Enzymes: Catalysis, Kinetics & Classification – Biochemistry | Lecturio

[Music] the ability of enzymes to speed up reactions is actually mind boggling the subject of this presentation is to talk about the ways in which we study the kinetics or the ways that enzymes speed up reactions in this presentation I will give a little bit of background about the process of catalysis talk about the flexibility of enzymes and how that enables them to do what they do talk about activation energy which is another consideration for enzymatic catalysis I'll talk about the mechanism of a specific reaction for an enzyme called a serine protease and then I'll give the kinetic considerations that we have during that analytical process and finally talk about the overall overview of using what are called Michaelis Menten kinetics now when we think about enzymatic reactions there's actually a series of different ways that that molecules can react in interacting with an enzyme we can have for example reaction that's a single substrate reaction and a single product a is converted into B we can have a reaction in which a single substrate is converted into multiple products so for example if I took a and I split it into two molecules I would make B and C I could take multiple substrates and make single products which is the opposite which would mean I would be putting two things together to make a third that third being C is shown here and last I could have multiple substrates and multiple products in which a and B are converted into two different things C and D now enzymes are as I said magical in their ability to catalyze reactions and they are so much faster than a chemical catalyst that it's important to think about the ways in which they're able to accomplish what they accomplish and so this illustration of an enzymatic reaction goes step by step into some of the considerations for the ways that enzymes accomplish what they do chemical catalysts I want you to remember are things that are very fixed a platinum catalyst for example has no breathing it has no movement to it it simply is a surface on which something can happen and enzymes are fundamentally different from that in this illustration we see an enzyme shown in green and we see the active site of the enzyme that is the place where the reaction is catalyzed shown in light green now the enzyme in this reaction I'm showing you is a reaction of multiple substrates multiple products so we will have a and B as you can see here that will be converted into two other molecules we start with the enzyme unloaded no products contain no products and the enzyme and of course no substrates the substrates are the molecules that bind to the enzyme and they will bind so as to position be positioned at the place where the reaction occurs the active site we can see here their substrates have started to bind to the enzyme we see the enzyme again in green we see substrate a that has bound the top portion of the enzyme and substrate B that has bound the bottom now the interaction of the substrates with the enzyme will actually cause the enzyme to start to change this is the coastland induced fit model of an enzyme in the coastland induced fit it says that not only does the enzyme change the substrates into products but transiently during the catalytic process the substrates change the enzyme and as we will see that's essential for this reaction to occur so the substrate binding has happened we have formed at this point what we call the es complex enzyme substrate complex now in the next rate step you see right here what has happened is we see the reaction going on and the enzyme has actually changed its shape slightly from the initial binding to bring a and B into closer proximity well of course for a chemical reaction closeness is an absolutely essential or requirement for the reaction to occur so the slight change in the shape of the enzyme has converted a and B from being a part to being slightly closer together these changes of shape can be very large on enzyme terms or it can be very very subtle but nonetheless the change happens with every reaction now the reaction is occurring again as we can see because they have been brought into close proximity at this point as the reaction is going on we have something called the es star complex and we can just simply think about this as the place where the reaction is now able to occur as we look at this reaction closer we see during the reaction a part of a has moved from A to B and this has been a transfer of a part of one substrate to another a is no longer a and B is no longer B at this point we have made what we call the EP complex we've made the products but we haven't released the products yet so a has become C and B has become D now the products are still contained within the enzyme but the products are different than a and B were so just as a and B caused the enzyme to change shape so too will C and D cause the enzyme to change shape and you can probably see where this is headed the enzymes going to go back to where it was and that's what happens right here we see in this reaction now that the enzyme has been changed and it's changed back to its initial state in the initial state we can think of its fingers being opened like my hand is open and C and D are ready to go flying away the enzyme now being back in its original state is able to go and bind more substrate it's ready for the next process now if we think about this our definition of a catalyst that everybody learns in freshman chemistry is a molecule or an entity that catalyzes a reaction but it's unchanged in the process that's a principle that is hammered into every freshman chemistry student now we see that enzymes are actually slightly violating that principle they're being changed transiently during the process but they end up in this in the end in the same way they started so overall they're not violating it but they cheat a bit we see in this slide then a summary of all the reactions or the steps in the process that you've seen before and I don't want to go through those again but I do want to make the point that you notice that the arrows are going both ways and that means that this reaction and every step in this reaction is reversible now reversibility of a reaction is a very important thing to keep in mind when we're talking about metabolic processes or for that matter even non metabolic processes but especially for metabolic processes because we have to think that is what are the conditions that would make something go backward we've seen how enzyme flexibility enables enzymes to accomplish what they accomplish but enzymes do have constraints that they have to work in I've mentioned in these presentations numerous times now that enzymes and cells all are governed by the rules rules of the universe that is they can't change the energies of reactions and so those are true for cells and those are also true for enzymes now enzymes as we will see are tricky little things I've mentioned how enzymes cheat and enzymes are going to cheat with respect to energy as well so let's consider a reaction of a going to be in a going to be this is plotted from an energy perspective on the screen what you see here on the left side of the screen we see a dot placed on the graph showing free energy that is the energy that's associated with molecule a in the process of going from A to B we see that there is a change in the energy that the energy is actually increased and we call this increase in energy activation energy that's necessary to get a reaction going the reaction proceeds and as the reaction proceeds we can see that the free energy Falls that we make a product B that down by the end has a lower free energy than a hat that meant that energy was released