I engineers in the last video If you guys remembered in part one we talked about the michis Menon equation we basically went through derive the entire michelos and meon equation algebraically and got some big big points out of that one was the steady state assumption the other one was the Michaelis mean equation and the other one was the Michaela um Michaela constant and its relationship to substrate affinity and substrate concentration at half V Max what we're going to do in this video is we're going to take we're going to be applying a lot of these Concepts in the third video when we talk about certain graphs and plots in this video I'm going to introduce what's called Inhibitors so if you remember this equation we talked about it in the last video we said enzyme reacts with the substrate to produce an enzyme substrate complex and then it disassociates to form enzyme and product and then we said that the constant for this forward direction is k1 the constant for the reverse direction is k1 and the constant for going towards the product is K2 well all I'm doing here is I'm just drawing in this a little bit more of a uh conceptual matter to under to explain something called inhibition so if you look here in green I have my enzyme in red I have my substrate I have my double arrows and again what would this one be this would be K1 this would be K1 and this would be K2 this this green right here is the enzyme the red right there is the substrate so this whole thing is the enzyme substrate this green right here is the enzyme and this blue structure right there is the product what we're going to do in this video is we're going to talk specifically about how Inhibitors can affect this normal process so in the first one we're going to talk about what's called competitive inhibition so what happens in competitive inhibition let's say right here I draw in Black a competitive inhibitor now a competitive inhibitor is structurally similar to the normal substrate so here in the red is our substrate I'll put substrate above that and here in black is going to be our competitive inhibitor if you look their structures appear appear almost exactly similar what happens in this reaction is as the substrate is trying to react with the enzyme the inhibitor might have more affinity for the enzyme or the en I might have full more affinity for the inhibitor than the substrate and so for example what will happen is the inhibitor will come in here and bind into the active site of this enzyme and then look what happens can the substrate get in there if the inhibitor is bound no so then what does it look like afterwards if I were to draw the product of this reaction here let's say I have one Arrow going this way and this is a reversible reaction what would it look like I would have something like this so now I would have my my enzyme here and again what's going to be bound to the active site of this enzyme it's going to be a inhibitor and when the inhibitor binds so now let's draw the inhibitor fitting into that active sight pocket now what can't happen the substrate can't bind so let's draw the substrate over here the substrate it can't bind into that active site so because the substrate can't bind into the active site the enzyme's affinity for the substrate goes down and its affinity for the inhibitor goes up now the only way that this reaction can continue to proceed is if you do something so if you think about this let's think about what's called later's principle so later's principle says in this case what's happening here to the the formation of my enzyme substrate well if the inhibitor is binding there to the enzyme I'm not making as much enzyme substrate right so my enzyme substrate concentration isn't really going up but if you think about this with my my my enzyme the enzyme is binding a lot to this inhibitor right if it's binding a lot to this inhibitor it's not binding with the substrate so if you think about this let's think about like a a seaa here so let's say here I have a seaw let's say on one end I have the enzyme substrate and let's say over here on this end I have the enzyme and we also have the substrate in this case I'm not making enough enzyme substrate so what's happening to this this like you can think about this is going down right now the amount of enzyme that's reacting with the substrate also is decreasing right so again this is going to decrease now lad's principle says that whenever this side of the equation is decreasing you're going to want to shift the reaction to the right to increase the concentration of the enzyme substrate well how I do that by increasing the substrate concentration so if I increase the substrate concentration enough to where the Affinity that the enzyme has for the inhibitor might go down a little bit and the sub the substrate might uh compete and outbeat the inhibitor to that active site then what can happen then I can form more enzyme substrat so again one more time a competitive Inhibitors competitive inhibitor will bind onto the active side of this enzyme block the sub from binding so now you have this structure here the only way that I can form more enzyme substrate cuz what's happening according to L chat's principle the amount of enzyme substrate that I'm making is decreasing if the amount that I'm making is decreasing lat's principle says I have to shift this reaction to the right to increase the concentration of my enzyme substrate so what do I have to do I need to increase the amount of substrate if I increase the amount of substrate less of the inhibitor will bind to the active site and more of the substrate will bind into the active site and form our enzyme