Proteins do not exist in a vacuum. The body is mostly water and as a result all proteins will be surrounded by this medium. Therefore, amino acid residues at the surface of proteins must interact with water molecules. Water is a highly polar compound which forms strong hydrogen bonds. Thus, water would be expected to form strong hydrogen bonds to the hydrogen bonding amino acids previously mentioned.
Water can also accept a proton to become positively charged and can form ionic bonds to aspartic and glutamic acids Fig.
These polar amino acids are termed hydrophilic. As a result, they are repelled by water. Therefore, the most stable tertiary structure of a protein will be the one where most of the hydrophilic groups are on the surface so that they can interact with water, and most of the hydrophobic groups are in the centre so that they can avoid the water and interact with each other. The hydrophobic amino acids in the centre have no choice in the matter and must interact with each other. Thus, the hydrophobic interactions within the structure outweigh the hydrophilic interactions and control the shape taken up by the protein.
One important feature of this tertiary structure is that the centre of the proteins is hydrophobic and non-polar. This has important consequences as far as the action of enzymes is concerned and helps to explain why reactions which should be impossible in an aqueous environment can take place in the presence of enzymes. The enzyme can provide a non-aqueous environment for the reaction to take place.
For example, haemoglobin is made up of four protein molecules—two identical alpha subunits and two identical beta subunits not to be confused with the alpha and beta terminology used in secondary structure. The quaternary structure of haemoglobin is the way in which these four protein units associate with each other.
Since this must inevitably involve interactions between the exterior surfaces of proteins, ionic bonding can be more important to quaternary structure than it is to tertiary structure. Nevertheless, hydrophobic van der Waals interactions have a role to play. It is not possible for a protein to fold up such that all hydrophobic groups are placed to the centre. Some such groups may be stranded on the surface.
If they form a small hydrophobic area on the protein surface, there would be a distinct advantage for two protein molecules to form a dimer such that the two hydrophobic areas face each other rather than be exposed to an aqueous environment.
The tertiary structure is the most important feature as far as drug action is concerned, although it must be emphasized Fig. Tertiary structure is a result of interactions between different amino acid residues and interactions between amino acids and water.
We are now ready to discuss the two types of protein with which drugs interact— enzymes and receptors. Without them, the cell's chemical reactions would be too slow to be useful, and many would not occur at all. A catalyst is an agent which speeds up a chemical reaction without being changed itself. An example of a catalyst used frequently in the laboratory is palladium on activated charcoal Fig.
It is therefore more correct to describe a catalyst as an agent which can speed up the approach to equilibrium. In an equilibrium reaction, a catalyst will speed up the reverse reaction just as efficiently as the forward reaction. Consequently, the final equilibrium concentrations of the starting materials and products are unaffected by a catalyst. How do catalysts affect the rate of a reaction without affecting the equilibrium? The answer lies in the existence of a high-energy intermediate or transition state which must be formed before the starting material can be converted to the product.
The difference in energy between the transition state and the starting material is the activation energy, and it is the size of this activation energy which determines the rate of a reaction rather than the difference in energy between the starting material and the product Fig.
A catalyst acts to reduce the activation energy by helping to stabilize the transition state. The energy of the starting material and products are unaffected, and therefore the equilibrium ratio of starting material to product is unaffected.
We have stated that catalysts and enzymes speed up reaction rates by lowering the activation energy, but we have still to explain how. There are several factors at work. We can see these factors at work in our example of hydrogenation with a palladium-charcoal catalyst Fig. In this reaction, the catalyst surface interacts with the hydrogen molecule and in doing so weakens the H-H bond. The bond is then broken and the hydrogen atoms are bound to the catalyst.
The catalyst can then interact with the alkene molecule, so weakening the pi bond of the double bond. The hydrogen atoms and the alkene molecule are positioned on the catalyst conveniently close to each other to allow easy transfer of the hydrogens from catalyst to alkene.
The alkane product then departs, leaving the catalyst as it was before the reaction. How do catalysts lower activation energies? Therefore, the catalyst helps the reaction by providing a surface to bring the two substrates together. It participates in the reaction by binding the substrates and breaking high-energy bonds, and then it holds the reagents close together to increase the chance of them reacting with each other.
Enzymes may have more complicated structures than, say, a palladium surface, but they catalyse reactions in the same way. They act as a surface or focus for the reaction, bringing the substrate or substrates together and holding them in the best position for reaction.