in the process of going from A to B and this makes this energy this this reaction process favorable now it's important to note that this change in free energy that's shown right here this change in free energy cannot be changed by an enzyme that is there's no change between the starting and ending points of the enzyme the enzyme does some other things however it's also important to note here that this height of the peak is really a critical place the height of this peak is the place where the reaction can reverse and go backwards from where it came that is a can start and then go back or be if it got enough energy could climb that curve and then go back to a otherwise a is going to go forward to B and the reaction is going to be is going to be favored it is going to be occurring now enzymes cheat okay enzymes can change the activation energy there are no rules about activation energy okay there are rules about beginning and ending energies but what activation energies changes do is they enable an enzyme to make more molecules more easily go through that transition that is the magic of enzymes how do they accomplish that well they accomplish this in a couple of ways one of the ways that they do it is by the fact that they have binding sites that are very precisely oriented so that the molecules are placed in the close proximity that they randomly would not be in too close proximity so easily so easily all right and that means that it takes less energy for them to go through the next step in the process by doing this enzymes can actually lower the activation energy and make it possible for a reaction to go easier and also to go faster meaning it's therefore much more likely that the reaction from A to B will be catalyzed you notice again enzymes have had no change in for overall free energy the energy of a is still a the energy of B is still be okay only that transitional state has made a difference now I want to go through and spend some time talking about the mechanism of an enzymatic reaction mechanism is important to consider because with mechanism we can begin to see how enzymes are facilitating electronic changes necessary for a chemical reaction to occur the example I will use is an example of a serine protease a serine proteases are a class of enzymes that cut proteins they break peptide bonds that's what they do and they break not every peptide bond they see but they break specific peptide bonds at specific places within the proteins that they bind to all right so that means that they have binding specificity they don't they don't cut everything that they see serine protease is have flexibility so we saw in the initial illustration the flexibility of an enzyme and we're going to see it occurring again here as we talk about the mechanism of the serine protease the electronic environment is very critical for a reaction in a chemical reaction electrons are being manipulated electrons are being moved around and to be able to do that one must have the environment for those electrons to readily be able to move around and we'll see that happening in the active site of the serine protease enzymes also use coenzymes now in this reaction in this example I'm going to give I won't show a coenzyme but I will say that coenzymes actually help an enzyme to accomplish what it accomplishes no serine protease is there they said cleave peptide bonds that's the catalytic action a catalytic thing that they do they have specificity of cutting again by binding only to certain molecule or certain proteins they only cut those proteins that they bind they have a common active site all the serine proteases the different serine proteases have a three-dimensional configuration of the place in them where the reaction occurs now we'll see that that is important because that configuration is what creates the electronic environment necessary for the reaction to take place and last of all the serine proteases are very well studied so we understand the mechanism of their action quite well so let's take a look now at the mechanism of a serine proteases i've shown on the screen here a substrate for the enzyme this is a polypeptide chain or protein that these subs that the serine protease will cut the specific cut that's going to occur here will occur between the carbon and the nitrogen on this molecule and of course you know from you know the structures we've talked about in other presentations this is the location of the peptide bond now on the right side of this of this image you can see the central part of a serine protease now the central part is the place here where the reaction is going to be catalyzed now it's a little hard to get our head around some of these things so you're gonna see in some cases I'm gonna stretch bonds and stretch molecules a little bit to actually make things fit so you can understand this please understand that in an enzyme itself of course they're already better positioned but it's hard with figures to make things fit as we would like to serine proteases all have a common feature of their active site and the common feature that they have of their active site is that they all contain these three amino acid side chains that you can see located in close proximity of each other now I always like to remind students that when we see something like this it reminds us that protein folding does occur that is that serine and histidine and aspartic acid which are the three side chains that we see here are not located close to each other in primary sequence they're brought into close proximity of each other by the folding of the enzyme to make them physically close to each other as we see here and the closeness of these is important to start but more importantly the flexibility of the enzyme with these side chains is absolutely essential to the catalytic function that will happen okay so we imagine now that we see this folded enzyme and that the rest of the enzyme is shown in yellow we're looking right now specifically at the active site near the active site we have a place where the protein is going to bind and the protein that's going to be cut is going to be interacted with this catalytic triad of serine histidine and aspartic acid The Binding of the substrate to the enzyme occurs in a specialized site on the enzyme called the s-1 pocket so we've shown here the s-1 pocket is a sort of a semicircle that's holding on to a part of that protein we can see the protein that's going to be cut now is at the active site now in the binding of this protein to the active site you notice that the nitrogen on the histidine has an arrow pointing towards the oxygen towards the hydroxide we also note that the oxygen that's on the sidechain of aspartic acid is has a little dot next to the hydrogen on the histidine what's happened here well I'm going from the previous slide to this slide we can see that what's happened is the enzyme has changed shape very slightly The Binding of the substrate and remember that binding of substrate changes enzymes has changed the enzyme very slightly so that the proximity of aspartic acids sidechain 2 histidines has changed that's very important aspartic acid here the oxygen has a negative charge and the negative charge has moved a little bit closer to the ring of the histidine is shown here by this small action the electronic configuration of the ring of histidine is changed and it's that change which is causing now the nitrogen to be reaching out and what it's going to do is it's going to grab that hydrogen that's on serine okay so this tiny change in shape that happened on the binding of the enzyme is starting the process by which the reaction is going to occur so we can see here that the s-1 pocket has facilitated all this happening I should say in the s-1 pocket