26,710 So you can never isolate a transition state. 558 00:32:26,710 --> 00:32:28,420 Everything needs to be aligned. 559 00:32:28,420 --> 00:32:30,010 That doesn't come free of charge. 560 00:32:30,010 --> 00:32:32,170 You have to do a lot of work to get to the stage 561 00:32:32,170 --> 00:32:34,420 where you can get this chemistry to happen. 562 00:32:34,420 --> 00:32:36,430 That's what our catalysts are doing. 563 00:32:36,430 --> 00:32:42,020 And then bang, the reaction is over at that time. 564 00:32:42,020 --> 00:32:45,130 So this is another way of describing the transition 565 00:32:45,130 --> 00:32:46,870 state of the reaction. 566 00:32:46,870 --> 00:32:50,260 And in reality, this is the cartoon 567 00:32:50,260 --> 00:32:58,270 you see in most introductory textbooks that are describing 568 00:32:58,270 --> 00:32:59,590 rates of reaction. 569 00:32:59,590 --> 00:33:03,100 But the reaction coordinate is much, much more complicated. 570 00:33:03,100 --> 00:33:06,250 And that's true in enzymatic reactions as well. 571 00:33:06,250 --> 00:33:08,320 So it's true of chemical reactions, 572 00:33:08,320 --> 00:33:10,001 it's true enzymatic reactions. 573 00:33:13,360 --> 00:33:16,570 So you might have a plus b, and they 574 00:33:16,570 --> 00:33:22,240 might form two or three intermediates 575 00:33:22,240 --> 00:33:27,580 along the reaction pathway where you 576 00:33:27,580 --> 00:33:32,410 have many transitions-- you have many transition states 577 00:33:32,410 --> 00:33:34,990 along the reaction pathway. 578 00:33:34,990 --> 00:33:39,610 And each of these transitions states would be non-isolable. 579 00:33:39,610 --> 00:33:41,360 But what about these little valleys? 580 00:33:41,360 --> 00:33:43,610 These little valleys are where you might have a chance 581 00:33:43,610 --> 00:33:46,810 to see an intermediate during the conversion of a plus 582 00:33:46,810 --> 00:33:49,450 b into p plus q. 583 00:33:49,450 --> 00:33:53,830 So an intermediate-- and if you're 584 00:33:53,830 --> 00:33:57,430 interested in studying catalysis and the chemistry 585 00:33:57,430 --> 00:33:59,470 of the reaction and you need to define 586 00:33:59,470 --> 00:34:03,350 what these intermediates are, they can be high 587 00:34:03,350 --> 00:34:04,930 or that could be lower in energy. 588 00:34:04,930 --> 00:34:07,900 They may be easy to isolate, not easy to isolate. 589 00:34:07,900 --> 00:34:14,230 But they have all covalent bonds intact. 590 00:34:14,230 --> 00:34:17,489 So in contrast to the transition state where 591 00:34:17,489 --> 00:34:19,466 the bonds are being made and broken, 592 00:34:19,466 --> 00:34:20,590 you can never isolate this. 593 00:34:20,590 --> 00:34:23,980 You have a chance to be able-- if you're clever and creative, 594 00:34:23,980 --> 00:34:27,130 which people that study mechanisms are, 595 00:34:27,130 --> 00:34:29,920 you can actually look at the intermediates 596 00:34:29,920 --> 00:34:32,409 along the reaction coordinate. 597 00:34:32,409 --> 00:34:34,485 So that's a reaction coordinate diagram. 598 00:34:34,485 --> 00:34:35,860 We're going to come back to these 599 00:34:35,860 --> 00:34:41,290 because I think they really help us to conceptualize how enzymes 600 00:34:41,290 --> 00:34:46,510 can go about achieving these fantastic rate accelerations. 601 00:34:46,510 --> 00:34:52,780 So from transition state theory, one 602 00:34:52,780 --> 00:34:54,400 assumes the following-- I'm not going 603 00:34:54,400 --> 00:34:56,889 to go through the details of this at all. 604 00:34:56,889 --> 00:34:59,980 But the key point that one needs to think about 605 00:34:59,980 --> 00:35:02,880 in transition state theory is that-- 606 00:35:02,880 --> 00:35:06,250 and this was first put forth by Linus Pauling. 607 00:35:06,250 --> 00:35:07,270 Who's Linus Pauling? 608 00:35:07,270 --> 00:35:08,730 He's my hero. 609 00:35:08,730 --> 00:35:10,920 OK, Linus Pauling, he's the vitamin C guy. 610 00:35:10,920 --> 00:35:13,630 He lost it when he got old, but in the early days, 611 00:35:13,630 --> 00:35:16,540 he's the one that could take a polypeptide chain-- just 612 00:35:16,540 --> 00:35:19,510 a string of amino acids-- and he sat there 613 00:35:19,510 --> 00:35:20,950 and he played with it. 614 00:35:20,950 --> 00:35:22,510 And lo and behold, he says, we're 615 00:35:22,510 --> 00:35:24,880 going to have alpha helices in proteins. 616 00:35:24,880 --> 00:35:25,930 How amazing is that? 617 00:35:25,930 --> 00:35:27,820 You've heard me talk about him before. 618 00:35:27,820 --> 00:35:31,750 He was the one that I think conceptualized-- first 619 00:35:31,750 --> 00:35:36,260 conceptualized-- how an enzyme might catalyze a reaction. 620 00:35:36,260 --> 00:35:39,610 What do you want to do to catalyze a reaction? 621 00:35:39,610 --> 00:35:42,640 You want to lower this barrier. 622 00:35:42,640 --> 00:35:45,100 So how do you lower the barrier? 623 00:35:45,100 --> 00:35:48,640 You don't want the enzyme to bind the substrates tightly, 624 00:35:48,640 --> 00:35:50,320 and I'll come back to this in a minute. 625 00:35:50,320 --> 00:35:54,790 You want to bind the transition state tightly. 626 00:35:54,790 --> 00:35:59,170 So he put forth in the 1940s that the way enzymes 627 00:35:59,170 --> 00:36:01,510 might be able to catalyze their reactions is 628 00:36:01,510 --> 00:36:05,320 by tightly binding-- uniquely and tightly binding 629 00:36:05,320 --> 00:36:07,850 the transition state of the reaction. 