The reaction takes place, aided by the enzyme, to give products which are then released Fig. Note again that it is a reversible process. Enzymes can catalyse both forward and backward reactions. The final equilibrium mixture will, however, be the same, regardless of whether we supply the enzyme with substrate or product.
The active site is usually quite a small part of the overall protein structure, but in considering the mechanism of enzymes we can make a useful simplification by concentrating on what happens at that site. It has to be on or near the surface of the enzyme if substrates are to reach it.
However, the site could be a groove, hollow, or gully allowing the substrate to 'sink into' the enzyme. Active Site Active Site Fig. Because of the overall folding of the enzyme, the amino acid residues which are close together in the active site may be extremely far apart in the primary structure. For example, the important amino acids at the active site of lactate dehydrogenase are shown in Fig.
The numbers refer to their positions in the primary structure of the enzyme. Substrate binding at an active site 31 The amino acids present in the active site play an important role in enzyme function and this can be demonstrated by comparing the primary structures of the same enzyme from different organisms. In such a study, we would find that the primary structure would differ from species to species as a result of mutations lasting over millions of years.
The variability would be proportional to how far apart the organisms are on the evolutionary ladder and this is one method of determining such a relationship.
However, that does not concern us here. What does, is the fact that there are certain amino acids which remain constant, no matter the source of the enzyme. These are amino acids which are crucial to the enzyme's function and, as such, are often the amino acids which make up the active site.
If one of these amino acids should be lost through mutation, the enzyme would become useless and an animal bearing this mutation would have a poor chance of survival. Thus, the mutation would not be preserved. The only exception to this would be if the mutation either introduced an amino acid which could perform the same task as the original amino acid, or improved substrate binding.
This consistency of active site amino acids can often help scientists determine which amino acids are present in an active site if this is not known already. Amino acids present in the active site can have one of two roles. Binding—the amino acid residue is involved in binding the substrate to the active site. Catalytic—the amino acid is involved in the mechanism of the reaction. We shall study these in turn.
However, whereas ionic bonding plays a relatively minor role in protein tertiary structure compared to hydrogen bonding or van der Waals bonding, it can play a crucial role in the binding of a substrate to an active site—not too surprising since active sites are located on or near the surface of the enzyme.
Since we know the three bonding forces involved in substrate binding, it is possible to look at the structure of a substrate and postulate the probable interactions which it will have with its active site. As an example, let us consider the substrate for lactate dehydrogenase—an enzyme which catalyses the reduction of pyruvic acid to lactic acid Fig.
If these postulates are correct, then it means that there must be suitable amino acids at the active site to take part in these bonds. A lysine residue, serine residue, and phenylalanine residue would fit the bill respectively. If there were no interactions holding the substrate to the active site, then the substrate would drift in and drift out again before there was a chance for it to react.
Therefore, the more binding interactions there are, the better the substrate will be bound, and the better the chance of reaction. But there is a catch! What would happen if a substrate bound so strongly to the active site that it was not released again Fig. The answer, of course, is that the enzyme would become 'clogged up' and would be L Substrate. Therefore, the bonding interactions between substrate and enzyme have to be properly balanced such that they are strong enough to keep the substrate s at the active site to allow reaction, but weak enough to allow the product s to depart.
This bonding balancing act can be turned to great advantage by the medicinal chemist wishing to inhibit a particular enzyme or to switch it off altogether. A molecule can be designed which is similar to the natural substrate and can fit the active site, but which binds more strongly. It may not undergo any reaction when it is in the active site, but as long as it is there, it blocks access to the natural substrate and the enzymatic reaction stops Fig 4.
This is known as competitive inhibition since the drug is competing with the natural substrate for the active site. The longer the inhibitor is present in the active site, the greater the inhibition. Therefore, if the medicinal chemist has a good idea which binding groups are present in an active site and where they are, a range of molecules can be designed with different inhibitory strengths. No Reaction Fig. Competitive inhibitors can generally be displaced by increasing the level of natural substrate.
This feature has been useful in the treatment of accidental poisoning by antifreeze. The main constituent of antifreeze is ethylene glycol which is oxidized in a series of enzymatic reactions to oxalic acid Fig. It is the oxalic acid which is responsible for toxicity and if its synthesis can be blocked, recovery is possible. The first step in this enzymatic process is the oxidation of ethylene glycol by alcohol dehydrogenase. Ethylene glycol is acting here as a substrate, but we can view it as a competitive inhibitor since it is competing with the natural substrate for the enzyme.
