hello everybody my name is Iman welcome back to my YouTube channel today our discussion for biochemistry is going to be about enzymes and kinetics and of course we want to start this conversation first by explaining and defining what are enzymes now enzymes are incredibly important biological catalysts and the thing about catalyst is that they do not impact the thermodynamics of a biological reaction what that means is that the enthalpy or the Gibbs free energy do not change instead they help the reaction proceed at a much faster rate as a catalyst the enzyme is not changed during the course of the reaction but the reaction proceeds faster so it affects the kinetics now of course I think there's a better definition for enzymes usually one that involves the way that they work but that will come here shortly so just hold on a minute the one thing that I do want to introduce before that is are the different classifications for enzymes the majority of enzymes are classified into the following categories that we see here in this table we have things like oxidor reductase this is an enzyme that catalyzes oxidation reactions in which electrons travel from one molecule to another so a really good example of this are alcohol dehydrogenases these are enzymes that convert alcohols to either aldehydes or ketones this enzyme makes alcohol less toxic as it breaks it down and it also plays a key role in things like the fermentation process so that's one class for enzymes another common class for enzymes are transferases these enzymes catalyze the transportation of a functional group from one molecule to another so one example of this is aminotransferases which catalyze amino acid degradation by removing the amino group another enzyme class that we should be familiar with are hydrolase these enzymes catalyze hydrolysis where single bonds are broken down broken down upon the exposure to water all right another common enzyme class are liaise liases these catalyze the breakdown of various chemical bonds by means other than hydrolysis and oxidation they often often forming new double bonds or ring structures so a good example of this is pyruvate decarbox decarboxylase it's an example of eliase that removes carbon dioxide from pyruvate so we've actually encountered this when we learned cellular respiration another class of enzymes are isomerase all right these enzymes catalyze structural shifts in molecules that cause changes in shape so an example of this is ribolose phosphate eppermarase these catalyze the inter-conversion of ribolose biphosphate to xylose 5-phosphate now another common category that actually isn't on this table so we're going to add it here is ligase all right this kind of enzyme what it does is it catalyzes ligation so the combination of pairs of substraces all right and then the last one that is on here is a synthetasis these are join two molecules by the formation of new bonds all right so these are all common enzyme classes it's good to get familiar with the different classes of enzymes and with that what we can begin to transition into is ask further what enzymes are and how do they work all right so again enzymes do not affect thermodynamic quantities instead they affect and change the kinetics of a reaction how fast a reaction proceeds and they act as catalysts things that help speed up the reaction but they're not used up they are not changed in the process all right so what are enzymes what do they do and how they work now to kind of motivate why it's important we discuss this living organisms like you and I with we seeds with metabolic activity I mean I I really like this figure because it shows a lot of metabolic pathways and and and processes that occur in our body that enzymes help um help proceed help function help speeding up the reaction to the appropriate sort of time frame that the body may need it to work under thousands of chemical reactions are proceeding very rapidly at any given time in our bodies and enzymes play a crucial role in all of these processes and so this is one of the main reasons why it's important that we understand that enzymes are agents of metabolic function they they help um reactions proceed forward and one of a good example of this is we've encountered enzymes before for example if you recall cellular respiration at each step of converting glucose to pyruvate every step requires an enzyme to help change and convert glucose to the different intermediates it needs to go through to finally form the final product of pyruvate now while enzyme mechanisms will vary depending on the reaction that is being catalyzed they do tend to share some common features enzymes may act to provide favorable micro environments in terms of charge or pH they can stabilize the transition state or bring reactive groups closer to each other near the active site and the formation of the enzyme substrate complex in the active site of an enzyme is the key catalytic activity of the enzyme it reduces it's where it reduces the activation energy of the reaction now essentially an enzyme is a protein all enzymes are proteins the opposite is not true by the way okay not all proteins are enzymes enzymes are proteins an enzyme is a protein that facilitates a cellular metabolic process how does it do this it does it by lowering the activation energy so we've seen this before when we've talked about Gibbs free energy right your energy