630 00:36:07,850 --> 00:36:11,410 And I think that turns out to be a really good way 631 00:36:11,410 --> 00:36:15,010 to conceptualize most enzymatic reactions. 632 00:36:15,010 --> 00:36:17,950 Now, transition state theory tells 633 00:36:17,950 --> 00:36:22,430 us, which again is not so appealing to me 634 00:36:22,430 --> 00:36:26,060 but it works to describe most experimental data, 635 00:36:26,060 --> 00:36:30,050 that the ground state-- so this would be the ground state-- 636 00:36:30,050 --> 00:36:32,565 is in equilibrium with the transition state. 637 00:36:32,565 --> 00:36:34,190 So you might ask yourself, how the heck 638 00:36:34,190 --> 00:36:36,064 can you ever be in equilibrium with something 639 00:36:36,064 --> 00:36:37,880 with such a short half life? 640 00:36:37,880 --> 00:36:39,920 That's a good question to ask. 641 00:36:39,920 --> 00:36:44,240 But in fact, this framework-- transition state theory-- 642 00:36:44,240 --> 00:36:47,030 allows us to able to explain almost 643 00:36:47,030 --> 00:36:49,280 all the experimental observations 644 00:36:49,280 --> 00:36:52,850 that we make as both chemists and biochemists. 645 00:36:52,850 --> 00:36:55,607 So this goes through and derives that equation, which 646 00:36:55,607 --> 00:36:56,690 I'm not going to do today. 647 00:36:56,690 --> 00:36:58,606 In the old days, I used to spend a lot of time 648 00:36:58,606 --> 00:37:00,260 deriving equations. 649 00:37:00,260 --> 00:37:03,860 Nowadays, I don't derive equations anymore. 650 00:37:03,860 --> 00:37:06,590 But the key equation that you need to think about 651 00:37:06,590 --> 00:37:09,580 is shown here. 652 00:37:09,580 --> 00:37:16,550 And the consequences of this equation are quite simple. 653 00:37:16,550 --> 00:37:22,850 It tells you that the rate constant for the reaction-- so 654 00:37:22,850 --> 00:37:29,820 from transition state theory, the rate 655 00:37:29,820 --> 00:37:31,151 constant for the reaction. 656 00:37:39,420 --> 00:37:41,140 And where is this rate constant? 657 00:37:41,140 --> 00:37:44,350 Where does this rate constant come from? 658 00:37:44,350 --> 00:37:45,670 A is going to some product p. 659 00:37:45,670 --> 00:37:47,090 You can measure it experimentally. 660 00:37:47,090 --> 00:37:50,560 So k observed is an experimentally measurable 661 00:37:50,560 --> 00:37:56,650 parameter is equal to a bunch of constants called 662 00:37:56,650 --> 00:37:58,110 the transmission coefficient. 663 00:37:58,110 --> 00:38:00,130 This should be a cappa. 664 00:38:00,130 --> 00:38:03,460 Boltzmann's constant, temperature 665 00:38:03,460 --> 00:38:08,050 in degrees Kelvin, Planck's constant times 666 00:38:08,050 --> 00:38:14,440 e to the minus delta g dagger over rg. 667 00:38:14,440 --> 00:38:15,624 So this is the equation. 668 00:38:15,624 --> 00:38:16,415 This is a constant. 669 00:38:18,920 --> 00:38:21,970 This is Planck's constant, Boltzmann's constant. 670 00:38:21,970 --> 00:38:23,590 This you can measure experimentally. 671 00:38:23,590 --> 00:38:27,730 Cappa is telling you basically-- the transmission coefficient 672 00:38:27,730 --> 00:38:30,940 is telling you the frequency that this transition state 673 00:38:30,940 --> 00:38:34,210 breaks down to form products versus going back 674 00:38:34,210 --> 00:38:38,130 to starting materials and in general, is on the order of one 675 00:38:38,130 --> 00:38:39,850 in most reactions. 676 00:38:39,850 --> 00:38:43,120 And so the key thing to remember from this equation, which 677 00:38:43,120 --> 00:38:48,280 explains the data and helps us to think about catalysis, 678 00:38:48,280 --> 00:38:54,910 is that as you increase the rate of the reaction, 679 00:38:54,910 --> 00:38:58,510 it's inversely related to the activation barrier. 680 00:38:58,510 --> 00:39:01,120 So what you want to do, this equation tells you, 681 00:39:01,120 --> 00:39:04,840 is you lower this barrier. 682 00:39:04,840 --> 00:39:08,140 The rate of your reaction becomes faster and faster. 683 00:39:08,140 --> 00:39:10,450 So the whole goal is, then, to figure out 684 00:39:10,450 --> 00:39:11,740 how to lower the barrier. 685 00:39:11,740 --> 00:39:14,290 If you can lower the barrier, this theory 686 00:39:14,290 --> 00:39:18,310 predicts that the rate of your reaction will be faster. 687 00:39:18,310 --> 00:39:20,980 So that's what we want to be able to do. 688 00:39:20,980 --> 00:39:24,760 The rate constant is inversely related 689 00:39:24,760 --> 00:39:28,880 to the activation barrier. 690 00:39:28,880 --> 00:39:33,324 And so now let's look at an enzyme system specifically. 691 00:39:36,700 --> 00:39:39,130 So I'm going to draw the same kind of reaction 692 00:39:39,130 --> 00:39:45,040 coordinate that we've drawn over there for a chemical reaction. 693 00:39:45,040 --> 00:39:49,520 And I'm going to use a simple equation. 694 00:39:49,520 --> 00:39:56,890 E is the enzyme, s is the substrate forms 695 00:39:56,890 --> 00:39:58,750 an enzyme substrate complex. 696 00:39:58,750 --> 00:40:00,400 The substrate binds in the region 697 00:40:00,400 --> 00:40:04,340 that we call the active site over here. 698 00:40:04,340 --> 00:40:11,110 Somehow, the enzyme is able to convert itself into product. 699 00:40:11,110 --> 00:40:13,560 Now, most reactions are much more complicated than this. 700 00:40:13,560 --> 00:40:14,601 You have many substrates. 701 00:40:14,601 --> 00:40:17,260 You have many products. 702 00:40:17,260 --> 00:40:19,540 But it doesn't affect anything in terms 703 00:40:19,540 --> 00:40:21,550 of thinking about the problem. 