that the s-1 pocket gives the specificity of the enzyme the s-1 pocket will not bind to everything it will bind to specific proteins with specific sequences within them very very important concept if it doesn't encounter those specific things it won't bind them and if it won't bind them of course there's nothing to react and the end of this process will not occur okay so the slight chart structural changes have happened and we now see the result of this starting to come into play the things that the the entities have moved closer into each other the electronic environment has definitely changed by this point and what we see is that that proton that was on the Oh H of serine is now associated with the nitrogen of the histidine ring now this is the first step in this catalytic process or actually the second step if we count the binding of the substrate this making of the oxygen with a negative charge on the end of serine is fundamental to this reaction occurring we call this negatively charged oxygen on serine and alkoxide ion okay that alkoxide ion that's on serine is extraordinarily reactive it's ready to go do business now we've stretched that s-1 pocket little bit too that again we're bringing things into closer proximity and that is important because the alkoxide ion is looking for something to bind to it's looking for a nucleus it's what we call a nucleophile and the nucleus that it's looking for here is this carbon which is the arrow that's being pointed from the oxygen - down to the orange carbon so there is actually what's called a chemical attack a nucleophilic attack that's occurring on that carbon we can see that the electrons that are double bonded to the oxygen are rearranging as we see the arrow being pointed and the next step of the process what will happen is that we're going to see a rearrangement in the molecule okay so we went from this position to this position notice that we had a carbon with a double bond to an oxygen that now is a carbon with a single bond to an oxygen that molecule is chemically unstable it's chemically unstable and a chemically unstable molecule has to be dealt with because if it's not dealt with it's going to cost problems well the enzyme has another pocket in it to deal with that unstable molecule it's called the oxyanion hole and the oxyanion hole helps that unstable molecule to fall apart without problem that's pretty cool okay it's going to fall apart without problem and what's going to happen here as you can see is the nitrogen in blue is going to reach up and grab that hydrogen that was originally grabbed by the histidine side chain okay so this intermediate that's in the oxyanion hole is what we call a tetrahedral okay and tetrahedral as we know from organic chemistry or what happens when carbon has those four bonds that you can see here okay the peptide bond which is between the carbon and the nitrogen okay is is going to be broken as a result of nitrogen grabbing that hydrogen here nitrogen has grabbed the hydrogen the grabbing of the hydrogen from the histidine caused the bond between the carbon and the nitrogen to break so we've broken the peptide bond and so part of the protein the part of the protein shown in blue is now free to go and do its business it's released there's nothing attaching it to the enzyme and it goes and it exits what we have done here is we have actually gone through the first part of the reaction and in this part of the reaction is what we call the rapid part of the reaction okay the other part of the protein is attached to serine it's physically attached to syrian it's a covalent bond at this point now that covalent bond has to be broken in order for the other part of the original protein to be released and that's what's gonna happen in the slow step of catalysis now the slow step of catalysis actually has about the same number of steps as the fast step of catalysis but other things have to happen including the movement of water into the active site in order for the this peptide to be released well we see that happening here water now has physically moved into the active site there's a wall a molecule of water and that process that we saw of the nitrogen on histidine taking a proton is going to repeat itself we see it happening here we see the arrow from the nitrogen on the histidine pointing to the hydrogen on water so it's going to take that hydrogen instead of taking the hydrogen that it originally took which is no longer there on Cirie what's going to happen in that process is now we're gonna have an activated oxygen like we had with the alkoxide ion except for here it's going to be a hydroxide we're gonna have an activated oxygen that's gonna make a nucleophilic attack on carbon just like we saw before so there's a nucleophilic attack that's going to happen in the process of this moving forward here's the attack of the hydroxide and look what happens we see that the electrons on oxygen are going to rearrange we create at a tetrahedral intermediate as we created before and now there's the oxyanion hole stabilizing that intermediate we now see that what happens is that oxygen is going to attack the hydrogen on that group and it's going to pull it away just like the first peptide did when it does that what happens is the molecules released so we see the second half of the polypeptide chain released and in addition we have the enzyme returned back to its original state gone and as it were the cycle is now complete there's about 10 steps going through what I've described here and the important thing to understand about this is that the enzyme started in one state it went through a transition and then went back to the original state it was in very much like the process I've already described but now you've seen it in mechanistic terms when we saw the image of the reaction occurring we saw these various states that you see on the screen the enzyme plus the substrate bound together to make the es complex which converted upon the change in the enzyme to the es star complex which created the EP or the enzyme product complex which ultimately resulted in the release of the enzyme in the product now I come back to this because we are going to need to consider some things about the kinetic parameters that is the Meccan of the speed parameters of the reactions that we're going to study now this rate of formation of product is really what we're interested in when we talk about how fast an enzyme can make a reaction occur this is the guts of what we're after we want to know how fast is the enzyme able to do this well if to do this we need to make some simple assumptions and so we assume in the simple case that the enzyme substrate complex proceeds directly to enzyme plus product okay so we've simplified this more complicated equation above to a simpler equation below and this is done to help us better understand what's going on in the price and the overall mechanism now these constants that are here won't really enter into our consideration but the cake at that you see in the enzyme going to be plus P will in fact be an important consideration for us as we talk about the kinetic parameters the k-kat as we shall see is the rate with which product is forming now let's consider what's happening inside of a couple of different scenarios of a reaction we can imagine that we have enzymes for example shown in yellow and we have substrates as little red colored balls that are there we could have a situation first of all where we have a reaction going on in a condition of low substrate and if we have a low amount of substrate in a solution we could imagine that there's very few enzymes that are going to be bound to substrate because the chances of encountering a substrate are reduced in the middle of course we have an