Lecture 17 : Concept of Enzyme Inhibition (Contd.)

After the completion of the reversible inhibition cases, now we will go to the irreversible inhibition Remember the basic difference between reversible and irreversible inhibition is the formation of a covalent bond in case of irreversible inhibition And, there is no covalent bond formation in case of reversible, for irreversible there is a covalent bond formation Now, I have already told you that there are two types of irreversible inhibition one what is called Suicide Inhibition And the other what is called active site directed inhibition Let me see before we go on to the suicide, I think we will talk about the Active Site Directed Irreversible Inhibition; it is abbreviated as ASDII, ok Now, what happens here the name suggest something active site directed; that means, you have a molecule which is having a directional property, which is having a property and that property is directly utilized to inhibit the enzyme Basically I can give an analogy that it is like a that if in, if somebody wants to kill another person So, he has a gun and he shoots the person straight away that is what is the What is the active site? The active site in a person is mostly the heart, if the heart stops then everything stops So, heart is the active site if you consider then this active site directed irreversible inhibition means you now should straight at the active site Schematically how it is shown? It is shown that E plus S that is the normal goes to the ES complex Then in actual actually it goes to enzyme product complex, but and then that goes to E plus P. Usually for Michaelis Menten equation, we generally do not consider this fact because we assume that is a very fast dissociation In active site directed inhibition what happens, you have a molecule which is represented as IX; this X is the one which is attached to the inhibitor that is extremely reactive And I is the part or the whole assemble is complementary to the active site of the enzyme although it looks IX, but the whole the complete inhibitor is IX not only I This is the inhibitor, but the inhibitor already has a very reactive functionality, but it has got again electronic and geometric complementarity with the active site of the enzyme So, now what happens this IX will go because it has got this complementarity So, it goes and binds to the enzyme at the active site, but at that point after binding the enzyme realizes the mistake It has got lot of reactive functionalities say like lysine NH2 or thiol cysteine So, or OH which comes from threonine or serine So, lot of reactive functionalities you can think of or acid functionality as carboxylic So, this is now, it is a very reactive X functional group So, that reacts with any with one of the amino acid residues that are present in the enzyme active site So, this is and then there is a covalent bond formation between E and X because you are doing a reaction now and, but this So, even if I goes tries to go out, but it is now bound to the enzyme via this X-E bond So, that is what is active site directed inhibition; that means, here the molecule goes binds and it already has a very reactive functionality and then while it binds, the enzyme amino acids residue which are extremely say reactive functionality So, that immediately reacts with X and in the process E gets tied up covalently with

this IX molecule So, even if this portion is broken now, but your enzyme is, now covalently attached to this IX What are some of the examples? See some of the examples are like this that if it is a continuing a carbon with a leaving group attached to another a carbonyl like COCH2X Usually these molecules from organic chemistry we know that any leaving group attached to a carbon which is adjacent to a carbonyl, SN2 displacement is very high, rate of SN2 displacement because this is a very reactive system So, based on this part COCH2X you can have different Xs these are all different X s that are given here So, you can have different reactive functionalities attached to this inhibitor and the inhibition can take place But remember the enzyme has not done any normal reaction that it was doing to the substrate, that reaction has not taken place; it is just the like a magic bullet it went in and then it forms a cross link with any of these amino acid residues What is the a better example like, if you have ice I told you that most of these inhibitors are based on systems like this And this is R; they will be all inhibitors because this is very reactive Now, if you want to design a particular inhibitor for a particular enzyme, then you have to think of what could be my R, ok Now, suppose if we have chymotrypsin in mind We know chymotrypsin hydrolyses amide bond where from the carboxy site, where the carboxyl belongs to an aromatic amino acid like phenylalanine, tyrosine or tryptophan Now, see if you have it was also found by people that chymotrypsin can hydrolyze also esters It not only hydrolyses amides the peptides, but it can hydrolyze esters like the ethyl esters of this, but you have to maintain that that factor that it only recognizes aromatic amino acids So, in general what happens, you have a system like this what is required for hydrolysis of by chymotrypsin You have a system like this and this is also important that that should be in the L configuration then phenyl this chymotrypsin as I told you know the mechanism now of chymotrypsin that it is a serine based enzyme; serine, histidine and aspartate that triad should be there But, apart from that it must have some pocket which is the hydrophobic pocket where phenyl or aromatic groups can be stabilized by π by stacking interactions or what are called this hydrophobic interactions And because of the presence of this pocket which only recognizes aromatic groups, chymotrypsin is specific for aromatic amino acids For lysine which hydrolyses only basic amino acids like when sorry for trypsin which hydrolyses only the basic amino acids like lysine or arginine; why it is so, specific then this pocket is no longer hydrophobic Because trypsin recognizes groups like say NH3 plus a lysine amino acid or an arginine here so, I can just represent by BH plus because they will be all protonated So, they go there they first fit to the enzyme So, the enzyme must have a trypsin enzyme must be having a site where lot of carboxylates are there so, that you can make salt bridges that NH3 plus and CO2 minus So, there will be lot of salt bridges So, that is why trypsin recognizes basic amino acids because of the presence of this salt, because of the presence of a pocket which ensures salt bridge formation or electrostatic