of your reactants and the energy of your products can can vary right but it goes through a process and it has to be able to overcome some sort of activation energy for the reactants to become products all right if the activation energy is small then the reaction can proceed more easily and faster and if the activation energy is very large and this reaction is going to require a lot of input energy and it could be a much slower process because of this enzymes how they work is they lower the activation energy so that the reaction may proceed faster all right enzymes facilitate a cellular metabolic process by lowering the activation energy levels in order to catalyze the chemical reactions between biomolecules now some enzymes reduce the activation energy to such low levels that they can actually even reverse cellular reactions but in all cases enzymes facilitate reactions without being altered like the way fuel burns when it's used all right for reactions to occur molecules have to collide under appropriate conditions and enzymes can help create those collisions so that the reaction proceeds faster all right now how do they exactly work well enzymes bind to a sub to a specific substrate or reactant molecule in a way that stabilizes its transition state and lowers the activation energy making it easier for the reaction to occur all right enzymes are highly specific all right let's write that down they're highly specific that means they won't just bind to anything all right they have to they they're only they're they only bind to specific substrates all right so this enzyme will only bite into a specific substrate so if the right substrate comes along all right it will bind to the ant to the enzymes active site all right they're highly specific in their binding and the shape of the enzymes active site is going to be complementary to the shape of the substrate molecule it will bind and this specificity it ensures that only it ensures that only the correct substrate will bind to the enzyme and that the reaction will proceed in the right direction all right so then once the right substrate binds to the enzyme in the active site all right the enzyme is going to catalyze the reaction by bringing the substrate molecule into close proximity orienting them in a way that promotes the reaction and therefore forming the enzyme substrate complex enzymes what they can do is they can modify the chemical properties of the substrate molecule think things like adding or removing functional groups to facilitate the reaction all right so once they form this enzyme substrate complex then the enzyme can do what it needs to to that substrate so that the products are formed and then the the products are released all right enzymes can also be regulated to ensure that they function at the right time and in the right place so for example enzymes can be activated or inhibited by other molecules things like Inhibitors or activators which by the way will cover near the end of this chapter all right these um activators or an inhibitor Inhibitors I'm so sorry I get excited and then I stumble upon my words these activators or Inhibitors that bind to the enzyme what they can do is Alter its activities so that the enzymes can be regulated in the manner that they need to all right they can also be regulated besides just you know activators and inhibit Inhibitors they can also be regulated by post-translational modifications things like phosphorylation that can alter their activity stability or subcellular localization all right so essentially that's how enzymes work all right now I want to step back I want to make mention of two competing theories that really explain how enzymes and substrates interact there's two competing theories your first one is the key lock and key Theory what it suggests is is that the enzymes active site is already in the appropriate conformation for the substrate to bind it's just waiting for the substrate to come and bind because it's in the perfect appropriate conformation all right so essentially this this is why it's called the lock and key Theory because it assumes that the enzyme has its active site in the right conformation and then the substrate can come and lock into that because it's already perfect for the substrate all right so that's the lock and key Theory the other competing theories that induced fit model and it hypothesizes that the enzyme and the substrate they undergo conformational changes together to interact fully all right so these are the two competing theories on explaining how the enzyme substrate complex comes to form now some enzymes also require metal cation cofactors or other small organic coenzymes to even become active all right so that's an important thing to keep in mind that sometimes the enzyme itself requires some modification through other molecules to then be ready for a substrate with that note then we can move into enzyme kinetics all right now the concentration of this substrate and the enzyme greatly affect how quickly a reaction will occur for most enzymes the mycolis mentin equation describes how the rate of a reaction depends on the concentration of both the enzyme and the substrate which forms the product what that means is we can think of it with this equation right here all right we have our substrate and our enzyme and together they can come and form the enzyme substrate complex and through that