intermediate state where we have a little bit higher concentration of substrate than we did before and so we can see here that there are more enzyme molecules bound to and engaged in the process of making the product and the third scenario we could imagine is high substrate and when we have a situation of high substrate we notice here that every enzyme is bound to a substrate and that's important because at high substrate concentrations we have enzymes that are what we call saturated with substrate meaning that once it is bound to substrate made a product and released it almost instantaneously it grabs another substrate it's not sitting around and waiting for things now so enzymes interestingly have some kinetic considerations which is of course what we're interested in studying here but we see now for the first time a projection of the way that the enzyme is working so I need to explain some things on the graph that you see first of all we're plotting on this graph a reaction the reaction is plotting the velocity of the reaction on the y axis versus the substrate concentration that's used in the reaction on the x axis now you notice the V has a little 0 beneath that and the 0 beneath that I'll explain later but it's called the initial velocity for our purposes the velocity of a reaction is measured as the concentration of product made divided by time the concentration of product made for time well we measure concentration in molar milli molar micro Moeller etc so that would be some molarity per time that is how velocity is measured the substrate concentration varies because to generate a curve like this I do not one reaction but I do a series of reactions so let me set that up we could imagine for example that I'm setting up a series of 20 reactions 20 different test tubes I want to measure the velocity in each one of those test tubes and what I do is I take into that test tube I place the buffer that holds the substrate I place the substrate and I place the enzyme now when I'm doing an experiment I want to have one variable because one variable is the only thing I can really manipulate and measure the effect of that the variable I have here is substrate concentration I use the same amount of enzyme in every tube all twenty tubes have the same amount of enzyme they all have the same amount of buffer and they have varying amounts of substrate starting from very small amounts to very high amounts I take and I let each one react for an exact same time and then I measure the amount of product so by doing that I can see the effect of measuring of changing substrate on the velocity and then I plot it so what you see on the screen is the sum of those plots that is each each point on that lot on that dot came from a series of reactions that I did and each one of those individual reactions had a specific substrate concentration and a specific velocity that was reached well not surprising as we look at this what do we see well on the far left were at low substrate concentration what's the velocity it's very low and that's what I showed on the original image low substrate concentration enzyme is sitting there waiting for substrate there's not going to be much velocity when I get to a high substrate concentration such as they see on the right side of the screen I've got a high velocity makes sense okay low substrate low velocity high substrate high velocity I want you to remember that now I'm showing another plot here to illustrate a principle of a reaction on that y-axis I have the concentration of product we could think of that again it's velocity but on the x-axis now I'm plotting the time of reaction so I could take one of the tubes that I used in the previous one and for example look at how fast the product is being accumulated and what happens to that product over time well we can see on this plot that over the early range of the reaction there's a linear relationship between the production of product and time okay but after a while what happens is that that curve flattens off now what that means is that the longer that we let a reaction go it doesn't stay linear forever and the reason it doesn't stay linear forever because remember enzymes catalyze reversible reactions so the more we let product accumulate the more likely product will start being converted back into substrate well that's not what we're interested in studying we want to study how fast the enzyme makes product so if we're going to study an enzymatic reaction we have to study what's called initial velocity we don't want to wait too long in order to study the concentration of product because if we wait too long we're actually starting to study the reverse reaction and that's not what we're after so that's why we use vo or the initial velocity in our measurements now this is kind of complicated so I want to step you through it but these are considerations for doing reactions in what are called Michaelis Menten kinetics we can see here that on the y axis again we have concentration and on the x axis we have time and before we saw simply the accumulation of product is shown in the orange icon here at the very beginning of a reaction what is are the circumstances well we have four different things to think about that can be measured we have the concentration of substrate we have the concentration of the enzyme we have the concentration of the enzyme substrate complex and ultimately we're going to have concentration of product which is what we're interested in studying okay at the beginning the concentration of prod as low as you can see and that's not surprising because the reaction hasn't had a chance to get started the concentration of es is low because there hasn't been an opportunity for the substrate to really encounter the enzyme very much the concentration of the free enzyme that is the enzyme not bound to substrate is relatively high and you see it's coming down from the y-axis and finally the concentration of substrate is high because none of the substrate has reacted so at the time zero we have these circumstances going on and these circumstances turn out not to be ideal for us to measure the enzymatic reaction now as the reaction proceeds we see changes to these entities we see first of all that the concentration of substrate by the end of the reaction is low and it's falling during the entire process the concentration of the es substrate which started out at zero is going higher and we'll see that it will eventually sort of level off we also see that the concentration of the free enzyme which started out at a relatively high position is falling and it too will sort of level off in time and finally we see of course that the concentration of product is going to start at the low and go to high by the very end well I show you this graph not to complicate the picture too much hopefully but rather to demonstrate what we try to do in studying enzymatic reactions in the very initial phase I hope I've made the case for you that we're in a set of conditions called pre steady state now I'll explain what steady state means in a minute but we have a circumstance where the reaction hasn't had a chance to get started the enzyme isn't doing its thing and everything in there is changing pretty rapidly the change in substrate the change in enzyme the change in enzyme substrate complex and the change in product this is going to give us a lot of variability in a reaction now I said we want to study initial velocity but we want to be careful if we do it too soon we may not get what we're after here so it's important to think about really studying or studying reaction at a place where these things have sort of leveled off now under conditions of steady state what's actually happening is that these other quantities that were