interactions and chymotrypsin has a hydrophobic pocket, but which recognizes only aromatic amino acids Now, I want to design an a active site directed inhibitor of chymotrypsin How do I do it? This is the mechanism of that charged that relay process; carboxylate that aspartate takes up the hydrogen from the imidazole and that activates this nitrogen as a base that takes up this hydrogen and this is supposed to go and attack the carbonyl But if you do not take the amide here, if you take a CH2Cl instead of taking an ester or an amide then what happens two things that are important One is now this serine if it attacks the carbonyl you cannot break a carbon carbon bond that is difficult to break; earlier there was carbon nitrogen bond that is one point, but the more important point is now this imidazole is very close to this CH2Cl this carbon which is extremely susceptible to nucleophilic attack, it is very close to that So, now the charge relay will be like this, this O minus takes the hydrogen this goes here and the nitrogen is activated So, instead of taking the proton from serine because that is now useless even if it takes the proton there is no productivity, because the serine cannot attack this carbonyl Because there is no meaningful reaction here, so the nitrogen instead of taking the hydrogen it goes and attacks this carbon and breaks the carbon chlorine bond So, what will happen or what is the result of this? The result of this is that you are forming a carbon nitrogen covalent bond with this molecule, ok So, this is and a very good example of active site directed irreversible inhibition Why active site directed? Because your molecule has a system which is recognized by the enzyme, so which is recognized by the enzyme, so the inhibitor goes and goes binds to the active site Then the normal cycle wants to take place, but that cannot happen here what is then, then because it has got a reactive functionality COCH2Cl, now the imidazole goes directly acts as a nucleophile and attacks this carbon and forming a carbon nitrogen bond So, this is the first example is the compound is called Tosyl Phenyl Chloro Ketone TPCK abbreviated as TPCK This is a very text book chemistry, this chemistry now has appeared in many textbooks And if you want to now you can, because you have this knowledge now if I ask that how to now make a irreversible inhibitor active site directed of course, of trypsin So, what you do, you now replace this phenyl alanine group and put instead of taking this TPCK; that means, tosyl phenyl chloro ketone, you take tosyl lysyl chloro ketone so; that means, that will be called TLCK; TLCK is tosyl this l stands for lysyl and then chloro ketone; TLCK This is an inhibitor for trypsin and the other one, this one is an inhibitor of chymotrypsin; both are active site directed, because both the molecules have reactive functionality number one number two they also have the structural feature that is necessary which is recognized by the pocket that is present in the active site So, this is active site The next one is Suicide Inhibition; this is another interesting very interesting topic Here what happens is that the inhibitor looks like very similar to the substrate And enzyme what the enzyme can do, because it is very similar to the substrate So, the inhibitor also goes and binds to the enzyme like the substrate After binding what happens usually the enzymes wants to do the reaction that is supposed to, it is supposed to catalyze So, it tries to do the reaction on the inhibitor and it does that reaction what is it is doing with the substrate original substrate It does the same reaction and, but in the process ok, now it is no longer I; like it