704 00:40:21,550 --> 00:40:25,900 And then in the end, the product dissociates. 705 00:40:25,900 --> 00:40:27,640 So that's a simple reaction. 706 00:40:27,640 --> 00:40:30,640 You get something in there, a catalyst works on it, 707 00:40:30,640 --> 00:40:32,350 it gets converted to the desired product, 708 00:40:32,350 --> 00:40:35,170 and the product is released. 709 00:40:35,170 --> 00:40:38,470 So what I've told you now a couple of times 710 00:40:38,470 --> 00:40:42,730 is that enzymes have evolved to such an extent 711 00:40:42,730 --> 00:40:46,090 that often the physical steps and not the chemistry 712 00:40:46,090 --> 00:40:47,140 is rate limiting. 713 00:40:47,140 --> 00:40:50,290 So what are the physical steps? 714 00:40:50,290 --> 00:40:53,920 Here are the physical steps. 715 00:40:53,920 --> 00:40:56,770 Enzyme finding substrate and solution, 716 00:40:56,770 --> 00:40:58,250 that's a physical step. 717 00:40:58,250 --> 00:40:59,800 What is limited by? 718 00:40:59,800 --> 00:41:01,900 It's limited by diffusion control. 719 00:41:01,900 --> 00:41:05,230 How fast can they find each other in solution? 720 00:41:05,230 --> 00:41:07,840 That's the number 10 to the 8 per molar 721 00:41:07,840 --> 00:41:11,500 per second that limits most enzyme-based reactions that I 722 00:41:11,500 --> 00:41:14,020 showed you several slides ago. 723 00:41:14,020 --> 00:41:15,670 What about this? 724 00:41:15,670 --> 00:41:21,100 We have product dissociation. 725 00:41:21,100 --> 00:41:22,510 What about product dissociation? 726 00:41:22,510 --> 00:41:23,670 That's a physical step too. 727 00:41:23,670 --> 00:41:25,640 You made the product sitting around, 728 00:41:25,640 --> 00:41:29,320 but in order for the enzyme to turn over, again, 729 00:41:29,320 --> 00:41:31,240 to free up the active site, the product 730 00:41:31,240 --> 00:41:35,410 has to come off so it can bind another substrate. 731 00:41:35,410 --> 00:41:40,390 And here is the chemistry. 732 00:41:40,390 --> 00:41:42,810 Ah, that's what I care about. 733 00:41:42,810 --> 00:41:47,430 But what happens, now, is that if these steps are 734 00:41:47,430 --> 00:41:51,120 rate limiting, then you can't see the chemistry. 735 00:41:51,120 --> 00:41:54,240 So it's really challenging, often really challenging, 736 00:41:54,240 --> 00:41:57,600 to study the chemistry of a reaction 737 00:41:57,600 --> 00:42:00,420 because the rate limiting steps have nothing 738 00:42:00,420 --> 00:42:01,800 to do with the chemistry. 739 00:42:01,800 --> 00:42:04,560 So let me just draw a diagram. 740 00:42:04,560 --> 00:42:06,754 So you can draw a reaction coordinate diagram. 741 00:42:09,320 --> 00:42:16,530 And so what you have is some enzyme plus substrate 742 00:42:16,530 --> 00:42:22,164 and it can form an enzyme substrate complex. 743 00:42:26,430 --> 00:42:29,160 You have a transition state of your reaction. 744 00:42:29,160 --> 00:42:32,490 The enzyme product complex can then 745 00:42:32,490 --> 00:42:37,080 dissociate to form enzyme plus product. 746 00:42:37,080 --> 00:42:38,940 So what you need to think about if you're 747 00:42:38,940 --> 00:42:41,940 thinking about how to accelerate the reaction is 748 00:42:41,940 --> 00:42:44,797 what is the bottleneck in the overall reaction? 749 00:42:44,797 --> 00:42:46,380 You don't want to start mucking around 750 00:42:46,380 --> 00:42:47,910 with something that doesn't control 751 00:42:47,910 --> 00:42:48,980 the rate of the reaction. 752 00:42:48,980 --> 00:42:51,900 So you need to know what the rate limiting step 753 00:42:51,900 --> 00:42:53,670 is in the reaction. 754 00:42:53,670 --> 00:42:59,580 And the rate limiting step is the highest barrier 755 00:42:59,580 --> 00:43:01,121 along the reaction coordinate. 756 00:43:11,090 --> 00:43:17,760 OK, now I've already told you that this is a simple case. 757 00:43:17,760 --> 00:43:21,210 We have one substrate getting converted into product. 758 00:43:21,210 --> 00:43:26,280 Most enzymatic reactions are going to have many barriers. 759 00:43:26,280 --> 00:43:31,380 And so in order to affect the overall rate of the reaction, 760 00:43:31,380 --> 00:43:33,690 you need to figure out what's rate limiting, 761 00:43:33,690 --> 00:43:37,200 and somehow the enzyme has figured out 762 00:43:37,200 --> 00:43:41,710 how to lower the barrier to make this reaction easier to occur. 763 00:43:41,710 --> 00:43:46,560 Remember, I just told you that the rate constant is inversely 764 00:43:46,560 --> 00:43:48,880 related to this activation barrier. 765 00:43:48,880 --> 00:43:54,060 So if we can lower this barrier somehow, 766 00:43:54,060 --> 00:43:56,310 what we're going to see, if we can lower this barrier, 767 00:43:56,310 --> 00:44:01,460 now we have a lower overall rate of the reaction. 768 00:44:01,460 --> 00:44:05,430 So this theory allows us to think 769 00:44:05,430 --> 00:44:10,200 about what we need to do to make these catalysts actually 770 00:44:10,200 --> 00:44:14,070 work with rate constants of 10 to 6 to 10 to 15 times 771 00:44:14,070 --> 00:44:18,000 faster than non-catalyzed reactions. 772 00:44:18,000 --> 00:44:20,970 And I want to say one other thing before you move on. 773 00:44:20,970 --> 00:44:24,510 As with everything, I think it's good 774 00:44:24,510 --> 00:44:29,790 that we're in a field that's rapidly changing. 775 00:44:29,790 --> 00:44:33,370 Remember, I told you have to think about dynamics. 