 Okay, good morning everybody morning morning Let's see. I Put a couple of announcements on the board the office hours again are from 9 to 10 today and tomorrow and Unfortunately, we will not be able to use the labs for office hours. So if there's too many people We're just gonna have to squeeze into 2084. They don't don't seem to be any other rooms available in that area. So Sorry about that We never planned for so many people to come to office hours. I guess I've put up the first announcement about the midterm It's coming So I want to give you some warning about that You'll be hearing more about it as it as it approaches I will only remind you at this stage of the game that it's Wednesday September 29th from 8 to 9 a.m And and different sections will be in different rooms So not everybody will come to Pimentel and there'll be a handout that describes where you go And it's very important you go to the room that your Lecture discussion is assigned to not your lab Okay, your lecture discussion because if you're in the wrong room then basically your exam has the possibility or your exam Scantron of getting lost you don't want that to happen There will be at least one review by me on Saturday the 25th from 11 to 1 and We'll tell you more about that where it is, but I'm telling you nice and early so if you were planning on going away that weekend you can go away, but you're going to miss the review the review is not webcast and And there'll be more details on that later. Okay, so it's two weeks from Wednesday. That's the bottom line Okay today. I'm going to talk Some more about enzymes enzyme structure and function and probably get it bit into the regulation of enzyme activity And it's very convenient in terms of the lab this week because you're doing the enzymes lab Which involves the use of an enzyme salivary amylase an enzyme isolated from saliva Which is involved in the breakdown of starch Amylose and amylopectin to maltose and you will be studying various properties of that enzyme Some of which may be discussed today, but certainly some of which will be discussed on Wednesday So I introduced the subject at the end of the hour and you can see they cleaned the bench off They always they're so efficient here. They cleaned the bench off every time and I gave you that Very dramatic demonstration of what happened to the glucose plus oxygen Which has a negative free energy change And if you put the glucose in the presence of oxygen on a bench Nothing happens. That's because the thermodynamic parameters that govern the reaction. That is Delta g Delta h Delta s those things Do not deal with the rates of the reactions. There's no kinetic component in that analysis There are thermodynamicists who deal with sort of free energy and there are that thermodynamicists who deal with kinetics but the two have to meet at some point and The way cells overcome this thermodynamic Barrier, I guess I should call it is by using catalysts catalysts are compounds that are Make reactions go faster Although they're not consumed during the course of the reaction and I discussed at some length what? biological catalysts are they are either proteins or RNA molecules ribosomes which have been discovered more much more recently than enzymes and They function by lowering the activation energy for a reaction. So now what we have to try to understand is How do enzymes we're talking about enzymes we're not talking about ribosome ribosomes here That's part of the second portion of the class So how do proteins do this and what are some of the properties of these enzymes that make them so special? So I put up here a very simple reaction Which is the way most people look at enzymes? An enzyme will react with a compound known as the substrate to form an enzyme substrate complex and then the substrate is converted into the product and It's released from the enzyme you don't want the product hanging around for long periods of time Because then another substrate model molecule cannot bind to the enzyme the substrates bind Transiently to what is called the active site of an enzyme if you look at figure 78 and 79 in your handout you can see the first one 78 simply shows what I've said here It shows a molecule binding to an enzyme the enzyme the product substrate being converted in the product But what's more important about figure 79 is it shows that there are many different? Amino acid side chains that are usually involved in the binding of the substrate and they come about When a protein folds into its native confirmation be it a tertiary structure or a quaternary structure what it's possible to achieve Through this folding is bringing lots of amino acid side chains that may not be close in the linear sequence of Amino acids, but they become close due to the folding of the protein and the folded protein has a region in which substrates can bind and you can see there are a number of interactions between the substrate and the protein in figure 79 and there it also says that Weak bonds are involved in this binding hydrogen bonds Ionic bonds because the substrate has to bind and then leave quickly Covalent bonding is not what you would expect to see in the binding of substrate to an enzyme I want you to jump ahead and look at figure 81 Which I