If the levels of natural substrate are increased, it will compete far better with ethylene glycol and prevent it from reacting. Toxic oxalic acid would no longer be formed and the unreacted ethylene glycol would eventually be excreted from the body Fig. CH2-OH Fig. The cure then is to administer high doses of the natural substrate—alcohol! Perhaps one of medicine's more acceptable cures? There are many examples of useful drugs which act as competitive inhibitors. For example, the sulfonamides inhibit bacterial enzymes Chapter 10 , while anticholinesterases inhibit the mammalian enzyme acetylcholinesterase Chapter Many diuretics used to control blood pressure are competitive inhibitors.
This would be a non-competitive form of inhibition since increased levels of natural substrate would not be able to budge the unwanted squatter. The most effective irreversible inhibitors are those which can react with an amino acid at the active site to form a covalent bond. Amino acids such as serine and cysteine which bear nucleophilic residues OH and SH respectively are common inhabitants of enzyme active sites since they are frequently involved in the mechanism of the enzyme reaction see later.
By designing an electrophilic drug which would fit the active site, it is possible to alkylate these particular groups and hence permanently clog up the active site Fig. In the same way, penicillin Chapter 10 is highly toxic to bacteria. We would therefore expect these inhibitors to have some sort of structural similarity to the natural substrate. We would also expect reversible inhibitors to be displaced by increased levels of natural substrate. However, there are many enzyme inhibitors which appear to have no structural similarity to the natural substrate.
Furthermore, increasing the amount of natural substrate has no effect on the inhibition. Such inhibitors are therefore noncompetitive inhibitors, but unlike the non-competitive inhibitors mentioned above, the inhibition is reversible. Non-competitive or allosteric inhibitors bind to a different region of the enzyme and are therefore not competing with the substrate for the active site. However, since binding is taking place, a moulding process takes place Fig.
If that change in shape hides the active site, then the substrate can no longer react. Adding more substrate will not reverse the situation, but that does not mean that the inhibition is irreversible.
Since the inhibitor uses non-covalent bonds to bind to the allosteric binding site, it will eventually depart in its own good time.
But why should there be this other binding site? The answer is that allosteric binding sites are important in the control of enzymes. A biosynthetic pathway to a particular product involves a series of enzymes, all working efficiently to convert raw materials into final product. Eventually, the cell will have enough of the required material and will want to stop production. Therefore, there has to be some sort of control which says enough is enough. It can do this by inhibiting the first enzyme in the biochemical pathway Fig.
Therefore, when there are low levels of final product in the cell, the first enzyme in the pathway is not inhibited and works normally. As the levels of final product increase, more and more of the enzyme is blocked and the rate of synthesis drops off in a graded fashion. We might wonder why the final product inhibits the enzyme at an allosteric site and not the active site itself. There are two explanations for this. First of all, the final product has undergone many transformations since the original starting material and is no longer 'recognized' by the active site.
It must therefore bind elsewhere on the enzyme. Secondly, binding to the active site itself would not be a very efficient method of feedback control, since it would then have to compete with the starting material. If levels of the latter increased, then the inhibitor would be displaced and feedback control would fail. An enzyme under feedback control offers the medicinal chemist an extra option in designing an inhibitor. The chemist can not only design drugs which are based on the structure of the substrate and which bind directly to the active site, but can also design drugs based on the structure of the final overall product and which bind to the allosteric binding site.
The drug 6-mercaptopurine, used in the treatment of leukaemia Fig. The catalytic role of enzymes 37 Fig. It inhibits the first enzyme involved in the synthesis of purines and therefore blocks purine synthesis.
This in turn blocks DNA synthesis. This results from the bonding interactions between substrate and enzyme. In the past, it was thought that a substrate fitted its active site in a similar way to a key fitting a lock.
Both the enzyme and the substrate were seen as rigid structures with the substrate the key fitting perfectly into the active site the lock Fig. However, such a scenario does not explain how some enzymes can catalyse a reaction on a range of different substrates. It implies instead that an enzyme has an optimum substrate which fits it perfectly and which can be catalysed very efficiently, whereas all other substrates are catalysed less efficiently. Since this is not the case, the lock and key analogy is invalid.