varying fairly rapidly in the very initial phase of the reaction will start to even out and that's very for our consideration so we can see for example that in that early state the concentration of free enzyme and es complex are changing the concentration of e is falling very rapidly in the concentration of es is rising very rapidly however in under steady state conditions as we can see here they have started to flatten out they're not exactly linear but they're much closer to linear than they were in that pre steady state condition that turns out to be important for us because what we're interested in studying is the conversion of enzyme substrate complex into product and so if we have a relatively constant concentration of enzyme substrate complex then that decay or that falling into product that's actually happened it's happening at a relatively constant rate that's the place we want to be and that's why it's important for us to be studying these reactions under steady state conditions steady state conditions of course again meaning that these quantities are not varying significantly now we can see now the overall plot of what's happening on here the steady state conditions are where we make our measurements and we see that this relatively linear portion of the plot for the concentration of free enzyme and concentration of es complex is happening under the conditions that we measure our enzymatic reactions okay under the Michaelis Menten kinetics we learned that it's important for us to study enzymatic reactions that are conditions where our steady states that is we have relatively constant amounts of es complex under Michaelis Menten kinetics the equation on the on the top applies and this equation tells us some very important things that we're going to learn in this lecture now vo that is the velocity of a reaction is equal to v-max and that's something that we'll discuss in a moment times the concentration of substrate divided by another quantity called km that we'll discuss plus the concentration of substrate so we've learned two terms here that are going to become important for us to understand and that's v-max the maximum velocity of a reaction and km which is a quantity that allows us to measure the affinity that an enzyme has for its substrate well first let's start with v-max with v-max it's important to and what it is and why it is and how that happens we saw when we plotted vo versus the concentration of substrate below that we saw that the curve grew and then it leveled off and the reason it levels off is due to the way that enzymes work and the way that they interact with substrates instead of an enzymatic reaction going and staying linear with increasing concentrations of substrate what happens is enzymes get saturated with substrate saturation of substrate means that the enzyme by is almost constantly bound to substrate meaning that we have almost everything in the es complex so at very high substrate concentrations the enzyme is continually releasing product and over time if we add more and more substrate we exceed the capacity of the enzyme to bind more substrate so under saturating conditions of substrate the enzyme is no longer able to stay linear and it flattens off so we see this hyperbolic plot now an example might be a factory that's making products a factory that's making products we'll have a lot of workers and that those workers are working on something but if they don't have enough materials to make product then the worker is going to be standing around a fair amount of the time waiting for material so they can make product on the other hand we could imagine that if we have those same workers working and they have all the problem is I need to make products they're going to turn out a certain number of products per day if we increase the amount of materials but we don't increase the number of workers we're not going to change that maximum amount they're going to get so we see the same thing happening in the real world that we see happening with enzymatic reactions if we want to increase the amount of product we have to get more workers perhaps get another factory in order to make more product now enzymes that don't follow Michaelis Menten kinetics and there are some include those that bind substrates cooperatively now in another presentation I talked about how hemoglobin binds to oxygen cooperatively and that means that the binding of one substrate is affecting the binding of others so when we this happens and of course this only happens for multi subunit proteins when this happens when the binding of one affects the others then of course we're going to see a change in the velocity because that's going to change the actual binding conditions of the enzyme when we have those things happen we can tell them pretty easily because what we will get is an s-shaped curve for the V versus s plot very much like what we saw with the hemoglobin binding to oxygen okay well let's now look at these parameters I've introduced the concept of v-max and we see that eventually the enzyme reaches a place where it's not going to make any more product over time because it's saturated with substrate v-max turns out to be an interesting quantity but v-max as we will see has some limitations nonetheless v-max allows us to study some things now the quantity v-max gives us a maximum amount and we could say well if we want to understand how much an enzyme interacts with a substrate maybe we should compare v-max 'as well that doesn't really tell us very much it tells us how fast a reaction goes but it doesn't tell us how well an enzyme interacts with a substrate because any enzyme will reach v-max as we add an infinite amount of substrate which is theoretically what v-max is occurring at when it's completely saturated that doesn't tell us much however the quantity v-max over to where we're getting an enzyme to a certain point of velocity but not the maximum amount of velocity actually allows us to measure that the affinity that enzyme has for its substrate if we compare a variety of enzymes and we compare how much substrate it requires the enzyme to get to v-max over two we get something very interesting we get a quantity called the km and the km is actually a measure of the enzymes affinity for its substrate so when I say affinity it's the desire to bind to how well does it bind to its substrate now km is interesting if we think about two enzymes one enzyme that catalyzes a reaction that has great affinity for its substrate it really likes that substrate it really grabs that substrate and we have another enzyme over here that doesn't like its substrate as well okay well which of the two we're going to bind substrate more readily the first one of course because it's got greater affinity which one is going to get to v-max over two with a lower substrate concentration well the one that grabs its substrate more easily so enzymes that have a greater affinity for their substrate are going to have a low km and those that have less affinity for their substrate are going to have a higher km okay greater affinity low km lower affinity high camp so km is inversely proportional to the enzymes affinity for its substrate okay so here we see high km low affinity you see low km high affinity a very important concept to remember with respect to km and I'd like to think about it about an enzyme that has low affinity we have to pound it on the head with substrate before it starts to bind it and by pounding on the head the way that we do that is by adding a lot more substrate now v-max as I said is a very interesting and important quantity but it actually is not the perfect quantity to measure the speed of a reaction it's good for the reaction but it's not so good for the enzyme so what does that