kind of like because figure 81 to the right shows the actual binding of a substrate Which is this molecule cyclic amp, which I briefly talked about when I talked about nucleotides It's not important what it is, but you can see that there are a number of amino acids involved They come from different portions of the chain and there are hydrogen bonds there are I'm looking to see if there are any charged bonding. Yes, there is if you look closely at that You can see they're charged amino acid side chains which are interacting with this molecule. They're hydrogen There's hydrogen bonding so you get some picture I hope of how the nature of the binding of a substrate at the active site of an enzyme now a Couple of features of this interaction enzymes have high Specificity by that I mean Because there is a specific interaction of the substrate with various amino acid side chains You might expect that certain compounds are going to bind very well and certain compounds are going to bind less well I didn't say there is perfect specificity. That is an enzyme May to some degree slight-degree react with compounds that are related to its main substrate and You'll hear later on when we talk about photosynthesis about an interesting problem that arises because of substrate Specificity with a very key enzyme in photosynthesis. So be aware. There's high specificity I Actually did give you the all the names of these enzymes in table 46 that I mentioned 4686 and and what you see for example in Molecules enzymes that are phosphatases that take off phosphates From various molecules that they may react with a large number of different Phosphated phosphorylated compounds, but they're only dealing with phosphate removal. So again, there is specificity the The efficiency of enzymes as I said they're catalysts they work over and over and over again the Efficiency of their operation is in some cases remarkable There's a table which is figure 82 That gives something known as the turnover number the turnover number represents the number of molecules of product formed or substrate altered degraded per protein molecule per second and At the top of that list is an enzyme which we'll talk about later carbonic anhydrase Does a very interesting reaction It takes it's present in red blood cells. It is involved in Maintaining the pH of the red blood cell because oxygen binding to hemoglobin is pH dependent And what it does is it takes carbon dioxide plus water And it is a reversible reaction and it forms carbonic acid. It's a relatively simple reaction 60,000 molecules of carbonic acid of carbonic acid are formed per second per enzyme molecule I think I'm not sure if that's the fastest enzyme the one of the enzymes I talked about during the cell structure the enzyme catalyze which Breaks down hydrogen peroxide and peroxyl zones is also at or near the top of that list But different enzymes have different turnover numbers Some of them don't work very well, but they still work better than the uncatalyzed rate Such as the enzyme lysozyme which is down at the bottom, but you can see numbers in the hundreds of thousands of Molecules being say produced product produced per second, so they are Little wonders I would say There have been various models proposed to describe how enzymes actually work The first one the sort of classic model in this field Is shown at the top of figure 80 this is called a lock-and-key mechanism and basically the lock-and-key mechanism Visualized that you have a static stable structure in the enzyme The substrate comes in and fits into that Structure there's no change in shape of the substrate there's no change in shape of the enzyme and then something through that interaction the Bonds of the substrate are altered. Maybe if something is being split off the substrate There's a weakening of the bonds through that interaction and then the product is released. So the enzyme in this Model was rigid and it's like putting your key into the door The lock of the door the lock of the door is not changing shape. The key is not changing shape They just fit together in the right way in the 1960s or so as It became known that enzymes can undergo Confirmational changes that is proteins in general and enzymes in particular can undergo changes in shape Some of them are small some of them are larger What this led to was a model? Which is known as the induced fit model for enzyme mechanism proposed by Daniel Koshland Who was a very prominent protein biochemist in his earlier days worked in in the on the east coast but came out to Berkeley in about 1968 or so 65 maybe and was in the biochemistry department That was the part when I was in so I knew Daniel Koshland Dan Koshland was a very distinguished as I said protein chemist and he proposed this idea that there is change there are changes in shape in Particularly the protein as the substrate binds to the protein the protein is shown in again in figure 80 the middle you can see the active site there, but as the Substrate starts to bind to the active site the protein is changing shape And that's inducing changes in the substrate molecule allowing products to be formed I think the most people would Accept the evidence that it was provided through the 60s and 70s for this idea of how enzymes