substrate complex which will disassociate into enzyme and product so again what is this one right here called this one is competitive competitive inhibition and this one is super super important a lot all of these are super important this one's all the most common usually but if we think about it um what could we do as because you know there's normal Inhibitors all around and everything that we do we can actually think of drugs actually that could act as um competitive Inhibitors that could actually be a good thing so for example what would be a normal example of a competitive inhibitor that would inhibit a specific enzyme maybe in our body so for example um we could say a Statin so statins are basically uh they they're trying to lower our normal cholesterol levels so they're good for hyper lipidemia and hypercholesterinemia what does statin do Statin actually binds on to the active site of specific enzyme that regulates cholesterol synthesis and that's called HMG COA reductase so we're just giving you some real life applications of competitive Inhibitors and what is the whole concept inhibitor is binding onto the active site of the enzyme blocking the substrate from binding but if you increase the substrate concentration you can overcome the amount of inhibitor that's binding and form more enzyme substrate and we're going to talk about km in just a second with this one another example let say I'll just give you another one um high blood pressure so they actually use another drug here it's called ACE inhibitors like captopril enalopril aoil so ACE inhibitors bind to the active site of Ace enzyme so it actually inhibits so statins inhibits HMG chor duct a ACE inhibitors would inhibit Ace which stands for Angiotensin converting enzyme okay so just some practical applications of competitive inhibition now one more thing I want to mention about competitive inhibition what did we have to do to get this enzyme and substrate to form more enzyme substrate we had to increase the substrate concentration well remember we said km is equal to the substrate concentration right with respect to half the Vmax well what what I did was I increase the substrate concentration so I threw off the normal equilibrium of this reaction so now what I have to do to account for this increasing substrate concentration is the km because there's more substrate what did I say remember that relationship I said that with increasing km the enzyme will have to take on more substrate so if there's an increase in km it's because there was a increase in substrate concentration so what's happening to the km or you can say the Affinity that that this enzyme has for the substrate it's increasing because we have to increase the substrate concentration super super important here so it competitive we increase the km but did we affect the amount of product that's being formed no because we increase the substrate concentration so we're still going to make our normal amount of products so Vmax which is the maximum velocity the the saturation point to where the enzyme can be no more saturated still occurs so we can still reach VMAX so we'll say Vmax is normal or unchanged okay these are two big Concepts that I need you guys to grasp from this inhibition okay so that covers competitive inhibition let's move on to the next one so the next one that we're going to do is let's talk about non-competitive inhibition a non-competitive inhibition it's a little different now non-competitive inhibition let's say again I draw here in Black this is my inhibitor but what happens is the inhibitor doesn't bind onto the active site there's a little pocket here on the enzyme there's a little pocket that's kind of always open here on the enzyme and what happens is this inhibitor right here so again what's this guy right there this is my substrate and then what's this black guy right there this is our inhibitor what can happen is this inhibitor can actually come on and B bed to this other site of the enzyme that's not the active site that site right there is called the aloe steric site okay and what happens is when the inhibitor comes in and it binds onto that free enzyme remember it's free enzyme what do we get out of this let's see if I draw this double arrow because this can be reversible I'm going to have something like this so I have my enzyme here and the inhibitor will I mean the enzyme will not be bound to the substrate because it's bound to the inhibitor right so if we look here what will happen it'll have this little pocket here right so I have this little pocket there and then what happens this inhibitor is bound onto that guy but now look what happens here this enzyme can still react with substrate it can still react with substrate that's not going to stop this reaction so if I go ahead in this reaction and I react this enzyme inhibitor right with my substrate so let's say now I reacted with my substrate the substrate can still bind it can still bind in there into that active site so now look what we have here so now look what we got here something not good right something not very good it's a beautiful thing but not a you know not necessarily good for this enzyme so what happens here so now look what we have here we have the substrate bound into the active site still and the inhibitor is still Bound in that little alisic pocket right there so it's still Bound in that alisic pocket now look what we have here here we had a enzyme inhibitor complex right here we have a enzyme substrate inhibitor complex holy crap can this guy ever get converted into this product no so this reaction will never produce product so there will be no product formed from this reaction but