complex and and the capabilities of the enzyme we can form the product and of course the enzyme doesn't get altered at all it just it just helps form the product and then it goes about it today all right now the reaction from substrate enzyme to enzyme substrate complex we can note this as K1 the reaction um the end forming the enzyme substrate complex forms at this rate that we're going to call K1 all right um now at the substrate all right this enzyme substrate complex it can either dissociate back at a rate of K2 or it can form the products at a rate of K3 all right ignore these I don't know why they're here all right so now we have the rates for the different directions that this can go about now under the condition that the enzyme is kept constant what we can do is we can relate the velocity of the enzyme to substrate con we can relate the velocity of the enzyme to substrate concentrations using this mycolis mentin equation all right and that's sort of um demonstrated and written here for us all right now we're going to make sense of this all right we're going to make sense of what this equation is really telling us all right but again under the right conditions under the condition that the enzyme is kept constant we can relate the velocity of the enzyme to substrate concentrations using the myculus mentus um equation all right so under these conditions we can write this equation where the rate of the reaction this velocity of the reaction you can think of it I I suppose is equal to the V Max of the substrate times the substrate over km plus the substrate concentration and we'll go over what each of these terms mean and how we can extract it from a Michaelis cementin curve all right now what this curve tells us all right starting from here so that we can extract some of these values all right so this is this is a curve all right of how the reaction proceeds all right in time in relation to the concentration of the substrate all right that's what the michaelismentin curve tells us this is what you can measure experimentally all right with a change in how how the change in the time of the time of the reaction in relation to the substrate concentration what you notice is as the amount of substrate increases all right as we increase substrate concentration the enzyme is able to increase its rate of reaction look at that that rate of reaction is increasing until until it reaches a maximum enzymatic reaction rate this maximum rate that this reaction can can go is called the V Max that's what this term is right here all right this is this substrate concentration so we figured out some of these terms already all right once you've reached the V Max adding more substrate will not increase the rate of the reaction all right fantastic so then what this reaction is saying we can relate the velocity of the enzyme to substrate concentration using this equation V Max times your substrate concentration over km we'll figure that out in one second hold your horses plus the substrate concentration all right km can be understood to be the substrate concentration at which half of the enzymes active sites are full all right once I'm going to repeat that km can be understood to be the substrate concentration at which half of the enzymes active sites are full and it has a name it's the Michaelis constant and it's often used to compare enzymes all right so that's what this value is right here now wherever your one-half V Max is all right that will give you your km so that's how you figure it out from the graph all right you can figure out your V Max because that's where the rate of the reaction no longer changes all right when you reach that plateau and the reaction of the rate no longer increases you know you've plateaued you've reached the V Max half of the V Max whatever that number is half of it will allow you to work back to find the km values from your Michaelis mantis curve all right fantastic now when the reaction rate is equal to half of the V Max right here all right you know that km will also be equal to your substrate whatever your substrate concentration is there all right so that's how we work from there fantastic now I do want to elaborate on what this km is because I think it's very very important all right like we said it's the substrate concentration at which half the enzyme uh and enzyme active sites are full under certain conditions km is the measure of the Affinity if the for the enzyme if the enzyme for its substrate all right I guess let me let me reword that km is a measure of the Affinity of the enzyme for its substrate all right now when you're comparing two enzymes the one with the higher cam all right let's pretend you have enzyme one enzyme two I'm just gonna one is open circle one is full okay if you're comparing these two enzymes all right one of them has 100K I'm I'm literally coming up with a random number this is not necessarily right one has 100 km this one has 200k all right that's what we're just gonna say when you're comparing these two enzymes the one with the higher km all right has the lower affinity for the substrate lower Affinity what does that mean and why all right it has a lower affinity for the substrate because it's going to require a higher substrate concentration to even be half saturated all right and this km value it's an intrinsic property of the enzyme substrate system it can't be altered by changing the