776 00:44:33,370 --> 00:44:37,170 We no longer think about a single reaction barrier. 777 00:44:37,170 --> 00:44:39,360 That's in most of the textbooks now. 778 00:44:39,360 --> 00:44:43,710 Really what we think about is we bring dynamics into this. 779 00:44:43,710 --> 00:44:46,950 I told you things outside the active site 780 00:44:46,950 --> 00:44:50,310 can modulate what's going on inside the active site. 781 00:44:50,310 --> 00:44:53,610 What we think about is a reaction landscape. 782 00:44:53,610 --> 00:44:57,750 And so one has many barriers that one has to get over. 783 00:44:57,750 --> 00:45:01,921 Almost all reactions involve multiple barriers. 784 00:45:01,921 --> 00:45:04,170 So you've got to figure out which one is rate limiting 785 00:45:04,170 --> 00:45:08,550 and lower that activation barrier. 786 00:45:08,550 --> 00:45:11,790 And enzymes, if you think about this, they're huge. 787 00:45:11,790 --> 00:45:13,920 Do they all fold exactly the same way? 788 00:45:13,920 --> 00:45:15,270 No. 789 00:45:15,270 --> 00:45:17,710 So we always think we have a homogeneous enzyme. 790 00:45:17,710 --> 00:45:18,480 No. 791 00:45:18,480 --> 00:45:20,310 If any of you work in UROPs, you'll 792 00:45:20,310 --> 00:45:21,630 find that out pretty fast. 793 00:45:21,630 --> 00:45:26,250 You use recombinant technology to fold things inside the cell. 794 00:45:26,250 --> 00:45:27,830 They don't all fold right. 795 00:45:27,830 --> 00:45:30,510 So you have all mixtures of things. 796 00:45:30,510 --> 00:45:32,700 And so you get a reaction landscape. 797 00:45:32,700 --> 00:45:38,970 And so this axis is bringing in the dynamics 798 00:45:38,970 --> 00:45:40,740 that I told you about earlier on that you 799 00:45:40,740 --> 00:45:43,560 need to think about-- the conformational changes that 800 00:45:43,560 --> 00:45:46,410 occur every step along the reaction pathway. 801 00:45:46,410 --> 00:45:51,000 The enzyme is moving at all kinds of steps, 802 00:45:51,000 --> 00:45:57,300 reorienting everything to get the chemistry exactly right. 803 00:45:57,300 --> 00:46:02,220 So what I want to do now is-- so that gives you 804 00:46:02,220 --> 00:46:07,110 a way to conceptualize rate accelerations. 805 00:46:07,110 --> 00:46:08,790 Now what I want to do is tell you 806 00:46:08,790 --> 00:46:13,440 what the major mechanisms are that the enzyme uses to enhance 807 00:46:13,440 --> 00:46:14,730 the rates of these reactions. 808 00:46:14,730 --> 00:46:18,660 How do we lower these energy barriers? 809 00:46:18,660 --> 00:46:19,490 So let me see. 810 00:46:19,490 --> 00:46:22,100 I need to start erasing somewhere. 811 00:46:26,490 --> 00:46:29,263 OK, so we're on the third bullet over here. 812 00:46:32,280 --> 00:46:34,540 Mechanisms of catalysis. 813 00:46:34,540 --> 00:46:36,300 And what we're going to be talking about 814 00:46:36,300 --> 00:46:39,070 is multiple mechanisms of catalysis. 815 00:46:39,070 --> 00:46:43,590 We're going to be talking about binding energy, which 816 00:46:43,590 --> 00:46:47,120 is the one people have most trouble thinking about. 817 00:46:47,120 --> 00:46:55,950 We're going to be talking about general acid, general base 818 00:46:55,950 --> 00:46:57,350 catalysis. 819 00:46:57,350 --> 00:47:02,580 And we're going to be talking about covalent catalysis. 820 00:47:02,580 --> 00:47:06,006 And we will see that over the course 821 00:47:06,006 --> 00:47:07,380 of the rest of the semester, when 822 00:47:07,380 --> 00:47:09,810 we start talking about metabolic pathways, 823 00:47:09,810 --> 00:47:13,590 all of these mechanisms are used in almost 824 00:47:13,590 --> 00:47:16,430 all enzyme catalyzed reactions to give us 825 00:47:16,430 --> 00:47:20,785 these tremendous rate accelerations. 826 00:47:20,785 --> 00:47:24,060 What I want to do-- that's the first time I did that. 827 00:47:24,060 --> 00:47:25,106 That wasn't too bad. 828 00:47:30,720 --> 00:47:33,490 OK, so what are the mechanisms of catalysis? 829 00:47:36,300 --> 00:47:46,154 How do we get 10 to the 6 to 10 to the 15 accelerations? 830 00:47:49,590 --> 00:47:51,870 And so the first thing, and I think 831 00:47:51,870 --> 00:47:56,250 the one that really is unique to enzyme catalysts compared 832 00:47:56,250 --> 00:47:59,640 to small inorganic or organic molecules, 833 00:47:59,640 --> 00:48:03,550 is the use of binding energy in catalysis. 834 00:48:03,550 --> 00:48:05,610 So this is the one-- and this is also 835 00:48:05,610 --> 00:48:08,450 the one that's thought to contribute the greatest 836 00:48:08,450 --> 00:48:13,700 amount to these factors of up to 10 to 15. 837 00:48:13,700 --> 00:48:23,200 So binding energy in catalysis, and what does that mean? 838 00:48:23,200 --> 00:48:26,100 What do we need to think about? 839 00:48:26,100 --> 00:48:29,370 So the enzyme binds to a substrate. 840 00:48:29,370 --> 00:48:34,470 If we take this simple case, we need it to bind. 841 00:48:34,470 --> 00:48:36,210 We need it to bind specifically. 842 00:48:36,210 --> 00:48:37,740 So that's a key part of the enzyme 843 00:48:37,740 --> 00:48:40,020 that we haven't gotten to yet-- specificity. 844 00:48:40,020 --> 00:48:43,280 But what if it bound its substrate really tightly? 