is no longer S; it is now it is now designated as product So, here the inhibitor is converted into another molecule, but ironically this molecule what the enzyme is doing, it is doing the same reaction like what it is doing on substrate, but in the process it makes a molecule which is highly reactive Before reaction it was not that reactive, after reaction it became very reactive And then as it is so, reactive it immediately forms actually this should be I star EI star So, covalent bond is super reactive covalent bond is formed So, basically it, I can take an analogy if you have read the story of Frankenstein, what happens a scientist wanted to bring life to a dead person So, he created the life into that dead person and the dead person wanted ultimately killed the scientists ok So, it is a very similar, the enzyme initially thinks that I is my friend like the substrate So, the inhibitor goes and enzyme embraces it with both arms and then what happens and then for friends we generally exchange words here, the enzymes that does the normal reactions, thinking that it is a very friendly molecule But, unfortunately after doing the reaction this molecule becomes an enemy and finally, kills the enzyme by forming a covalent bond So, that is why it says, that the enzyme is as if committing suicide and that is why this is called suicide inhibition It is also called mechanism based enzyme inhibition another name is given there, because the whole thing is depending on a particular mechanism that how I is converted to I star So, this is also called mechanism based inhibition, irreversible inhibition and, but the more common name is suicide inhibition Now, what is an example Before we go to this, I told you that enzyme inhibitors are very important from drug design concept many of our drugs 60 percent of the drugs are actually inhibitors of some kind of enzyme or receptor Now in case of cholesterol, when we have the disease high cholesterol, I want to modulate the activity of the enzyme, lower the activity of the enzymes which are involved in the biosynthesis of cholesterol But I do not want to shut off totally that cholesterol should not be produced in my body Because cholesterol has other roles to play, from cholesterol we get the steroid hormones in our body, ok Cholesterol also constitutes the that membrane that surrounds our that keeps the cell contents intact So, you cannot completely shut off cholesterol Like sugar, if somebody suffering from higher diabetes, but you have to maintain the level of the glucose at a certain value If it goes down, that is also a problem from the normal value, but in some cases So, in those cases a reversible inhibition is better because you take the drug it modulates the activity of the enzymes So, that your cholesterol level or sugar level is maintained at the optimum condition, optimum what is expected and then when the drug is metabolized you take the next day another pill to keep the concentration of the drug in your body But, the important thing is that you need reversible inhibition You do not want to completely shut off the cholesterol biosynthetic pathway or your sugar glucose metabolic pathway; you do not want to completely shut down But in some cases you need this irreversible inhibition Remember reversible inhibition can be reversed, the inhibition can be overcome by adding excess of the substrate But for irreversible inhibition you are destroying the enzyme, whatever molecule is attached covalently to the inhibitor, that enzyme you cannot recover it is activity, it is already gone It has formed a covalent bond, active site residue is gone So, but where it is required, it is required when we want to really kill the enzyme totally, like when we have a bacterial infection And suppose there is a particular enzyme in the bacteria which is very essential for the bacteria to grow

So, then we target that enzyme in the bacteria and it will be much better if you have this type of irreversible inhibition, because you want to kill the bacteria; that means, you want to kill the enzyme You want to completely shut off the pathway that the enzyme follows So, I will quickly go to the example; again a text book example, this is not a drug molecule, this is a text book example that whether how to design a suicide inhibitor This was this is a molecule you know again chymotrypsin was the model because chymotrypsin structure is well known it is mechanism is well known it is available in plenty at a cheap rate, so, all these studies initially were done with chymotrypsin We know chymotrypsin recognizes aromatic amino acids So, all these examples are based on phenylalanine that is the easier one to work So, phenylalanine with a this is nitrogen that is the carbonyl side of the phenylalanine You have the carbonyl as protected with isopropyl and here instead of the amine CONH what you have is that this nitrogen is also having another benzyl This is actually benzyl We call it phenylalanine because we think it is phenyl derivative of alanine, but you can also think of that this is a benzyl derivative of glycine So, that also you can think that this is called benzyl glycine, but unfortunately from the very beginning it was called phenylalanine, ok So, you have now 2 CH2Phs this is, this has a configuration, L configuration and this nitrogen is attached to a nitrosyl group NO So, what you have is N-nitroso N-benzyl phenylalanine derivative and show it to the enzyme What was the idea of the scientist who was doing this? The idea is that that we know that the enzyme because it is phenylalanine, the enzyme is having this same catalytic triad and it will come and attack this, and this bond will be broken, not this bond sorry I am sorry this is attacking the just one second I will erase that, ok So, again just take the pen from here, ok So, what was it supposed to do the OH is supposed to do an attack on the carbonyl and break the carbon nitrogen bond that what was the earlier the peptide bond, here it is N-nitroso N-benzyl Because you have this benzyl group in the correct configuration there was no problem So, it goes and it binds to the active site and then this via this catalytic triad mechanism this hydrolyzes this carbon nitrogen bond Now, what happens to this species? The species will be initially like this PhCH2N minus initially and So, this is basically nitroso benzyl amine anion So, that will now take the proton so, to make it NH So, what you get now N-nitroso benzyl amine Now this type of functionality in organic chemistry from organic if, you have read organic chemistry so, because this subject is all about organic chemistry and biology So, you see now the reactivity of molecules come in to play This NH-NO; that means, N nitroso amines are very unstable What happens they go to the, this other tautomeric form that is called the diazo compound and this diazo compound is also not very stable Now, this goes to the it releases the hydro this OH, the OH comes out as H water and this forms this, there are some mistakes here I think better is you write this way P hCH2N triple bond N and these nitrogen is plus Earlier structure did not this is one resonating structure, but this is not very stable one, because this is not having the octet fulfilled This is the better structure, where all the octets are present all the atoms are fulfilling the octet, but this is also not stable Now, these triple N triple bond N can come out as nitrogen, a stable very stable molecule So, in the process what you are making a PhCH2 plus which is nothing, but a very reactive electrophile Any alkyl group with plus will be a very reactive electrophile