if by some possible way this inhibitor is released so let's say I draw here an arrow coming up to the free to this enzyme substrate not free enzyme enzyme substrate so now look I have this reaction here and what did I let's say I release out out of this what did I release I release out this inhibitor by some way I get rid of the inhibitor so the inhibitor is binding onto the aleric site forming the enzyme inhibitor complex but the enzyme inhibitor can still bind with the substrate then when it binds with the substrate we have an enzyme substrate inhibitor complex what can happen then the inhibitor can be released and if the inhibitor is being released then what happens I form my enzyme substrate and then the reaction can occur and then make more product but look what's happening in this reaction so if you think about it non-competitive Inhibitors they can bind on to the free enzyme but they can also bind onto the enzyme substrate complex so again what is this one right here this is called non competitive inhibition okay now we have to look at something else here so when we look at non competitive inhibition as compared to competitive inhibition because we're still able to form enzyme substrate so in other words because this reaction is proceeding in this way that it's still making enzymes so this can go two ways it can go this way or can come this way technically our equilibrium is is not thrown off we we're still in equilibrium with these reactions so because this reaction can proceed this way and it can come all the way up through this way our equilibrium isn't thrown off so because the equilibrium isn't thrown off we did not change the km so the km for this reaction remains unchanged so I'm going to put normal right or unchanged so because again one more time why is the CM normal because the equilibrium for this reaction is not changed it's not shifting from one side to the other it's favoring a normal equilibrium however look what happens here because this inhibitor is binding onto that enzyme with the substrate can you form product all the time no some product isn't going to be formed and then look what happens some of it you can make enzyme substrate and some of them you can actually form product but at some point in time not all of this enzyme will be in the form of enzyme substrate complex so in other words you'll never be able to have all of your enzymes bound to substrate so what does that mean I'll never reach Vmax so Vmax decreases in this point so Vmax decreases so these are really important Concepts like me highlight these guys again these are really really important things again so what happens in non-competitive inhibition cm is normal why because the normal equilibrium of this reaction is not changed or it's not favoring one side of the other Vmax decreases why because some of this reaction will form no product and it's and you'll never have the enzyme all of our enzymes never never will all the enzymes be bound to substrate or it going to be completely saturated so it never reaches Vmax all right now that we've done that let's give some practical applications of certain types of non-competitive Inhibitors like physiologically so one of them um is actually used a lot in certain cases maybe certain types of depressions or certain things with catacol amines norepinephrine epinephrine and these can be called monoamine oxidase Inhibitors so monoamine oxidase Inhibitors there's many many different types of monomin oxidase Inhibitors we're not going to go into each one but basically what they can they do they can bind on to certain allosteric sites of monoamine oxidases and what do they do they inhibit the normal activity of that enzyme right so it inhibits the monoamine oxidases which are important for being able to break down norepinephrine epinephrine dopamine so on and so forth okay that's one example um another one could be what's called um oximate oximate or oxamic acid so I'm going to put oxamic acid and what this enzyme does is it inhibits a specific enzyme which is called lactate dehydrogenase so oxamic acid or oximate can basically inhibit this enzyme called lactate dehydrogenase how binding onto the alisic site and affecting the normal production of product how is that going to affect us physiologically it canect lactate dehydrogenase is actually important for being able to convert lactate um into pyruvate and pyruvate can actually get converted into glucose and if lactate is going into forming glucose that's called glucon neogenesis so oxamic acid is a normal non-competitive inhibitor of lactate dehydrogenase or an overall pathway called gluconeogenesis which is needed to be able to put glucose into our bloodstream all right so that can affect our glucose levels all right so that's an example there let's do the next one here and this is going to be called uncompetitive inhibition so uncompetitive inhibition is kind of like non-competitive but here's where the difference is the the the uncompetitive inhibitor only binds on to the enzyme substrate complex whereas the non-competitive the inhibitor could bind on to the enzyme substrate complex or the free enzyme in uncompetitive when the enzyme binds the substrate then they make an alisic site okay so there's an allosteric site that's only opened up whenever the enzy is bound to the substrate and then look who comes in and plugs in here now we have our uncompetitive inhibitor if the uncompetitive inhibitor binds onto this what do you get then we'll get a look at this new enzyme over here we'll get a new enzyme over here and this is going to be bound to the substrate right which is going to be bound in the active site and then when