function So this idea that the that the molecules rigid Inflexible it has been pretty much abandoned. It seems now to be widely accepted that The molecule it's more like the molecule starts like this and as the substrate approaches it Becomes more available to that substrate molecule allowing binding when the products for leave The molecule relaxes again back to its native confirmation. These are not big structural changes They're small structural changes because they have to happen quickly so induced fit model is generally accepted I would say and This was a sort of a major advance this idea of the flexibility of proteins and enzymes in their catalytic activity I'd also mentioned that Dan Koshland and his wife who was in the immunology department were Good friends of this campus by that I mean They donated a lot of money to this campus the building that I am presently located in where my office is is called Koshland Hall named after Professor Koshland the Bioscience library down in LSB. How many how many people have been to the bioscience? That's named after Miriam Koshland. So they Consistently were major donors to to the activities of the campus okay Okay, now we're going to come back to something that I talked about way back when The first or second lecture and that is prosthetic groups and I said I would talk about prosthetic groups again And now's the time What many enzymes contain are Prosthetic groups Low molecular weight groups that are bound at the active site of an enzyme By that immediately you should recognize that these prosthetic groups are required for the activity And I'll give you an example or two of Systems showing how a prosthetic group is actually involved in the activity of a particular enzyme Now you're going to find different terms to describe prosthetic groups in the textbook and in Biochemistry books you're going to find words like cofactors and coenzymes and I find these terms to be very Fuzzy, I don't know what a cofactor is. I don't know what a coenzyme is I do know that a prosthetic group that name gives me some information about what this thing is doing in assisting Reactions to occur so I prefer to squall these types of things that are bound to proteins about the enzymes and Function in their catalytic activity prosthetic groups they can be they come in two varieties variety one is organic compounds variety two inorganic compounds The organic compounds are related to vitamins. How many people here take a vitamin pill in the morning? A fair number now. I thought there'd be more When you take a vitamin pill and you look if you look at the label what you see Part of the example of what you see is you see vitamin B1 B2 B3 B6 and B12 and These are what this these are vitamin B2. It says riboflavin Vitamin B1. It says thiamine vitamin B3. It says niacin vitamin B6. It says pure doxyl vitamin B12 It says cobalt the organic Molecules that are prosthetic groups in proteins are derivatives of these vitamins. For example thiamine is Replace is modified to a compound known as I Don't know why I want to write this out It's a good example thiamine pyrophosphate. It's a phosphorylated Derivative of thiamine and that is then inserted into various enzymes and functions in the catalytic activity of the enzyme so what is shown in Table 383 is The quote vitamins such as thiamine and then the derivative that is the prosthetic group thiamine pyrophosphate niacin is converted into not nicotinamide adenine di-nucleotide Pyridoxyl phosphate comes from vitamin B6, etc. So the organic Prosthetic groups are related to vitamins. This is why you need to take a vitamin You know, maybe you never understood this. I mean everybody says vitamins are good. You don't get them in your food Well, these vitamins become Part of the active component of various enzymes in the cell and that's why they are required if we look at figure 84 try to draw this because This is an example of a prosthetic group and how it functions in the Activity of an enzyme so we've got this compound Again, I do not expect you to remember these structures. I certainly can't remember the these structures This is biotin You have this in your handout what happens to biotin is it is attached to an enzyme through this carboxyl group So what happens is you form a a bond here like this To a protein so clearly This is not going to come off the protein it's going to stay on the protein What is important for you to know is the kind of enzymes that use biotin are known as carboxylases And in this case These are enzymes that are taking CO2 and adding it to another molecule when they do this the CO2 actually binds to biotin like this and then reacts with a Different substrate. This is a these are enzymes that take a substrate Plus CO2 to form a product and the function of biotin in this enzyme is to actually bind the CO2 one of the two substrates almost all Enzymes known as carboxylases contain biotin in them so I think this is just a good example of How a molecule functions at the active site of an enzyme obviously this is the active site of the enzyme Because this is the substrate one of the substrates of the molecule Okay, so this thing has to be sitting at the active site of this particular enzyme You can find similar Mechanisms involving