845 00:48:43,280 --> 00:48:46,220 Do you think that would be good? 846 00:48:46,220 --> 00:48:47,220 No. 847 00:48:47,220 --> 00:48:49,500 So it's not good because what does it do? 848 00:48:49,500 --> 00:48:57,090 If it took all of the spinach changing off of your substrate 849 00:48:57,090 --> 00:48:59,820 and made hydrogen bonds and Van Der Waals interactions, 850 00:48:59,820 --> 00:49:03,000 all the weak non-covalent interactions 851 00:49:03,000 --> 00:49:05,280 we spent a half a lecture talking 852 00:49:05,280 --> 00:49:11,691 about four or five lectures ago, what would happen is you 853 00:49:11,691 --> 00:49:12,690 would have type binding. 854 00:49:12,690 --> 00:49:14,520 You would have lower energy. 855 00:49:14,520 --> 00:49:20,100 But what does that then do to the activation barrier? 856 00:49:20,100 --> 00:49:23,580 It increases the activation barrier. 857 00:49:23,580 --> 00:49:37,510 So the binding energy is the free energy released when 858 00:49:37,510 --> 00:49:39,400 enzyme combines with substrate. 859 00:49:45,380 --> 00:49:49,080 But the key is that this bind energy is not 860 00:49:49,080 --> 00:49:51,420 used to bind completely. 861 00:49:51,420 --> 00:49:53,410 It's used for catalysis. 862 00:49:53,410 --> 00:50:06,690 So this energy is used both to bind substrate 863 00:50:06,690 --> 00:50:09,524 and-- and this is the key thing-- for catalysis. 864 00:50:15,660 --> 00:50:19,100 So what do we want to be able to do and how does it do this? 865 00:50:19,100 --> 00:50:23,100 So if we look at this, if we look at our reaction 866 00:50:23,100 --> 00:50:27,870 coordinate diagram over here, we don't want to bind substrate 867 00:50:27,870 --> 00:50:30,510 tightly because this is the biggest barrier-- the rate 868 00:50:30,510 --> 00:50:33,450 limiting step along the reaction pathway. 869 00:50:33,450 --> 00:50:36,000 What we want to do is lower this barrier. 870 00:50:36,000 --> 00:50:38,460 So how can we lower the barrier? 871 00:50:38,460 --> 00:50:43,020 We can lower the barrier by stabilizing the transition 872 00:50:43,020 --> 00:50:44,060 state. 873 00:50:44,060 --> 00:50:48,350 That now makes this barrier-- probably 874 00:50:48,350 --> 00:50:51,365 can't read anything now, but that makes this barrier lower. 875 00:50:51,365 --> 00:50:53,790 How's another way you could lower the barrier? 876 00:50:53,790 --> 00:50:57,350 You could lower the barrier by straining the substrate 877 00:50:57,350 --> 00:50:59,520 to look more like the transition state. 878 00:50:59,520 --> 00:51:03,100 So you could strain the substrate in this form, 879 00:51:03,100 --> 00:51:07,260 and now, again, you would have a lower barrier compared 880 00:51:07,260 --> 00:51:08,640 to that barrier. 881 00:51:08,640 --> 00:51:14,610 So you're going to use this binding energy to stabilize 882 00:51:14,610 --> 00:51:17,460 a transition state. 883 00:51:17,460 --> 00:51:31,590 So we want to use binding energy to stabilize the transition 884 00:51:31,590 --> 00:51:39,700 state to de-stabilize-- any of these or all of these 885 00:51:39,700 --> 00:51:51,490 could be true-- de-stabilize the ground state-- G-S. 886 00:51:51,490 --> 00:51:55,060 Or what else do you need to do to get a reaction 887 00:51:55,060 --> 00:51:58,030 to work if you have one or two substrates or even one 888 00:51:58,030 --> 00:51:59,040 substrate? 889 00:51:59,040 --> 00:52:02,075 Your molecules in solution are all solvated. 890 00:52:02,075 --> 00:52:04,450 What you need to be able to do is get rid of the solvent. 891 00:52:04,450 --> 00:52:07,240 If you have two substrates, you have to bring them together. 892 00:52:07,240 --> 00:52:10,420 You have to bring them together at the right orientation. 893 00:52:10,420 --> 00:52:11,940 That doesn't come free of charge. 894 00:52:11,940 --> 00:52:14,110 You have to get the energy from somewhere, 895 00:52:14,110 --> 00:52:17,540 and the energy is proposed to come from this binding energy. 896 00:52:17,540 --> 00:52:19,270 So the binding energy is not used 897 00:52:19,270 --> 00:52:21,250 to completely bind the substrate, 898 00:52:21,250 --> 00:52:24,820 but to do all of these things to get your substrates 899 00:52:24,820 --> 00:52:26,680 ready to form product. 900 00:52:26,680 --> 00:52:39,380 So you can dissolvate and bring reactants together. 901 00:52:45,040 --> 00:52:49,620 And you can freeze out rotational, translational 902 00:52:49,620 --> 00:52:50,200 entropy. 903 00:52:50,200 --> 00:52:53,610 So you're getting everything ready for the reaction 904 00:52:53,610 --> 00:52:54,790 to happen. 905 00:52:54,790 --> 00:52:57,900 So in this case, then, let me just erase this and make this 906 00:52:57,900 --> 00:53:00,150 so that this is clearer. 907 00:53:00,150 --> 00:53:06,000 What you could have, now, is you can-- so in the beginning, 908 00:53:06,000 --> 00:53:07,050 this is the barrier. 909 00:53:07,050 --> 00:53:11,820 If you stabilize the transition state, this becomes a barrier. 910 00:53:11,820 --> 00:53:14,760 If you de-stabilize the ground state, 911 00:53:14,760 --> 00:53:17,580 then this becomes the barrier. 912 00:53:17,580 --> 00:53:21,000 So what we're trying to do is lower this barrier 913 00:53:21,000 --> 00:53:24,630 to get the reaction to work. 914 00:53:24,630 --> 00:53:28,980 And so the major way that we do this 915 00:53:28,980 --> 00:53:33,330 is by using the interactions between the enzymes 916 00:53:33,330 --> 00:53:36,240 of the weak non-covalent interactions between the enzyme 917 00:53:36,240 --> 00:53:39,300 and substrate to help us do these things 918 00:53:39,300 --> 00:53:42,570 to enhance catalysis. 919 00:53:42,570 --> 00:53:48,350 So that's one of the major mechanisms of catalysis. 920 00:53:48,350 --> 00:53:51,510 A second-- and this type of catalysis 921 00:53:51,510 --> 00:53:55,120 is unique to proteins. 