it's bound into the active site again what happens then it changes its shape to form a little alisic pocket there and then the uncompetitive inhibitor comes in and binds into that alisic site and then the substrate is still bound into this active site here now where's the problem with this this enzyme is going to have a hard time turning the turnover rate in other words so in other words the product formation is going to decrease so me being able to form product from this reaction is not going to happen same thing like this one what do you get no product okay so with this one it only the inhibitor only binds on to the enzyme substrate complex why because when the enzymes bound to the substrate opens up an alisic pocket for the uncompetitive inhibitor to come in and bind and then you form this enzyme substrate inhibitor complex so again what is this guy here enzyme substrate inhibitor complex there which will form no product but if by some way you're able to release that inhibitor from the allosteric site so I say here's your inhibitor and you're able to release that inhibitor from the allosteric site you can form let me actually get this guy out of here so now we have this free enzyme here so we can actually have this guy coming in this way so we can have the inhibitor coming in into this reaction so going this way or we we can have the inhibitor released coming to forming our enzyme substrate so if the inhibitor is released then what do you get you get your enzyme substrate which can progress to forming product now with the non-competitive we said that the normal km like the equilibrium of going from enzyme substrate to enzyme substrate complex wasn't affected however in this reaction it is why if you think about it if the inhibitor is binding onto the aleric site on the enzyme substrate complex and making enzyme substrate inhibitor what's happening to the normal concentration of our enzyme substrate our normal enzyme substrate it's decreasing so if the normal concentration of our enzyme substrate not bound to the inhibitor not this one if this amount is decreasing right because a lot of this is bound in this form then what does l principle say it kind of goes back to this the reaction is going to proceed to the right so now when the reaction starts proceeding to the right what starts happening to the concentration of my enzyme and substrate well the substrate concentration starts going down and as the substrate concentration starts going down as you're working this way right so again what's happening to the substrate concentration as this reaction is progressing this way so I'm going to draw it with uh red here as this reaction is progressing this way according to lat's principle because again what's happening the normal enzyme substrate concentration is decreasing the reaction is going to shift to the right so what happens to the substrate concentration it starts decreasing so as the substrate concentration starts decreasing what starts happening to the km what goes back to that concept Whenever there is low substrate concentration we said in the miches Menon equation video that it does what to the km it decreases the km right so now because the substrate's moving to form enzyme substrate what's happening to the km of this enzyme it's decreasing well decreasing km means what it means it will accommodate less substrate so whenever this event changes because the substrate concentration is going down as the re reaction is proceeding to the right the substrate concentration go goes down and now the km goes down because the enzyme is dealing with less substrate okay so this is again a measure of infinity so now what will happen to the Affinity again if the km is going down it's going to have more affinity for the remaining substrate all right so what happens in this reaction and this one is uncompetitive let's write this one down this one is uncompetitive and I'm I going to put inhibition here so what is uncompetitive inhibition happening here so if we look what happened to the km the km decreased okay that's looking good then what remember we said when we form no product what else are we forming here no product so that means that the normal amount of enzyme and product that we're going to form our turnover is going to start decreasing right so we're not going to make the full amount of enzyme substrate complex so in other words we will never have a point in the reaction in which the enzyme is completely saturated and bound with substrate why because some of it will always be bound to this enzyme substrate inhibitor some of it will be in the enzyme substrate inhibitor form so if that's the case then are we going to be able to make product with this reaction no so what happens to the Vmax the same thing that happened with the non-competitive it decreases so in this case the Vmax decreases okay and again this is another important concept here so out of all these let's let's quick recap cuz we're going to do the last one here competitive is increase in the km and then the Vmax is remaining the same non-competitive inhibition the km is staying the same but the V-Max is decreasing uncompetitive inhibition the km and the Vmax are decreasing let me give you an example real quick of um again two just uncompetitive Inhibitors so um one of them is actually important within the glycolysis pathway and this this one is called glyceride three phosphate dehydrogenate so this is the enzyme this enzyme is normally important for being able to convert glyceride 3 phosphate into uh 1 comma 3 by phosphoglycerate but what happens here is I can there's a certain type of uh like a like a poison it's called arsenic or arsenate