Reversible Inhibitors For Enzymes

Reversible Inhibitors For Enzymes What characterizes reversible inhibitors that they 1) bind enzymes noncovalently while irreversible inhibitors bind enzymes in a covalent manner 2) dissociate/associate rapidly with enzymes. What binds the enzyme more (substrate or inhibitor ) depends on the affinity of the inhibitor and the conc. of the inhibitor in the solution. Competitive Inhibition This competition occurs between the substrate and the inhibitor itself but what will determine which one will bind the active site is : 1) the affinity: which one has higher affinity toward the active site 2) the conc.: if we have higher conc. of the substrate it will eliminate the effect of the inhibitor even if the affinity is higher for the inhibitor. According to this, if we will plot Michaelis Menten plot for competitive inhibitors and have the [S] against the reaction rate without having the inhibitor (black curve) we will have the hyperbolic plot. While in the presence of competitive inhibition, the reaction rate will decrease because now there are fewer active sites available to produce product per unit of time. to overcome this decrease in reaction rate, if we want to keep the reaction rate as is, we must increase [S], therefore we see in the Michaelis Menten plot in the presence of competitive inhibitor in the solution a shift to the right in the plot (red curve) 1|Page So to achieve the same point on the y axis (for example rate of 20) we were using 10 MM (micro molar) of [S]… while in the presence of competitive inhibition we use 40 MM of [S], so we increase the [S] of the substrate to reach the same rate that we have it without inhibitor. So the more we add from the substrate the more neglected the effect of competitive inhibition in the solution will be, until the reaction reaches its maximum velocity • In these reactions maximum velocity needs more and more [S] in order to be reached, but this [V max] is constant and doesn’t change under the effect of the competitive inhibitor (what changes is the amount of substrate needed to reach the [V max]) • Km gets increased in the presence of the competitive inhibitor If you want to plot on a double reciprocal plot (Lineweaver-Burk plot) : 1/V versus 1/[S] Since V max does’t change in competitive inhibition, lines in the plot (that represent : inhibited enzyme and uninhibited enzyme) will have the same cross point on Y axis but differ in Km which is represented on the X axis, so lines differ in the cross point with the X axis Since Km/V max = the slope and V max is the same for all enzymes regardless the type of the competitive inhibitors or substrates that bind on the active sites, when Km increase the amount of (1/Km) will decrease so ( -1/Km) will increase, which means that the line become closer to the zero point on the plot, so the slope increases 2|Page Noncompetitive Inhibition The inhibitor binds at a site other than the active site changing the geometry (the 3D shape) of the active site so as now the catalytic activity of the enzyme is less( The complex does not proceed to form product or has a lower efficiency ), accordingly there is no competition between the inhibitor and the substrate, accordingly if we keep increasing [S] on a Michaelis Menten plot we will not eliminate the effect of the inhibitor but we will have a new V max value which is less than what we started with. If I have 10 molecules of the enzyme, to achieve V max there should be 10 molecules of the substrate binding the active sites of the enzymes. Km is the substrate needed to reach 50% of the old velocity now in the presence of the non competitive inhibitor (changing the geometry of the active site) for sure I will have a new V max which is less, because the capability of the active site of changing the substrate into product per unit of time is less, but how many molecules should I bind to the active site of the enzymes so as to get the new V max ? 10 So less velocity (for example 50%) will be achieved by having the same number of the molecules how much the new Km? >> it is the substrate needed to achieve 50% of the new maximum velocity The needed amount of the substrate is the same so Km is the same in both situations *numbers in the box are assumptions, for further understanding only : In this figure assume that the old V max = 100 Km (remember Km = substrate needed to reach half the maximum velocity) = for example 2 The new V max = 40, To achieve V = 20 (half the V max), Km = 2 also 3|Page Now on a Lineweaver-Burk plot : The slope will increase in the case of having inhibited enzyme because V max is decreasing while Km is the same V max is getting less, 1/V max is getting higher Uninhibited enzyme The X intercept, if Km is not changing then the interception between the line that has inhibited enzyme and the line that has uninhibited enzyme should occur on the X axis not the Y The Y intercept Regulation Through Conformational Changes We regulate enzymes through changing the conformation of the enzyme These regulatory mechanisms include: A. Allosteric activation and inhibition; B. Phosphorylation or other covalent modification; C. Protein-protein interactions between regulatory & catalytic subunits or between two proteins; D. Proteolytic cleavage 4|Page These types of regulation can rapidly change an enzyme from an inactive form to a fully active conformation. As we discussed before : when Michaelis Menten tried to study the enzyme behavior he saw some enzymes that follow hyperbolic act (simple enzymes), and certain enzymes that follow a sigmoidal