thiamine pyrophosphate in enzymes That's another very good example if you're interested in looking at this in detail Okay, so these are the organic prosthetic groups The inorganic prosthetic groups. What are they? Well you oh incidentally You are probably aware of the kind of deficiencies that you can have if you don't have sufficient vitamins Berry berry thiamine deficiency scurvy I didn't mention a scorbic acid, but a scorbic acid is not exactly What we want to talk about here, but things like that So if you have a vitamin deficiency you generally will end up with a fairly serious illness Okay, the inorganic prosthetic groups are metal ions For example iron copper zinc Magnesium manganese all of these metal ions are Functioning as prosthetic groups already. I've given you the example of heme heme is an iron-containing compound Which is put into proteins like hemoglobin and a whole another range of proteins. We'll talk about next week and They some of these function as Components that transfer electrons fe3 plus plus an electron To fe2 plus that's an oxidation reduction reaction This The other example is copper 2 plus plus an electron To Cooper Cooper cyan so there are a bunch of proteins bunch of enzymes Where the transition metal ion is actually undergoing oxidation reduction there are Additionally transition metal ions which are not undergoing oxidation reduction, but are still directly involved in the catalytic reaction Let's see if I can put this okay. You can look at figure 85 But I'll also put this on the board on the overhead if I can Blow it up a little hard to see Getting better Okay, I'll have to stop there This is carbonic anhydrase the enzyme. I was just talking about in this enzyme. That is a zinc ion this is also Shown in figure 85 the zinc ion sits there. It's surrounded by histidines three histidine residues Figure the figure to the right shows the actual three-dimensional structure the x-ray structure of carbonic anhydrase carbonic anhydrase is Rather small protein. I think it's about 30,000 molecular weight and This is all protein. You can see there's not a lot of alpha. He looks in this protein There's some beta sheet, but a lot of it is just sort of random structure, but in the middle of it is This zinc bound to three histidine and when you look at The right hand part of figure 85 that shows you the mechanism of this very simple reaction that I had on the board and I Erased that is it shows the histidine actually binds water and The water then reacts with carbon dioxide to form carbonic acid so the histidine is in carbonic carbonic anhydrase is Finding one of the substrates of the reaction again. It's a very simple reaction but the Prosthetic group is carrying out participating directly in the catalytic activity Okay, so that is sort of the structural aspects of Enzymes does does anybody have any questions because we're going to sort of change directions a little not that much Okay, you had your chance Okay, the next thing I want to talk about which is certainly relevant to the lab this week Are factors that regulate activity of enzymes? the important point here there's an important point that I Have to make it the outset of this and that point is That not all enzymes in a cell are active all the time one of the sort of big take-home lessons I think the first two parts of this course is Why are why what differentiates the different types of cells? We have say in various organisms and multicellular organisms We have blood cells muscle cells liver cells skin cells on and on and on and on All of those cells have the same DNA in them, right? You don't have different kinds of DNA in your blood cell from your liver cell yet those cells are different and There are I think two reasons why for these differences one of them relates to the molecular biology of the system that is You've got genes in DNA and not all the genes in DNA are Expressed at the same time in every cell so some proteins are they're never synthesized they're shut off But you also have the possibility of when you make proteins in particular enzymes that we're talking about now Not all of these enzymes are active in every cell at the same time and this allows for this differentiation of cells for specialization of cells certain cells have certain activities and functions they will Use a subset of the proteins that are synthesized so regulation of enzyme activity is very important and again once Sort of our understanding of protein structure came about in the the detailed understanding we have today I think the area of Regulation of activity jumped up in terms of being coming more important because of the structural information that we had the first thing you can do or see To regulate the activity of an enzyme is you can vary the substrate concentration So this is substrate and this is activity and this is an experiment you'll do in lab this week and one sees a curve that looks like that the Activity increases with substrate and then the activity levels off at some optimal Rate remember this is just This region up here the activity the enzymes still working working very well, but it's Activity is not increasing as I increase the substrate concentration You saw this kind of figure when I talked about active transport