concentration of a substrate or an enzyme all right so small km numerically tiny or low value of KM represents a strong substrate enzyme affinity and large is the opposite low substrate enzyme affinity all right fantastic now staying on this topic we also want to introduce something called the called the line Weaver Burke plots let me write that down all right we're going to come up here let's try to hit line Weaver Burke plots all right what is that all right this plot is a way of graphing enzyme kinetic data that allows for the determination of kinetic properties like Vmax and km in a different way the plot is what is just a double reciprocal graph okay so you notice how in your Michael mentis graph let's go back here let me just do some erasing what's the y-axis here the y-axis is your velocity of reaction your rate of reaction all right and what's your x-axis substrate concentration all right now what we can do is we can plot the same information just just a slightly different way how we're going to do a a double reciprocal all that means now is that our y-axis is no longer just V naught it's one over V naught all right and our x-axis is not just substrate concentration it's one over substrate concentration all right it's really we're not collecting any new data we're just conveying it in a different form and this form is called a line Weaver Burke plot all right your y-axis is 1 over concentration your x-axis is just one over um substrate concentrate I'm sorry your y-axis is one over your rate of reaction one over V naught all right it's just the reciprocal of the initial reaction velocity and it's plotted against in your x-axis the reciprocal of the substrate concentration all right and now what you're going to get all right when you plot this is some line instead of a curve right because you're doing this double reciprocal all right that's the mathematical relationship you're going to get a line a straight line and the slope of this line is going to be equal to km over V Max your y-intercept wherever it touches on the y-intercept right here that y-intercept all right let's do this in a different color that is your y-intercept y-intercept what does our y-intercept tell us this is your 1 over V Max all right and what about your x-intercept all right your x-intercept is going to be is going to give you information about the km it's going to be 1 over your k m all right so then we have this different means of plotting things and we can still extract our V Max and our km values all right so you can extract it from this kind of graph you can extract it from your Michael mentus curve it's important to know how to do both and understand to look at each either plots recognize they contain the same information just in different ways all right so that's how we understand enzyme kinetics now what we can also discuss that's equally important is the effects of local conditions on enzyme activities all right there are certain variables that will affect our enzyme activity these are temperature pH and salinity in some cases how much salts are presents all right now enzymes enzyme catalyze reactions tend to double in velocity all right they tend to double in velocity velocity for every 10 degrees Celsius increased in temperature of course until it reaches an Optimum temperature all right above such temperatures you can get things like denaturation all right but enzyme catalyzed reactions they tend to double in velocity for every 10 degrees Celsius increase all right so temperature has an effect of course there are temperatures that are much too high all right that that this fact no longer holds and the pro and the enzyme can denature same goes for pH most enzymes also depend on ph in order to function properly not only because pH affects ionization of the active site but also because changes in PH lead to denaturation now for enzymes in the human blood the optimal pH is 7.4 and then in vitro salinity also affects uh enzyme activity because it increases levels of salts that can disrupt things like hydrogen and ionic bonds and when those are disrupted disrupted they can cause partial changes in the conformation of the enzyme and in some cases again denaturation all right now as a final topic for this chapter something also that we need to talk about and we made earlier mention of is how to regulate enzyme activity all right because it's important to be able to stop enzyme substrate complexes from forming when you have enough product or to start initiating that when you don't have enough product right you have to be able to regulate it in both directions right and although enzymes are useful like we said the body must be able to control when they work and so enzymes are often subject to regulation by products further down a given metabolic pathway and this is usually through a process called feedback regulation now less often than not enzymes may be regulated by intermediates that precede the enzyme in the pathway and while there are examples of feedback activation what's more prominent and what we're going to start with discussing first is feedback inhibition all right feedback feedback inhibition or negative feedback helps maintain homeostasis once we have enough of a given product we want to be able to turn off that pathway that creates the product rather than creating so much more especially because there are some things where too much of a