behavior Whenever you see a sigmoidal behavior this tells you that there is a cooperative behavior between materials that you are dealing with (eg. this is how hemoglobin works) A small change of the [S] gives a huge increase in the velocity (that’s why we have a first order in the beginning then a curve ending by a zero order reaction where a large increase in [S] will give a small increase in velocity) Chymotrypsin follows hyperbolic behavior : Specificity for aromatic residues mainly. Also, hydrolysis of ester bonds In vivo chymotrypsin is an enzyme acting in the digestive systems of many organisms facilitating the cleavage of peptide bonds by a hydrolysis reaction Large increase in the [S] at the beginning will cause a small increase in the velocity then there will be a term where small increase in [S] will give us a huge increase in velocity, then this is followed at the end by a plateau when the enzyme is fully saturated where a large increase in the X axis will give a small increase in the Y axis Aspartate transcarbamoylase (ATCase) follows the sigmoidal behavior : an enzyme that has more than one subunit in its structure: synthesis of CTP & UTP for RNA and DNA synthesis All enzymes that has a sigmoidal plot we call them Allosteric. Allosteric Regulation *Allosteric regulation : is to control the enzymatic activity from away and this needs an enzyme that has more than one subunit 5|Page *What are allosteric enzymes? A multisubunit enzyme with catalytic subunit(s) -with active site(s)- and regulatory subunit(s)-without active sites*The catalytic subunit can be considered regulatory to the other subunit ¬ Homotropic vs. Heterotropic For examples: 1) Oxygen is considered as homotropic effector for hemoglobin, because it is the same ligand that binds to hemoglobin as a substrate in The catalytic subunit, as well as binds to The regulatory subunit, and changes hemoglobin activity.(when same material binds to the active site and regulatory site) 2) CO2 is considered as heterotropic effector for hemoglobin, it doesn’t bind as a substrate in active site, another material does that. But rather it binds to regulatory subunit and changes hemoglobin activity. (Different materials bind to the active and regulatory sites) ¬ Binding triggers a conformational change in the active site - once we have a material that binds to another place other than the active site (catalytic subunit), it will cause a change to the active site. So as the active site now either will behave better and we will call the material activator, but if the material will make the active site behaviour less efficient, we would call it inhibitor. ¬ The Michaelis-Menten model can’t explain the kinetic properties ¬ The substrate concentration at half of the Vmax is called (K0.5) Same concept of The Michaelis-Menten model is applied to allosteric enzymes, but we don’t have quietly the same parameters (KM and Vmax), so it cannot explain them. In other words, we have for sure (Vmax), since we have saturation for all active sites. But we have (K0.5) instead of (KM), which means that (KM) is Michaelis constant, and whenever you hear "Michaelis", it referred to simple enzymes not for allosteric ones, that’s why they derived another value called (K0.5) for allosteric enzymes, and it has the same concept of (KM) but in different name, which is The substrate concentration that is needed to achieve half of (Vmax) for allosteric enzyme. ¬ Allosteric inhibitors have a much stronger effect on enzyme velocity. • This curve represents uninhibited allosteric enzymes action. • Inhibitors and activators change the parameters of enzymatic action: 6|Page 1) (Vmax) will not change, it will stay constant either we put inhibitor or activator. the difference is that in activator solution, we will reach (Vmax) faster. While in inhibitor solution, we will reach the (Vmax) slower, there will be a delay. 2) (K0.5) will change, in the activator solution, it will become less. While in inhibitor solution, it will become higher. Activator inhibitor The effect of modifiers on Vmax& K0.5 -Allosteric inhibitors will change the conformation of active site, accordingly the enzyme will catalyst less reactions per unit of time, so we need more substrates to achieve same velocity, in other words we need more reactions per unit of time to achieve same velocity of the middle curve(without effectors),and less activity. That’s why we see a right shift in the curve under inhibitors effect. Now, if we increase the concentration of inhibitors we will have another line Under the right-shifted inhibitor curve, and as we add more inhibitors we will have another lines downwards shifted to the right, becoming more sigmoidal and less hyperbolic. Inhibitors->right shift curve in sigmoidal way->more concentration->more sigmoidal curve. -Allosteric activators will change the conformation of active site, accordingly the enzyme will catalyse more reactions per unit of time, so we need less substrates to achieve same velocity of the middle curve, in other words we need less reactions per unit of time to achieve same velocity, resulting in more activity. That’s why the curve will shift to the left, becoming more hyperbolic and less sigmoidal. BUT it will never be hyperbolic, because hyperbolic shapes are for simple enzymes. allosteric enzymes have multiple subunits cooperating between each other to produce the best action, and regardless how much is good the activator you put in a solution, it will never reach the hyperbolic curve, it will behave in cooperative manner.so if you zoom in and make the x-axis so large, you will realize the cooperative manner clearly in the beginning of activator curve. 