or transport facilitated or active these systems show saturation behavior That is the activity will not increase when you increase the substrate concentration the explanation for this is that You've got a limited amount of enzyme you've got a fair amount of substrate around eventually you reach a point where every enzyme Molecule in the cell isn't it's working as little hard out as fast as it can okay So increasing substrate is not going to increase the rate of the Reaction when one sees this type of behavior. This is known as Mikaela's menton kinetics Because we'll distinguish it from a different kind of kinetics later and It is the kind of enzymatic behavior that one sees for I wouldn't say all enzymes It's certainly not all enzymes many enzymes You will admit you will some of you will make this kind of measurement in lab There are two parameters that are associated with the Mikaela's menton kinetics one is the maximum rate Which is known as V? max and the other is a Factor where you go down so this is a hundred percent you go down fifty percent in rate and You determine the substrate concentration that gives fifty percent of the maximum rate Okay, which would be this and that is known as the Km for the Reaction so the Km is a concentration the V max is a rate now the Km is Related in some way loose way to how tightly a substrate binds to the enzyme But it's very loose It is a number that is distinctive for every particular enzyme. So if you go into the lab and you're measuring You measure doing some measurements on Glycolysis and you measure you're studying hexokinase the first reaction in glycolysis Glucose plus ATP Converted into glucose 6-phosphate Probably the first measurement you would make in the characteristic characterization of that enzyme is the determination of the Km and the V max because Those two numbers do give you some useful information if you determine a if you determine actually this is supposed to be little Am but it doesn't if you determine the Mikaela's menton constant the Km for an enzyme and it turns out to be very high That is in concentration is like a hundred millimolar Some very high concentration and you know that the cell doesn't have anything like that concentration of substrate There's a problem Very high, you know if the if you know the cell only has one millimolar of the substrate Then it becomes a problem as to how that enzyme could function at any significant rate in the process similarly if the maximum rate of a reaction is Very slow then again that presents a mechanistic problem And there are ways around this because these numbers can be changed Depending on whether an enzyme can be activated or something like that But there there are there are some reasons for determining the Km and V max for an enzyme. That's under investigation All enzymes have different Kms and V maxes So you might have one that has a Km of 1 millimolar and the next enzyme you look at it may be a tenth that may be You know less than 1 millimolar So there's no fixed Km the Km is determined by the nature of the active site of the enzyme and the nature of the Substrate and how well it binds to that enzyme the second way You can affect the activity of an enzyme Is by changing the temperature So most enzymes will have temperature profiles that look like this So this is temperature That's wrong erase that this is activity As a function of temperature This is something you will certainly do in the lab and you generally see bell-shaped curves like this You might expect that for an enzyme like amylase Which functions in our saliva that this temperature would be somewhere around? 38.6 degrees And you'll see what it is okay Maybe it is maybe it isn't the These kind of bell-shaped curves show that the activity of the enzyme increases Goes to a certain temperature and then that activity starts to decrease What what what's going on? Suggestions why does the temperature? Why does the enzyme lose activity as the temperature increases? It's denaturing right so I mean you know if you heat proteins they denature So the enzyme starts to lose activity as it is becoming unfolded Presumably you're increasing the number of collisions that are occurring When you are below that temperature and you come to an optimum or you've optimized the possibility of the enzyme and Substrate colliding with each other interestingly You can take enzymes Say that are in liver cells that have optima at thirty eight point six degrees And you can look at the same enzyme in a bacterium that grows in a rather harsh environment I may have mentioned that I was up in Yellowstone a few months ago early June and They have all these you know Gurgling ponds and smoky smelly things. It's a great place to go But these these ponds are generally almost boiling or near boiling or slightly below boiling in temperature and there are organisms that are growing in these ponds and Obviously they have temperature Optima that have been adapted They have so you can look at the same enzyme that we have which has an optimum at thirty eight point six And you look at it in some bacterium, and it has an optimum of 95 degrees Centigrade, which is you know unreal But it is real and it's a very interesting question as to what Changes in this enzyme are