922 00:53:55,120 --> 00:54:01,410 So the two types of catalysis are used widely 923 00:54:01,410 --> 00:54:03,600 in organic or inorganic chemistry 924 00:54:03,600 --> 00:54:06,270 when you're designing your catalyst. 925 00:54:06,270 --> 00:54:10,590 I mean, when you're designing a catalyst substrate binding, 926 00:54:10,590 --> 00:54:14,130 a small molecule, a big product release is still an issue. 927 00:54:14,130 --> 00:54:17,070 If you go and read the organometallic literature, 928 00:54:17,070 --> 00:54:19,350 people have trouble with product release all the time. 929 00:54:19,350 --> 00:54:23,180 So the issues in catalysis are exactly the same 930 00:54:23,180 --> 00:54:26,790 in biochemistry as they are in organic and inorganic. 931 00:54:26,790 --> 00:54:29,460 But now we have to deal with this big protein, which 932 00:54:29,460 --> 00:54:32,070 has these unique properties, one of which 933 00:54:32,070 --> 00:54:36,210 is that the whole protein is playing a key role in catalysis 934 00:54:36,210 --> 00:54:40,770 and allowing everything to align within tenths of angstroms 935 00:54:40,770 --> 00:54:43,770 to make these reactions work really efficiently, which 936 00:54:43,770 --> 00:54:46,560 chemists can't do yet. 937 00:54:46,560 --> 00:54:49,380 And I don't think we'll ever be able to design it, 938 00:54:49,380 --> 00:54:53,340 but we can evolve catalysts to become better and better 939 00:54:53,340 --> 00:54:54,930 so that they can do the same thing. 940 00:54:54,930 --> 00:54:58,560 That's the beauty of proteins is you can evolve them to become 941 00:54:58,560 --> 00:55:01,830 better and better catalysts. 942 00:55:01,830 --> 00:55:05,790 So the second mechanism-- so the first mechanism 943 00:55:05,790 --> 00:55:07,390 is binding energy. 944 00:55:07,390 --> 00:55:10,980 The second mechanism-- I can't remember whether they're using 945 00:55:10,980 --> 00:55:11,940 I's or 2's. 946 00:55:11,940 --> 00:55:22,960 The second mechanism is general acid, general base catalysis. 947 00:55:28,440 --> 00:55:31,050 Now, as a chemist, what do you learn 948 00:55:31,050 --> 00:55:32,790 about catalyzing reactions? 949 00:55:32,790 --> 00:55:36,270 Well, one way you could do it is with a big fat proton. 950 00:55:36,270 --> 00:55:39,510 Protons are pretty good at helping you catalyze reactions 951 00:55:39,510 --> 00:55:44,310 if you go back and think about chemical transformations 952 00:55:44,310 --> 00:55:47,580 or hydroxide ions. 953 00:55:47,580 --> 00:55:51,270 What are the concentration of protons and hydroxide ions 954 00:55:51,270 --> 00:55:54,540 in aqueous solution of pH 7? 955 00:55:54,540 --> 00:55:56,790 10 to the minus 7 molar. 956 00:55:56,790 --> 00:56:00,300 So you don't have much protons and hydroxide 957 00:56:00,300 --> 00:56:01,620 ions in the active site. 958 00:56:01,620 --> 00:56:04,380 So even though these are very good catalysts 959 00:56:04,380 --> 00:56:06,510 that organic chemists and inorganic chemists 960 00:56:06,510 --> 00:56:09,579 use all the time, they're using them in organic solvents, 961 00:56:09,579 --> 00:56:11,370 you can argue the active site of the enzyme 962 00:56:11,370 --> 00:56:13,530 is more like an organic solvent. 963 00:56:13,530 --> 00:56:18,730 But anyhow, this type of catalysis is called specific. 964 00:56:18,730 --> 00:56:23,360 So when you see specific acid or based catalysis-- 965 00:56:23,360 --> 00:56:27,560 where does the general acid and base catalysis come from? 966 00:56:27,560 --> 00:56:30,200 It comes from the side chain of your amino acids. 967 00:56:30,200 --> 00:56:33,980 So remember, the second or third lecture I said, 968 00:56:33,980 --> 00:56:35,710 oh, here are all the amino acids. 969 00:56:35,710 --> 00:56:37,520 Here are all the side chains. 970 00:56:37,520 --> 00:56:40,310 You really shouldn't know all of your amino acids. 971 00:56:40,310 --> 00:56:42,680 It's a basic vocabulary of all of biochemistry, 972 00:56:42,680 --> 00:56:44,710 and the pKas of all the side chains. 973 00:56:44,710 --> 00:56:45,830 Why? 974 00:56:45,830 --> 00:56:46,900 That's why. 975 00:56:46,900 --> 00:56:50,020 Because you can't understand anything about catalysis 976 00:56:50,020 --> 00:56:53,540 without knowing what these side chains of the enzymes 977 00:56:53,540 --> 00:56:54,790 are actually doing. 978 00:56:54,790 --> 00:56:57,500 So the general acid and base catalysis 979 00:56:57,500 --> 00:57:03,930 come from the side chains of your amino acids. 980 00:57:03,930 --> 00:57:07,710 So what side chains do you have? 981 00:57:07,710 --> 00:57:09,400 You can have carboxylates. 982 00:57:09,400 --> 00:57:12,225 Anybody know what the pKa of a carboxylate is? 983 00:57:14,810 --> 00:57:16,280 Hey, Boggin, what is it? 984 00:57:16,280 --> 00:57:17,894 STUDENT: Four to five. 985 00:57:17,894 --> 00:57:19,310 JOANNE STUBBE: Good, four to five. 986 00:57:19,310 --> 00:57:20,879 See, he remembers. 987 00:57:20,879 --> 00:57:21,920 You could have imidazole. 988 00:57:27,770 --> 00:57:31,850 This has a pKa at neutral pH. 989 00:57:31,850 --> 00:57:33,580 Anyhow, you need to go back and look 990 00:57:33,580 --> 00:57:36,770 at what the groups are that can be involved in catalysis. 991 00:57:36,770 --> 00:57:39,790 And chemists, for decades, have studied 992 00:57:39,790 --> 00:57:43,150 how you can use general acid and base catalysis 993 00:57:43,150 --> 00:57:45,160 to give you rate enhancements. 