So, whatever nucleophilic amino acids are there in the active site; now they can react with this enzyme active site and forms this is the covalent bond that is formed So, this is first of all irreversible inhibition because you are forming a covalent bond with the substrate, but this is with the molecule that it is bind; it is binding that is the inhibitor molecule Number 2 is that this is not active site directed, because there is a particular reaction that the enzyme has done what is that reaction the normal reaction The enzyme hydrolyzes this carbon nitrogen bond carbonyl carbon nitrogen bond So, that normal reaction has happened and ultimately what is created is a monster This is a monster that ultimately creates a very electrophilic benzylic cation And if the enzyme wherever whatever nucleophilic amino acids it has, it will now capture this electrophile and in the process the enzyme should be inhibited Now, the scientist who made this molecule he had this in mind, ok In research we have many things in mind, but that does not work Here apparently it was very well thought out proposal So, he made this molecule show it to the enzyme that is chymotrypsin; unfortunately he did not observe any inhibition Why is that? That was the question that why there was no inhibition whether there is this reaction failed No he found that, this reaction was going on; that means, this part was released and this part was forming the acyl enzyme complex and finally, this was converted to the CO2H But it failed to inhibit the enzyme What happened here? The interesting point because he was a brilliant scientist, so he realized what is happening here See here the part which is attached to the enzyme is the phenylalanine that benzyl part the phenylalanine L phenylalanine that Ph that is bound to the enzyme And the reactive species that was generated CH2 and then you have NH sorry NH and NO that is generated, that has no connection with the enzyme So, by the time all these reactions take place it just has come out of the enzyme pocket So, it has gone out of the enzyme because there was no connection of these with the enzyme The part which was attached to the enzyme hydrophobic pocket was this phenyl that was attached So, the other part was free to be released and by the time all these rearrangement reactions take place that reactive part is outside the enzyme If it is outside the enzyme, so, it cannot destroy the enzyme So, this did not work But as I said he was a brilliant scientist So, what he did? He did a slight change what is this slight change? This is the one I was telling that because this phenyl was being attached to the enzyme hydrophobic pocket; this part is free to escape from the enzyme active site So, then what he did, he changed the configuration of this carbon He made it a D alanine a D phenylalanine derivative This was L remember amino acids, proteins are all made of L amino acids and So, it is natural that the enzyme will also recognize only L amino acids containing molecules It will not recognize, or it will not recognize the D so; that means this phenyl now cannot bind to this hydrophobic pocket, why because the configuration is D. Earlier this hydrogen was alpha (below the plane) and you can think of this phenyl as above the plane, if you think that and the active site is somewhere here So, now when the phenyl is above the plane hydrogen is below it can the phenyl can go and fit in to the active site But when you take the other configuration now the hydrogen is up and the phenyl is down So, it cannot go and bind to this active site, but what happened here we are all the Now this benzyl was sitting by the side of this alpha carbon, when it was L because there

was perfect match in stereo chemistry So, it goes and binds and this is free to escape When you made it D then this molecule binds to this enzyme, but via the phenyl group of the N-nitroso part now And you know nitrogen what is the stereo chemistry, nitrogen can actually flip back and forth Nitrogen configuration is not a particular one whatever is required it will do that So, if it requires a up phenyl So, the nitrogen lone pair will be down and the phenyl will be up So, this phenyl now serves as the it replaces the other phenyl or phenylalanine So, that N-nitroso linked benzyl group the aromatic ring now is interacting Now what is the effect of that, now this is not free to escape from the active site because it is the phenyl is bound to this point So, if it is bound So, exact what will happen now this phenyl this is the phenyl of the N-nitroso part So, that is remaining so, ultimately this species So, whatever time it takes it will take, but this is already attached by weak interactions So, it cannot escape from the enzyme active site So, this goes out you make the electrophilic carbon here And now if there is a nucleophile in surrounding amino acid then that is going to react with the this benzylic carbon So, this is the mechanism of this reaction Now, this is an example of suicide inhibition Because as I said in suicide inhibition the substrate goes and the inhibitor goes and binds, then the enzyme does the normal reaction Normal reaction means chymotrypsin is supposed to hydrolyze an amide bound and that what has happened But after the hydrolysis, the species that is generated, that is extremely reactive And because of this switch over L to D this benzyl is now is bound to the hydrophobic pocket; that means, this reactive species is now bound So, whatever chemistry that takes place after this, even if it takes some time, that is that does not matter because the reactive species is still bound to the enzyme And then the enzyme nucleophile will attack this benzylic carbon and forms the carbon X bond So, this is the story of reversible and irreversible inhibition I think now, we have completed this inhibition part So, next day we will see more designed part because this forms the basis of our drug design the next module that is related to whatever we are talking now So, we have to clarify all doubts about enzyme inhibitions and then we will go to the nucleic acids once this is over Thank you very much