It is now believed that the substrate is nearly a good fit for the active site but not a perfect fit. It is thought instead that the substrate enters the active site and forces it to change shape—a kind of moulding process. This theory is known as Koshland's Theory of Induced Fit since the substrate induces the active site to take up the ideal shape to accommodate it Fig. For example, a substrate such as pyruvic acid might interact with its active site via one hydrogen bond, one ionic bond, and one van der Waals interaction.
In order to maximize the strength of these bonds, the enzyme would have to change shape so that the amino acid residues involved in the binding move closer to the substrate Fig. This theory of induced fit helps to explain why enzymes can catalyse a wide range of substrates. Each substrate induces the active site into a shape which is ideal for it, and as long as the moulding process does not distort the active site too much, such that the reaction mechanism proves impossible, then reaction can proceed.
But note this. The substrate is not just a passive spectator to the moulding process going on around it. As the enzyme changes shape to maximize bonding interactions, the same thing is going to happen to the substrate.
It too will alter shape. Bond rotation may occur to fix the substrate in a particular conformation—not necessarily the most stable conformation. Bonds may even be stretched and weakened. Consequently, this moulding process designed to maximize binding interactions may force the substrate into the ideal conformation for the reaction to follow i.
The catalytic role of enzymes 39 Once bound to an active site, the substrate is now held ready for the reaction to follow. Binding has fixed the Victim' so that it cannot evade attack and this same binding has weakened its defences bonds so that reaction is easier lower activation energy. Histidine is a weak base and can easily equilibrate between its protonated form and its free base form Fig. In doing so, it can act as a proton 'bank'; that is, it has the capability to accept and donate protons in the reaction mechanism.
This is important since active sites are frequently hydrophobic and will therefore have a low concentration of water and an even lower concentration of protons. HIS Fig. These amino acids have nucleophilic residues OH and SH respectively which are able to participate in the reaction mechanism. They do this by reacting with the substrate to form intermediates which would not be formed in the uncatalysed reaction.
These intermediates offer an alternative reaction pathway which may avoid a high-energy transition state and hence increase the rate of the reaction. Normally, an alcoholic group such as that on serine is not a good nucleophile.
However, there is usually a histidine residue close by to catalyse the reaction. For example, the mechanism by which chymotrypsin hydrolyses peptide bonds is shown in Fig. The presence of a nucleophilic serine residue means that water is not required in the initial stages of the mechanism. This is important since water is a poor nucleophile and may also find it difficult to penetrate a hydrophobic active site.
Secondly, a water molecule would have to drift into the active site, and search out the carboxyl group before it could attack it. This would be something similar to a game of blind man's buff. The enzyme, on the other hand, can provide a serine OH group, positioned in exactly the right spot to react with the substrate.
Therefore, the nucleophile has no need to search for its substrate. The substrate has been delivered to it. Water is required eventually to hydrolyse the acyl group attached to the serine residue.
However, this is a much easier step than the hydrolysis of a peptide link since esters are more reactive than amides.
Furthermore, the hydrolysis of the peptide link means that one half of the peptide can drift away from the active site and leave room for a water molecule to enter. As far as the medicinal chemist is concerned, an understanding of the mechanism can help in the design of more powerful inhibitors.
First of all, if the mechanism is known, it is possible to design antagonists which bind so strongly to the active site by non-covalent forces that they are effectively irreversible inhibitors—a bit like inviting the mother-in-law for dinner and finding her moving in on a permanent basis.
The logic is as follows. We have already seen that the enzyme alters the shape and the bond lengths of the substrate such that it effectively converts it to the transition state for the reaction. Therefore, if the enzyme was given a compound which was already the same shape as the transition state, then that compound should be an ideal binding group for the active site, and should bind very efficiently.
If the compound was unable to react further, then the enzyme would be strongly inhibited. Such compounds are known as transition-state analogues. The beauty of the tactic is that it can be used effectively against enzyme reactions involving two substrates.
With such enzymes, inhibitors based on one substrate or the other could be designed, but neither will be as good as an inhibitor based on a transition-state analogue where the two are linked together. The latter is bound to have more bonding interactions. The target enzyme is thymidylate synthetase which catalyses the conversion of 2'-deoxyuridylic acid to dTMP Figs 4.