product can lead to toxicity in our bodies all right feedback inhibition can actually fall under two big categories we're going to discuss both we have irreversible Inhibitors and reversible inhibitor Inhibitors irreversible Inhibitors they tend to form covalent or very tight permanent bonds at the ACT um with the active site of the enzyme and it renders it inactive and there's three groups or classifications that are really prominent we're not going to really focus on these classes so much for irreversible Inhibitors and but what we should know is that irreversible Inhibitors are going to form covalent or very tight bonds which render the enzyme pretty much inactive then we have reversible Inhibitors um and these can form uh complexes with the enzymes that actually can be dissociated back to the enzyme and free the inhibitor and the enzyme back to single entities and these come in there's three main groups here that we will cover competitive non-competitive and uncompetitive all right and we can we can actually visualize this in sort of a flow chart right we have feedback inhibition main two classes are reversible and irreversible these ones render the enzyme inactive by forming strong covalent bonds for example and reversible Inhibitors like their name suggest they can stop an enzyme from reacting with a substrate but they're reversible so when we need more product they can dissociate and the enzyme is free to react with substrates again all right these come in three categories all right we have competitive inhibition all right competitive inhibition this results when the inhibitor um this simply involves occupancy of the active site so substrates um cannot access and enzymatic binding sites if there is an inhibitor in the way um Can competitive inhibition actually can be overcome by just adding more substrates so that the substrate to inhibitor ratio is higher if more molecules of substrate are available then molecules of inhibitors for example then obviously the enzyme will be more likely to bind substrate uh substrates than inhibitors right and so in the case of competitive Inhibitors all right adding a com adding a competitive inhibitor will not affect V Max all right because if enough substrate is added it will out-compete the inhibitor and be able to run the reaction at maximum velocity what a competitive inhibitor does do is increase the measured value for km all right excuse me now something to keep in mind with competitive Inhibitors let's follow this Arrow over here all right this is an example of what the line Weaver Burke plot looks like comparing an enzyme with and without a competitive inhibitor all right this common trend is important to know this is without inhibitor notice that your x-intercept what do you know about this this is one over km all right and then with inhibitor this is where your 1 over km is so notice how there's an increase all right there's an increase there all right and also notice that it won't affect your Vmax they both have the same y-intercept all right so no changes to kit to to to V Max here but changes to km for competitive inhibition all right what about non-competitive inhibition non-competitive Inhibitors they're going to bind to an allosteric site instead of an active site so competitive Inhibitors come right into the active site non-competitive Inhibitors leave the active site they bind somewhere else to the enzyme and what happens though is that it induces a change in the enzyme conformation altogether though now allosteric sites they're non-catalytic regions of the enzyme that bind regulators excuse me the thing about non-competitive Inhibitors is that you cannot overcome it by adding more substrate it doesn't work that way what happens here though is adding a non-competitive inhibitor does decrease your Vmax that you measure it doesn't alter the km though no changes to cam so if we come and look at our line Weaver plot over here for non-competitive notice they have the the same y-intercept so the same km but they do have different intercept y-intercept so they have different V Maxes all right now what about uncompetitive inhibitors all right these bind only to the enzyme substrate complex and then they essentially lock the substrate in the enzyme preventing its release so they do not let this substrate be released they affect both the Vmax and the km all right they lower the km and the VMAX and we can look right here to see that with Inhibitors and no Inhibitors they have different km values they have different x-intercepts so they have different km values they have different y-intercepts so they have different V Max values as well excuse me um so that's um negative uh feedback feedback inhibition all right now you can also have regulatory enzymes that experience activation also less common than feedback inhibition but we have a couple examples like allosteric activation where allosteric sites can be occupied by activators increasing either efficiency or enzyme turnover you can have phosphorylation and you can have zymikins phosphorylation you have covalent modification that can alter the activity or selectivity of enzymes enzyme agains are secreted in an inactive form that can be activated by cleavage all right that's all I have for you for enzymes and kinetics let me know if you have any questions down below other than that good luck happy studying and have a beautiful beautiful day