994 00:57:45,160 --> 00:57:48,850 Now, what I haven't told you is the amount of rate enhancement 995 00:57:48,850 --> 00:57:53,290 And so people over the years have measured 996 00:57:53,290 --> 00:57:57,230 that with binding energy, you can get factors of 10 to the 8. 997 00:57:57,230 --> 00:58:01,100 If you look at general acid base catalysis from all 998 00:58:01,100 --> 00:58:03,080 the organic and inorganic reactions 999 00:58:03,080 --> 00:58:09,400 people have studied for decades, you get factors of 100 to 1,000 1000 00:58:09,400 --> 00:58:11,350 fold. 1001 00:58:11,350 --> 00:58:14,810 Now, we need to get to a factor of 10 to the 15 in some cases. 1002 00:58:14,810 --> 00:58:17,620 We've already gotten to the factor of 10 to 6. 1003 00:58:17,620 --> 00:58:19,250 So obviously, you're going to have 1004 00:58:19,250 --> 00:58:22,360 to use multiple combinations of these mechanisms 1005 00:58:22,360 --> 00:58:27,130 to give you these tremendous rate accelerations. 1006 00:58:27,130 --> 00:58:33,290 So you will see over the course of the rest of the semester 1007 00:58:33,290 --> 00:58:37,690 many active sites of enzymes with many amino acid side 1008 00:58:37,690 --> 00:58:42,440 chains that are playing roles in general acid and base 1009 00:58:42,440 --> 00:58:44,560 catalysis. 1010 00:58:44,560 --> 00:58:52,015 And the last type of catalysis is covalent catalysis. 1011 00:58:56,900 --> 00:59:01,750 And again, covalent catalysis means that you form-- 1012 00:59:01,750 --> 00:59:05,200 and where have you already seen covalent catalysis? 1013 00:59:05,200 --> 00:59:07,810 You've already seen this when we talked about, how 1014 00:59:07,810 --> 00:59:10,145 do you study the structure of the primary structure 1015 00:59:10,145 --> 00:59:11,880 of a protein? 1016 00:59:11,880 --> 00:59:15,300 We use proteases with tripsin or kimotripsan 1017 00:59:15,300 --> 00:59:18,220 that can break down the big protein into small pieces. 1018 00:59:18,220 --> 00:59:20,670 We went through the mechanism of that reaction. 1019 00:59:20,670 --> 00:59:22,570 In the active site of that enzyme, 1020 00:59:22,570 --> 00:59:26,220 there is a serine that forms a covalent bond. 1021 00:59:26,220 --> 00:59:28,900 So over the course of the semester, 1022 00:59:28,900 --> 00:59:30,970 you're going to see many examples. 1023 00:59:30,970 --> 00:59:33,130 And I'll just put in parentheses for those of you 1024 00:59:33,130 --> 00:59:38,360 who don't remember, go back and look at serine proteases. 1025 00:59:38,360 --> 00:59:42,660 This is a classic example that's in every textbook. 1026 00:59:42,660 --> 00:59:45,580 And how do we know how much rate acceleration 1027 00:59:45,580 --> 00:59:48,490 you get from covalent catalysis versus not having 1028 00:59:48,490 --> 00:59:49,560 covalent catalysis. 1029 00:59:49,560 --> 00:59:54,010 We know this, again, because of organic chemists studying 1030 00:59:54,010 --> 00:59:57,870 the detailed chemical mechanisms of these reactions, 1031 00:59:57,870 --> 01:00:00,520 and we find out that in this case, 1032 01:00:00,520 --> 01:00:07,890 we get rate accelerations of 100 to 1,000 fold. 1033 01:00:07,890 --> 01:00:09,930 So what you see is the enzymes. 1034 01:00:09,930 --> 01:00:13,680 And these are the three general mechanisms 1035 01:00:13,680 --> 01:00:17,280 by which all enzymes catalyze their reactions 1036 01:00:17,280 --> 01:00:18,870 in some variation. 1037 01:00:18,870 --> 01:00:24,150 Now, attributing out of this 10 to 15, 10 to the 8 1038 01:00:24,150 --> 01:00:26,320 is associated with this, and 10 squared 1039 01:00:26,320 --> 01:00:29,010 is associated with that is extremely challenging. 1040 01:00:29,010 --> 01:00:30,900 And there are a lot of people still trying 1041 01:00:30,900 --> 01:00:35,320 to dissect reaction mechanisms in detail. 1042 01:00:35,320 --> 01:00:39,070 And I would argue that understanding 1043 01:00:39,070 --> 01:00:42,280 how these different methods work and synergize to give you 1044 01:00:42,280 --> 01:00:45,820 these accelerations is a key to eventually designing 1045 01:00:45,820 --> 01:00:49,660 new catalysts that can do what you 1046 01:00:49,660 --> 01:00:52,710 want them to do that's distinct from biological 1047 01:00:52,710 --> 01:00:54,220 transformations. 1048 01:00:54,220 --> 01:00:56,074 And I think I'm probably over. 1049 01:00:56,074 --> 01:00:57,490 I just want to say one more thing. 1050 01:00:57,490 --> 01:00:59,550 I just want to give you a feeling 1051 01:00:59,550 --> 01:01:03,430 for what you have to do. 1052 01:01:03,430 --> 01:01:07,110 If you're thinking about this reaction coordinate, what 1053 01:01:07,110 --> 01:01:09,210 you need to do is think about how would you 1054 01:01:09,210 --> 01:01:14,110 stabilize the transition state relative to the ground state? 1055 01:01:14,110 --> 01:01:16,540 So what we're talking about is stabilization 1056 01:01:16,540 --> 01:01:21,550 that's unique to the transition state and not the ground state. 1057 01:01:21,550 --> 01:01:24,510 If you stabilized them both, what would happen? 1058 01:01:24,510 --> 01:01:44,220 If you stabilized them both-- if you stabilize this guy 1059 01:01:44,220 --> 01:01:48,450 and you stabilize this guy, the barrier 1060 01:01:48,450 --> 01:01:50,170 would be exactly the same. 1061 01:01:50,170 --> 01:01:54,880 So what you need is some way to uniquely stabilize 1062 01:01:54,880 --> 01:01:57,951 the transition state over the ground state. 