It is converted in the body to the fluorinated analogue of 2'-deoxyuridylic acid which then combines with a second substrate tetrahydrofolate to form a transition-state analogue in situ. Up until this point, nothing unusual has happened and the reaction mechanism has been proceeding normally. The tetrahydrofolate has formed a covalent bond to the uracil skeleton via the methylene unit which is to be transferred. At this stage, the loss of a proton from the 5-position is required. Further reaction is impossible since it would require fluorine to leave as a positive ion.
As far as the enzyme is concerned, the situation moves from bad to worse. Not only does it find it impossible to complete its task, it finds it impossible to get rid of the logjam. As part of the mechanism, the uracil skeleton is covalently linked to the enzyme.
This covalent bond would normally be cleaved to release the thymine product, but since the mechanism has jammed, this now proves impossible and the complex remains irreversibly bound to the active site.
Synthesis of thymidine is terminated, which is turn stops the synthesis of DNA. Result: replication and cell division are blocked. In the above example, the enzyme accepted the drug as a bona fide visitor, only to find that it gained an awkward squatter impossible to move. Other apparently harmless visitors can turn into lethal assassins which actively attack the enzyme.
Once again, it is the enzyme mechanism itself which causes the transformation. One example of this is provided by the irreversible inhibition of the enzyme alanine transaminase by trifluoroalanine Fig. The normal mechanism for the transamination reaction is shown in Fig. A proton is lost from the imine to give a dihydropyridine intermediate. This reaction is catalysed by a basic amino acid provided by the enzyme as well as the electron withdrawing effects of the protonated pyridine ring.
The dihydropyridine structure now formed is hydrolysed to give the products. Trifluoroalanine contains three fluorine atoms which are very similar in size to the hydrogen atoms in alanine.
The molecule is therefore able to fit into the active site of the enzyme and take alanine's place. The reaction mechanism proceeds as before to give the dihydropyridine intermediate. The growing peptide chain will then be grafted on to that amino acid Fig.
The rRNA will shift along the chain to reveal the next triplet and so the process continues until the whole strand is read. The new protein is then released from rRNA which is now available to start the process translation again. These are discussed in Chapter Summary 81 6. There are other drugs e. Since then, a large variety of biologically active compounds have been obtained and their structures determined e.
These natural products became the lead compounds for a major synthetic effort where chemists made literally thousands of analogues in an attempt to improve on what Nature had provided. The vast majority of this work was carried out with no real design or reason, but out of the results came an appreciation of certain tactics which generally worked. A pattern for drug development evolved. This chapter attempts to show what that pattern is and the useful tactics which can be employed for developing drugs.
Nowadays, the development of a novel drug from natural sources might follow the following pattern. Screening of natural compounds for biological activity. Isolation and purification of the active principle. Determination of structure. Structure-activity relationships SARs. Synthesis of analogues. Receptor theories.
Design and synthesis of novel drug structures. Plants, fungi, and bacterial strains were collected from all round the world in an effort to find other metabolites with useful biological activities. Download both the 2 parts to open the book. Respective File Sizes of Parts:. Part 1 - Part 2 - Download may show or may not show true file size but will continue till it reaches the respective file size as shown above.
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Who Posted This? Email This BlogThis! It has been discov- ence other reactions within the cell. By this process, nitric explore how the concepts of medicinal chemistry are ered that cells can generate the gas nitric oxide by the reac- tion sequence shown in Fig. Because nitric oxide is a gas, it can diffuse easily through immunological defence mechanisms.
However, not all compounds with similar chemical structures have the same biological action. Enzymes can be used in organic synthesis. For example, estradiol in the presence of the cofactor NADH. The initial End-of-chapter questions allow you to test your the reduction of an aldehyde is carried out using aldehyde dehydrogenase.
Unfortunately, this reaction requires the rate data for the enzyme-catalysed reaction in the absence of an inhibitor is as follows: understanding and apply concepts presented in the use of the cofactor NADH, which is expensive and is used up in the reaction. Suggest what sort of binding plot. Chemistry World. Navia, M. Current Opinion in Structural Biology 2, — Knowles, J. Science , — EMBED for wordpress. Want more? Advanced embedding details, examples, and help!
Instant Notes in Medicinal Chemistry provides concise yet comprehensive coverage for undergraduates studying medicinal chemistry as part of a science, pharmacy, or medical course. It is a truly multidisciplinary subject involving such subject specialities as organic chemistry, pharmacology, biochemistry, physiology, microbiology, toxicology, genetics and computer modelling.
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