mean well it means that v-max when we do a reaction the way I described doing a reaction as we set up 20 tubes and we have in those 20 tubes buffer we have substrate and we have enzyme and when we're doing a V versus s plot what we're doing is we're having one variable the one variable that we have is substrate which means that all 20 tubes have the same amount of enzyme that's great we don't want to have variable amounts of enzyme but imagine I were to do the same set of reactions and instead of using the amount I used in the first set let's say that I did the reaction now they used twice the amount of enzyme for the second set of reactions in each case constant however varying substrate but now with twice the amount of enzyme what would I see with respect to v-max well if I go back to my factory analogy and I think about what happened with the factory I said the factory got to a point where it's saturated it made a maximum amount of product that the workers are gonna put out per day and it wasn't gonna make anymore what if I had two factories well if I have two factories I would say well I'd probably expect that I would get twice as much product per day and so if I use a set of tubes that have twice as much enzyme the parallel follows I would get twice as much product so v-max is proportional to the amount of enzyme we used it's not a constant for an enzyme but it's a constant only for a reaction with a set amount of enzyme I'd like to be able to compare enzymes with a quantity that is independent of the amount of enzyme that I used well fortunately that's fairly easy to do okay v-max is a velocity and we measure velocity of a reaction as the concentration of the product produced divided by time if I take the quantity of enzyme that I used in the reaction and I divide v-max by that quantity and I say quantity in this case meaning concentration the concentration of enzyme that I used what will happen well the v-max was measured as a concentration of product and I divided by a concentration of enzyme as long as I use concentration and concentration consistently the concentrations actually drop out and so what happens is I get a number and the units on the number are per time so I get something that says a thousand per second what is a thousand per second mean well I've taken the enzyme out of the equation and now the number that I get corresponds to the number of molecules of product per enzyme per second so a thousand per second means every enzyme in that solution is making a thousand molecules of product per second and that's the fastest that's going to go because remember we started with v-max that quantity is called Kitcat k-kat is a number that's also called the turnover number but I can come the cake hats of two enzymes and have a much better understanding about the relative speeds of production of product that those enzymes have now the idea of cake aunt brings up another thing for us to think about and enzymes are really remarkable okay we've seen that enzymes can speed up reactions mind-boggling numbers of times and we've also introduced the concept here of an enzyme having affinity for its substrate the idea of what a perfect enzyme would mean okay starts to come into shape we think about what would be a perfect enzyme a perfect enzyme would be an enzyme that would have as much velocity as possible with as great of affinity for its substrate as possible meaning that to get to that maximum velocity it wouldn't take very much substrate because the ends that would be grabbing substrate and converting it into product very readily so a perfect enzyme would have a high velocity and a low km well we use k-kat as our measure of velocity and km is our measure of affinity for substrate high cap means high by k-kat means high velocity low km means high affinity the perfect enzyme will have a large ratio of k-kat to km so if we take those two numbers and we divide them by each other and we start comparing enzymes we see enzymes have widely varying ratios of k-kat over km but we also see that there's a sort of a top echelon beyond which enzymes really don't have a number that increases very much now these numbers vary a little bit from each other but these are really the top echelon enzymes they don't have a cake at over km value that's significantly different these are on the order of ten to the seventh two in one case ten to the ninth but most of them in the range of about 10 to the 8th we don't see enzymes that make it to 10 to the 15th for example why is that well what's happened with these enzymes is they've reached their maximum efficiency they can't get any more efficient there's two things that limb them one is they can't with shape and sequence of amino acids make a better active site than what they've made by evolution in that sense they literally are perfect mutations that change those will always make an enzyme that's less efficient there's a limit to what that efficiency can be and the second thing is really interesting it is believed that the reason that we reach a max with this in addition to what I've just mentioned is that there's something else that's limiting about the enzymatic reaction and the limiting thing for these enzymes in a solution is one one quantity and that's the rate with which the substrate can diffuse in water diffusion of course happens with the mixing that we see in its diffusion that's bringing substrate into the enzymes active site and though that process of diffusion can itself occur at mind-boggling rates that's what allows enzymes to do what they do it to has a limit and so these enzymes are so efficient that they're sitting there waiting on water to deliver substrate to them that's a remarkable thing all right let's take and use now some of these parameters that we've been talking about with respect to kinetics and understand enzymatic reactions I've shown several times now the plot of vo versus s and we saw that was a hyperbolic curve and you saw in that curve that at the very top of that we had something called v-max and if I'm eyeballing that curve I have to ask myself what I draw in v-max at the right place is it up a little bit is it down a little bit and I have to make a judgment call with that I'd like to have a more precise way of saying what is the v-max well one of the tricks or tools that we use to do this is to actually change the analysis of the data a little bit instead of plotting one instead of plotting vo versus the concentration of substrate that is the velocity versus the concentration of substrate I take the same data that I had for that vo versus s plot and I invert it I invert all the data so I do what's called a double reciprocal plot or a lineweaver-burk plot they were the people who came up with this and when I invert the data like that what I discover is that that hyperbolic plot becomes linear and that linear plot is much more easy for us to interpret to determine what these values are when I make such a double reciprocal plot I create a linear plot of the data and the linear plot of the data I can use a lock and draw a line through the points and extrapolate through the axes the y axis and the x axis when I do I create an intercept on a y axis and the y axis has the value of 1 over v-max I can very quickly of course invert that value and I've got B max on the x-axis the intercept is minus 1 over km so if I take whatever that value the intercept is and I take minus 1 over that I will get camp very simple plot so lineweaver-burk plot sand there are other manipulations that people do of grouse lineweaver-burk plot help me to write very readily determine v-max and km from a set of data I've described so far how enzymes are flexible around the active site and how that flexibility of the active site facilitates