1063 01:01:57,951 --> 01:01:59,700 So the question is, how much do you think? 1064 01:01:59,700 --> 01:02:01,116 How much rate acceleration do you 1065 01:02:01,116 --> 01:02:02,740 think you can get from a hydrogen bond? 1066 01:02:02,740 --> 01:02:04,070 Does anybody have any idea? 1067 01:02:04,070 --> 01:02:05,490 One hydrogen bond. 1068 01:02:05,490 --> 01:02:10,470 So here, you have a protein with 1,500 hydrogen bonds. 1069 01:02:10,470 --> 01:02:14,160 But if you can get one hydrogen bond that's 1070 01:02:14,160 --> 01:02:16,430 here in the transition state of the reaction, 1071 01:02:16,430 --> 01:02:18,760 that's not over here, how much rate acceleration 1072 01:02:18,760 --> 01:02:21,820 do you think you can get? 1073 01:02:21,820 --> 01:02:24,870 Anybody got any idea? 1074 01:02:24,870 --> 01:02:29,070 You can get almost 1,000 fold. 1075 01:02:29,070 --> 01:02:32,265 I mean, and you can do a very simple calculation. 1076 01:02:32,265 --> 01:02:37,320 I can't remember whether I have this on the-- OK, so that's it. 1077 01:02:40,590 --> 01:02:43,200 So we can do a very simple calculation, 1078 01:02:43,200 --> 01:02:46,840 and I'll use this to show you the calculation. 1079 01:02:46,840 --> 01:02:48,210 Here, we have our rate. 1080 01:02:48,210 --> 01:02:51,280 This should be Delta G. The dagger should be up in the air. 1081 01:02:51,280 --> 01:02:53,590 So this is the enzymatic reaction. 1082 01:02:53,590 --> 01:02:56,050 This is the [INAUDIBLE] equation. 1083 01:02:56,050 --> 01:03:00,490 One has the same equation for non-enzymatic reactions. 1084 01:03:00,490 --> 01:03:03,240 So here's a non-enzymatic reaction. 1085 01:03:03,240 --> 01:03:05,670 In general, the non-enzymatic reaction 1086 01:03:05,670 --> 01:03:08,260 can happen by some mechanism. 1087 01:03:08,260 --> 01:03:11,560 To the enzymatic reaction is just much, much slower. 1088 01:03:11,560 --> 01:03:15,760 So if we assume, for example, that the rate difference 1089 01:03:15,760 --> 01:03:18,820 between enzymatic and a non-enzymatic reaction 1090 01:03:18,820 --> 01:03:22,860 is a factor of 10, how much do you 1091 01:03:22,860 --> 01:03:25,810 get assuming that all of these terms 1092 01:03:25,810 --> 01:03:29,950 are the same in the enzymatic and the non-enzymatic reaction? 1093 01:03:29,950 --> 01:03:32,820 You can calculate a Delta Delta G dagger 1094 01:03:32,820 --> 01:03:35,550 of 1.38 kilocalories per mole. 1095 01:03:35,550 --> 01:03:37,740 For those of you who are modern, this 1096 01:03:37,740 --> 01:03:39,760 is 5.8 kilojoules per mole. 1097 01:03:39,760 --> 01:03:42,280 Sorry, I'm really old, so I still 1098 01:03:42,280 --> 01:03:45,180 think in kilocalories per mole. 1099 01:03:45,180 --> 01:03:49,110 But a hydrogen bond, one hydrogen bond is worth 2 to 7-- 1100 01:03:49,110 --> 01:03:51,810 compared to no hydrogen bond, is worth 2 1101 01:03:51,810 --> 01:03:53,980 to 7 kilocalories per mole. 1102 01:03:53,980 --> 01:03:59,020 So a factor of 10 is 1.4 kilocalories per mole. 1103 01:03:59,020 --> 01:04:02,290 So that shows you, then, that if you had 2 to 7 1104 01:04:02,290 --> 01:04:04,150 with one hydrogen bond, it can give you 1105 01:04:04,150 --> 01:04:06,180 these factors of 1,000. 1106 01:04:06,180 --> 01:04:09,100 So I think that's an observation that's 1107 01:04:09,100 --> 01:04:11,760 something you need to keep in the back of your mind. 1108 01:04:11,760 --> 01:04:14,160 Because you think about it over and over again. 1109 01:04:14,160 --> 01:04:18,340 It really doesn't take much to align everything 1110 01:04:18,340 --> 01:04:19,940 in exactly the right way. 1111 01:04:19,940 --> 01:04:24,930 And when I say hydrogen bond, these hydrogen bond strengths 1112 01:04:24,930 --> 01:04:29,010 are really dependent on how everything is aligned. 1113 01:04:29,010 --> 01:04:33,200 If they're exactly aligned, then you get much stronger bonds. 1114 01:04:33,200 --> 01:04:34,950 They can even approach-- in the gas phase, 1115 01:04:34,950 --> 01:04:38,560 they could approach 30 kilocalories per mole. 1116 01:04:38,560 --> 01:04:40,470 So having everything aligned, that's 1117 01:04:40,470 --> 01:04:44,220 the job of this whole big protein, 1118 01:04:44,220 --> 01:04:48,510 to actually give you catalysis. 1119 01:04:48,510 --> 01:04:51,840 And I think I'm at the end of my lecture now. 1120 01:04:51,840 --> 01:04:54,610 I won't have time to talk about-- I went over already 1121 01:04:54,610 --> 01:04:56,370 about the question of specificity. 1122 01:04:56,370 --> 01:04:59,590 But let me just say, I think enzymes are really 1123 01:04:59,590 --> 01:05:01,620 quite amazing. 1124 01:05:01,620 --> 01:05:03,090 There's nothing like them. 1125 01:05:03,090 --> 01:05:04,800 Faster than a speeding bullet. 1126 01:05:04,800 --> 01:05:09,210 They can catalyze the rates a million to 10 to the 12, 10 1127 01:05:09,210 --> 01:05:11,640 to the 15-fold. 1128 01:05:11,640 --> 01:05:14,880 And they use really the simple concepts 1129 01:05:14,880 --> 01:05:17,370 that chemists have developed over the years. 1130 01:05:17,370 --> 01:05:20,524 But the key to the enzyme is this big huge molecule, 1131 01:05:20,524 --> 01:05:24,490 and the dynamics within this molecule that gets everything 1132 01:05:24,490 --> 01:05:29,550 to align exactly right to be able to lower these barriers so 1133 01:05:29,550 --> 01:05:32,850 that you can convert your substrate into your product. 1134 01:05:32,850 --> 01:05:35,440 OK guys, see you next time. 1135 01:05:35,440 --> 01:05:37,290 The end.