so arsenic or arsonate basically what happens is that can actually bind on to the G3 pdh which is the glyceride 3 phosphate dehydrogenase where does it bind it binds when the enzyme is bound to the substrate what's the substrate glyceride 3 phosphate but when arsenic binds it affects the normal formation of the product okay so that's that's one example um another example of this one could be um in bipolar disorder um they give people what's called uh lithium so in bipolar disorders so they give this in individuals who have what's called bipolar uh disorder they treat them with lithium why because lithium binds on to a specific enzyme in this pathway the pathway um here I'll write it out it's called the phospho um inositide uh cycle or pathway but basically what lithium does is is it binds on to the the specific enzymes within this phosphoinositide pathway when it's bound to the alisic site right whenever the enzyme is bound to that substrate and he affects the normal reaction and km of that enzyme so these are just again some examples so again once a uncompetitive Arsenic and once another uncompetitive is lithium competitive non-competitive uncompetitive are all examples of reversible Inhibitors this last one we're going to talk about is an irreversible inhibitor so how does this happen all right so if we look here we have an enzyme and the enzyme is going to it's going to want to bind on to this substrate but let's say here in this case I put a inhibitor here so here's this inhibitor so here's the inhibitor and again this red guy is the substrate what happens is the inhibitor will bind into this enzyme when it binds into this enzyme it's acting as kind of like what's called a it's going to act kind of like a substrate but it's going to perform what's called a substrate analog so again let me actually get this one out of here and let's replace this one with blue so now he binds in here and when he binds in here he forms what's called a substrate analog but here's where the bad thing happens when this reaction occurs this this uh in inhibitor right here when it's bound into that active site the difference between this and competitive Inhibitors is the competitive Inhibitors through weak interactions weak Bond interactions but what happens with this one right here this irreversible inhibitor or what's called suicide inhibitor and we'll talk about it is that substrate analog forms what's called a Cove valent Bond so I'm going to draw this Cove valent bond in Red so look here it forms what's called a Cove valent Bond so so basically what happens here you have an enzyme it likes to bind a normal substrate but then this inhibitor binds into the active site and acts like a substrate analog when it acts like a substrate analog it forms what's called a CO valent bond which is a very strong bond and then look what happens is this reaction reversible no so this enzyme is stuck in this form and what is an example of this inhibition which is IR reversible this is called suicide inhibition okay so suicide inhibition so it under goes this coent modification other words and when it forms a coent bond via the substrate analog uh with calent bond to the enzyme this is not reversible and this is very very bad why so again this is substrate inhib suicide inhibition these ones are super dangerous Inhibitors I'll give you an example of one of the worst ones and we know it you know it's can be used in like certain types of chemical warfare right it's called sarin so sarin is a nerve gas and basically what this uh sarin does is Let's Pretend this enzyme is what's called acetylcholineesterase which is normally responsible for breaking down acetylcholine well if you give sarin sarin's act as the inhibitor it's going to bind in to this acetylcholine esterase act as a substrate analog and then form a Cove valent bond with the acetylcholine esterase and then what happens can the acetylcholine esterase break down the acetylcholine no and these individuals die a very painful and quick death okay so that's one example another one that's not a bad example it can be ACC it's not always it's not always bad it depends on the specific you know situation or physiological condition but they also can be good what what's an example of a good one um aspirin aspirin is a good example because um whenever you're having inflammation or certain types of blood clots and stuff like that aspirin has the ability to inhibit certain types of enzymes the enzymes are called cycle oxygenases so what could be this enzyme I'm going to put Cox but remember keep your minds clear it's not what you think it is and what does aspirin do aspirin can actually inhibit these cyc oxygenase enzymes and again how will it do it let's say that the um the normal substrate here for the cyc oxygenase is going to be some type of uh basically a radonic acid what happens the aspirin will actually come in bind on to a specific enzyme and then form this actual Cove valent Bond modification okay so these are examples these two are examples of what's called suicide inhibition but specifically I need to make sure I write this completely CLE clear this is a ear reversible inhibitor okay this is IR irreversible Inhibitors these three that I talked about here competitive non-competitive uncompetitive are reversible Inhibitors all right guys so in this video we talked about competitive non-competitive uncompetitive and suicide inhibition we gave you examples of certain types of uh um drugs or certain types of agents that can act as these Inhibitors we talked about their km and their vmx in the next video we're going to apply this to the Michaelis Menon curve and the lime Weaver BG plot