the catalytic process that happens but enzymes are flexible all over and that flexibility all around the enzyme gives the enzyme some interesting properties as regards its activity now we can see here on the left an enzyme that is getting ready to bind a substrate as we've seen before and on the right we see the enzyme after having bound this substrate has adapted itself to the shape of the enzyme this was the induced fit that I've been referring to this induced fit makes a lot of sense for the active site as I said but the rest of the enzyme is also affected by these things by these by these interactions now this is actually manifests itself in the plot that's shown on this figure right here on this plot we can see the V versus s binding for an enzyme that's allosteric now remind you that allosteric means that the enzyme is interacting with a small molecule and having a sec tivity affected in this case the small molecule that it's interacting with it's affecting it is actually its substrate so this happens with multi subunit enzymes and then what I'm getting ready to describe very much parallels what I talked about with hemoglobins finding of oxygen in another of the presentations when he would open binds to oxygen you may recall that the binding changed as the oxygen concentration increased as the oxygen concentration increased hemoglobins affinity for oxygen went from low to high and that was important for the action of hemoglobin the same thing can happen with an enzyme whose affinity can change depending on its binding of the allosteric in terms of binding of the substrate that affects it allosteric li now multi subunit enzymes have this happen because a part of one one part of the enzyme binds the substrate and affects the binding of the substrate on other parts of the enzyme now there this change that I described to you results in a change in the overall physical shape of the enzyme not just the catalytic site now this overall change of the enzyme is given a couple of names first we talked about a state that's relaxed it's called the our state the relaxed state of an enzyme is the state that really the enzyme is open to binding substrate and is very able to bind substrate the relaxed state of an enzyme corresponds to a more active state of the enzyme by contrast to the T state of an enzyme where T stands for tight is the enzyme is tense it is tight it is not flexible and it is not able to bind substrate as well on this plot for example we could see at the low substrate concentrations the enzyme is in the T state it's not binding substrate very well but once the substrate concentration gets high enough the enzyme flips and then it's able to bind substrate better so it's velocity change actually flips we don't see the hyperbolic plot that we saw before well there's a couple of ways people have studied and tried to explain this phenomenon going on so I want to spend a little time going through and explaining ways that we interpret this change they're called the concerted model and the sequential model so the first of these I'll talk about here is the concerted model the concerted model is conceptually a little hard to get one's head around we see the enzyme in this model existing in two states and for the purpose of this illustration we've assumed that this enzyme has four sub-units enzymes can have many many subunits --is up too easily at least a dozen subunits in some cases but for this illustration it has four in the t-state we have the enzyme shown in the squares on the top and the t-state we recall is the least favored of the states this circles below refer to the enzyme in the our state where the enzyme is relaxed and more able and likely to bind substrate what the concerted model says is that the flipping between the T state and the our state happens completely as as shown there as we go from the top to the bottom and there's no intermediate and what this model says is something that seems counterintuitive because this model says that the flipping from T to R is not caused by the binding of the substrate but rather the our state or the T state is favored by whatever the the state happens to be in when it binds substrate so if I have an enzyme that flips into the our state and it binds substrate the substrate will lock it in the our state so that it will tend to stay in the our state and consequently be more reactive if the enzyme binds it in the t-state it's likely to stay in the T state once the enzyme is in the our state it's going to stay there and keep producing and since the our state is producing more and more of the product anything that favors our locks in the our state is going to favor the reaction more so the concerted model is an all or none but the locking into one state or another is central to what it does you can see here that there's an equilibrium between the two and the equilibrium shifts as we get more of the substrate binding our locking it into a given state as we go further to the right the our state is favored because there's more enzymes now in that our state and more enzymes means more product the our states can flip as I said independently of each other but the bound state is favoring in this case the our state now the other model that we called sequential model is very much like what we saw when I described the flipping or the changing of hemoglobin we also refer the states our state and T State and hemoglobin but we more commonly used this as regards enzymes now in this model what happens is we have an enzyme that starts out in the T state is shown in the in the four squares on the left The Binding of the first substrate causes one of the subunits of the enzyme to flip and that's shown in blue in the second of a model from the left when that flipping occurs the blue interacts with the other two units of the enzyme and we can see that there's a sort of a purple that happens and a rounding of those two that's indicating that the blue circle which is in the our state is affecting the two units around it and causing them to start to flip into the our state well they're starting to flip favors the binding of more substrate and so we can see sequentially then that the Blues are becoming more dominant as we get farther to the right the binding of the substrate is a critical thing for this this enzyme because the binding of the substrate in this model says it's the cause of the flip now casually when we talk about it we frequently say well this causes the enzyme to do this or that and when we say that we're sort of loose in the language that we use in this case the cause is physically causing the flipping to occur and the concerted model the cause is not a direct but it's an indirect as a result of the locking that I described so the distinguishing difference between the concerted model and the sequential model is that cause that I mentioned causing the the flipping is a physical causing the two models that I've just described the concerted model and the sequential model or just that they're just models in terms of explaining how the T state in the our states come to be within enzymes it's very likely that no enzyme actually uses exclusively one of the other and there's a lot of evidence that enzymes may use as sort of a hybrid of these two models enzymes as I noted at the beginning can bind reactions in different ways and I talked about one substrate going to one product or to substrate going to one product or in the case I'm going to describe here two substrates going to two products now when we think of two substrates that can bind to an enzyme we realize that there's different ways that they could bind for example if we have the reaction a plus B goes to C plus D we can imagine that maybe an a would have to bind first and then B or maybe be