allowing it to function basically in a boiling water bath. I think that's a real interesting problem the next thing I want to mention and This will lead you to a little thought project is What is the effect of pH on enzymes? well, you might expect that For enzymes that are functioning in a nice normal happy healthy environment Like our cells this would be pH two four six Eight, you know somewhere around pH eight Would be the optimum for these temp for for these enzymes Although obviously enzymes that form it that function in unusual environments like in our stomach I think the pH of our stomach is one Okay, there are proteolytic enzymes and the enzymes that digest proteins that can function basically down here at pH one or two So this would be a stomach enzyme Now I'm not going to tell you what happens during this bell-shaped curve in relation to pH By that I mean I Want you to think about it. Why does activity increase up to an optimum and then decrease beyond that optimum? it has to do with again the structure of the protein the structure of the active site and if you if you can think about this and Come up with a reasonable idea about it. It will reflect your understanding of side chains in proteins and and Things that can happen to those side chains when you change the pH so it's got a lot to do with protein structure So those are those are three things Very substrate very temperature very pH that are generally done when one is Characterizing an enzyme and as I said you will you will do some of these things This week in lab okay another factor that comes in in sort of the regulation of the activity of an enzyme relates to Compounds that can inhibit enzymes again as I've said enzymes aren't perfect So there are compounds in cells Which are known as inhibitors and if you look at figure 88 It shows two kinds of inhibition that can occur in with enzymes one is Competitive inhibition and the second is non-competitive inhibition and and that figure Shows pretty well the difference between these two To the left you have competitive inhibition Which shows you have an active site of an enzyme and you have an inhibitor that inhibitor competes with the substrate It binds to the active site of the enzyme. So as I said Way back here enzymes. I have high specificity spot high specificity, but they're not perfect So there are compounds that can to some degree look like the substrate They'll bind to the active site of that enzyme and then the substrate can't bind So you will get no activity. So they compete with the substrate now, it's generally found that Competitive inhibition can be overcome by adding more substrate in other words You can it's if you've got two molecules and they're sort of similar in Their interaction with the protein if you increase this one, it's going to work better the classic example of a competitive inhibitor is The molecule carbon monoxide or hydrogen cyanide carbon monoxide or HCN Both of these as you probably realize are highly toxic you know if you Go into the Garage and turn the car on and close the door of the garage You produce a lot of carbon monoxide and you kill yourself. They're a common way of doing it. I guess cyanide is Used in things like gas chambers if they're still used. You know, these are bad compounds. Why do these compounds kill you? because they compete with oxygen for binding a heme In an enzyme which is known as cytochromoxidase which will talk of talk about much more in more detail later But it's the major enzyme in mitochondria that uses oxygen uses oxygen in the oxidative phosphorylation pathway you make ATP if you don't have this enzyme functioning then You stop ATP formation and of course you're going to die very quickly so carbon monoxide or HCN can be competitive inhibitors with molecular oxygen and They are lethal. Okay, put this bag up non competitive inhibition Okay, non competitive inhibition is shown to the right of the figure and what is shown there is you have The active site on the enzyme, but you have some other site on the enzyme not the active site an inhibitor binding site the inhibitor binds that site and It changes the structure of the active site Indirectly through some type of conformational change in the protein and that Does not allow the substrate to bind adding more substrate In a situation where you have a non competitive inhibitor has no effect on the inhibition now So that these two have to keep them straight competitive inhibitor inhibition non non-competitive inhibition and and you know I make sort of a big deal out of inhibitors because Unfortunately in our environment. We are exposed to lots of different kinds of compounds. Some of them are nebulous. They're negative I mean there's no effect But some of them can inhibit one of the many many kinds of reactions that are incurring in cells In which case you are going to have some metabolic problems So again, this is a way you will alter the activity of particular enzymes. You can inhibit them or you can non inhibit them Okay, I it's Five minutes early, but I'm going to stop because the next subject is more complicated and and it will just confuse you if I Go into it now. So if you have any questions, you can come up and ask me Otherwise, we'll continue with regulation on Wednesday