binds first and then a but what we find is that for some enzymes it really doesn't matter which one binds first this is called random binding as it's shown in the first example that I have on the screen random binding means it doesn't matter now some enzymes bind substrates randomly as I'm showing you here but a lot of enzymes do what's called ordered binding that means that either a must bind first or B must bind first now that model or that mechanism is significant and the reason it's significant is it's probably the best illustration that I can give you for the coastland induced fit model because what order binding tells us that is is that if one of these must bind first before the other one does that means then that the binding of the first one is actually changing the shape of the binding site for the second one because if the second one tries to bind first the change hasn't already happened and that's why the second one can't bind first so ordered binding reinforces the coastland induced fit model now that might seem to cover all the territory but there's actually a third model that enzymes use to catalyze reactions and this one's kind of interesting and it has a fun name we call it the ping pong mechanism and it's also called order a double displacement reaction but the point is the same the ping pong mechanism is an enzyme that actually exists in two covalently different states that means that the enzyme is actually physically binding to something and causing a change most of this happen in the next slide now this illustrates a reaction of a plus C going to B plus D and we're seeing it's split into two reactions all right in this reaction what's happening is a is starting out with an oxygen on it and in the reaction of A to B the oxygen is being replaced by an amine so we see this happening and where is the amine coming from the mean is coming from the enzyme so the enzyme is carrying the amine and it's carrying it to a so when a interacts was the enzyme the enzyme swaps the amine that it's carrying for the oxygen that's on a so on the right side of the top equation we see that a has become B because it now has an amine and the enzyme has grabbed the oxygen it no longer has an amine so I've colored it with green so that you can see that in the second part of the reaction C which has an amine is interacting with the enzyme that now has an oxygen and when that happens they trade places C becomes D where D has a double bond to the oxygen and the enzyme has become a linked to an amine right so the enzyme has returned to its original state so by this ping-pong mechanism the enzyme is continually going from a mean two oxygen to a mean to oxygen and depending upon which state it's in it determines which of the substrate it binds and swaps with now this type of reaction that I just described to you is a common reaction that's used by enzymes called transaminases transaminases are enzymes that do just what I've described they swap oxygens for amines and this is a very important reaction in the metabolism of amino acids because amino acids get their amines in some cases by the reaction that you see on the top they start out with a double bonded oxygen and they become an amine a really good example of this is the the molecule alpha ketoglutarate in the citric acid cycle alpha ketoglutarate can become glutamic acid if the oxygen on it is swapped for an amine and this way the cell can make an amino acid that it might need because of this mechanism on the other hand we might have the situation where a glutamic acid or even another amino acid is needed for energy people that go on low-carb diets for example don't have a lot of carbohydrates but they're not starving to death because they're eating plenty of protein proteins providing amino acids and amino acids provide energy as a result of what I'm showing you here the lower-left reaction has an amino acid that has the amine replaced by a double bonded oxygen so imagine if you will that the amino acid on the lower left side is actually aspartic acid aspartic acid can be converted by swapping its amine with an oxygen into exile o acetate and xlo acetate can be oxidized in the citric acid cycle so this transaminase reaction is important both for making amino acids and also for metabolizing amino acids for energy the last thing i want to talk about here are the classifications of enzymes enzymes according to a systematic scheme that has been developed by the ec commission the enzyme commission have broken all reactions that enzymes catalyze into six categories and this six category scheme is used to organize and named all enzymes that are in biology the first scheme a category scheme is that of an oxido reductase that is an oxidation and a reduction is happening in the reaction that's catalyzed by the first category of enzymes in this case you can see male 8 which is shown on the left that is being oxidized it's donating its electrons to nad to form X all acetate and NADH so oxidoreductases will always have transfers of electrons and will always have an electron carrier involved you can see the nad and the NADH here the second category of enzymes are those called transfer raises and transfer raises grab a part of one molecule and move it to another so we can see here for example that we're starting with glucose we're taking a phosphate off of ATP and we're putting it on to glucose this enzyme hexokinase catalyzes the first reaction in glycolysis and it's a transfer ace the next reaction involves hydrolysis and as the name would suggest these enzymes use water to break bonds so we can see in the schematic reaction here a molecule on the left that has a peptide bond is actually combining with water to break that peptide bond that's what happens with a serine protease for example water is being used to split a peptide bond in the fourth category we have enzymes called lyases and Ally A's as an enzyme that uses a non hydrolytic meaning no water non oxidative way of breaking bonds so on the Left we see for example isocitrate the enzyme isocitrate lyase which is found in plants breaks this six carbon molecule into a two-carbon piece called glyoxylate in a four carbon piece called succinate because water is not involved and because there is no oxidation involved this reaction is a lie ace the fifth category of enzyme is an isomerase and isomerases our enzymes that catalyze rearrangements without doing anything else to the structure of the molecule that they're acting on so in this case we see another reaction from glycolysis the enzyme converts glucose 6-phosphate which is a sugar with a phosphate on it to fructose 6-phosphate which is the different sugar with a phosphate on but all that has happened is spend simply a rearrangement of the molecule that's an isomerase the last category of reaction are those of ligases and ligases are molecules that put things together so instead of breaking things apart ligase is are making covalent bonds to join things together so in this case we're seeing ATP energy being used to join urea and bicarbonate to make your real one carboxylate ligases join things together well we've seen in the reactions here that enzymes have some pretty amazing abilities in terms of flexibility for catalyzing reactions flexibility as they affect the mechanism that they use to catalyze things and we've also learned about the different categories of enzymes that are there in other lectures I will talk about the ways that enzymes become inhibited [Music]

Channel: Lecturio Medical

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Keywords: Kinetics & Classification, Enzymes: Catalysis, Enzymes biochemistry, enzymes catalysts, biochemistry lecture

Id: -slPtkO2d9g

Length: 67min 8sec (4028 seconds)

Published: Tue Jun 12 2018