7|Page ¬ Two conformations: more active (R) & less active or inactive (T). We studied in hemoglobin lecture about relaxed conformation (R), when O2 have high affinity to haemoglobin, while in taut/tight conformation (T), O2 have less affinity to hemoglobin. Now, since enzymes are proteins, and they have more than one subunit cooperating with each other, same concept applied to enzymes. In the presence of activator, we will have more relaxed conformation (R), while in presence of inhibitor, we will have more taut conformation (T). ¬ The equilibrium ratio (T/R) is called L and assumed to be high. In the presence of an activator, we will have more relaxed confirmation of the enzyme. and in the presence of an inhibitor there will be more tight confirmation. The Ratio of how much tight conformation of enzyme compared to relax confirmation of it is called L ratio. ¬ As L (T/R) increases, the shape becomes more sigmoidal. # If you want to plot the normalized velocity or rate of the reaction from zero to 1 - where 1 is the maximum velocityagainst the substrate concentration, as we have presence of more and more inhibitors, we will get more tight conformation (T) compared to relaxed conformation (R), and the L ratio will increase,(T/R) increases.# If the L ratio is 1, the shape is almost hyperbolic. And with the L ratio of 10, which we will have more inhibitors and more tight confirmation, so the curve will shift to the right, and L value increases. And The shape becomes more and sigmoidal and less and less hyperbolic. 8|Page ¬ ATCase and Hb are allosteric proteins (cooperative behavior). ATCase: is called aspartate transcarbamoylase enzyme. the name implies, the substrate is aspartate, and it does transcarbamoylation reaction producing UTP&CTP as products. ¬ Catalytic can be separated from regulatory (hyperbolic) - ATCase is an enzyme that composed of 12 Subunits, those 6 which appeared in orange or deep Brown are regulatory subunits. And the other 6 subunits are catalytic subunits which appear in green, and they are three over another three subunits. -In the past, they did an experiment where regulatory subunits are separated from catalytic subunits, and they added aspartate to the enzyme. The enzyme behaved as simple enzyme, MichaelisMenten behaviour, showing hyperbolic curve. And that’s happened because we don’t have regulatory subunits where the material combine to and affect the enzyme activity, no allosteric manner, so the enzyme will behave in a simple manner. ¬ Cooperativity in relation to substrate 9|Page ¬ CTP is an inhibitor of ATCase (feedback inhibition), ATP is an activator -the inhibitor for the enzyme is the final product of the previous reaction (CTP), it is a heterotropic effector ( ) which behaves in a feedback manner. It comes back and inhibits the ATCase enzyme. Under the effect of (CTP), we can see the plot shift to the right. The degree of shifting to the right depends on the concentration of inhibitor (CTP). The curve become more sigmoidal and less hyperbolic. -the activator is ATP. Usually when we have a high concentration of ATP, that implies on DNA and RNA production. Accordingly we need a high concentration of UTP (U) and CTP (C) for their production. That’s activate aspartate reaction to give us more U and C , that’s why ATP is considered as allosteric activator for the enzyme. And the curve will shift to the left. 10 | P a g e • -Why is it effective? ¡ Adds two negative charges: new electrostatic interactions and accordingly conformation: Phosphate group has two negative charges or sometimes three negative charges, highly negative and that bulky to cause conformational changes in enzymes, either making activation or inhibition for enzymes. ¡ Can take place in less than a second or over a span of hours : It is so quick, it can make covalent bond and break it in less than a second. ¡ Often causes highly amplified effects : Things to keep in your mind: 1) a lot of enzymes can be regulated by phosphorylation. 2)phosphorylation doesn’t always activate the enzymes, sometimes leads to inhibition of enzymes and making it inactive. 3)phosphorylation is essential, because enzymes are those which are responsible for metabolism inside our cells. metabolism reactions are contradicting each other, which means when a message come to our cells, it activates certain enzyme and inactivate the contradicting enzyme for the first one, by telling it to shut down. For example, when a message comes to our cell via neurotransmitter to release glucose, protein kinase makes a second message by causing phosphorylation for enzymes. It activates One enzyme to breakdown glycogen (glycogenolysis) and inactivate the Other enzyme which make glycogen synthesis (glycogenesis). ¡ Can form three or more hydrogen bonds: specific interactions with hydrogen-bond donors A protein-linked phosphate group can form hydrogen bonds. Phosphate group binds to very specific areas on proteins, it binds hydroxyl group on serine, thyronine, or tyrosine. * 11 | P a g e * * * ¬ Rapid and transient regulation of enzyme activity - REVERSIBLE ¬ Phosphorylation: (Ser, Thr, & Tyr): *Phosphorylation does not lead always to activation of enzymes -Example of phosphorylation: *Glycogen phosphorylase-reaction (two forms; a & b). Ser is away from the active site. *Mostly, ATP is the donor. *Kinases vs. Phosphatases. The regulation of glycogen phosphorylase enzyme occurs by phosphorylation process, this enzyme has two serine residues, and more than one subunit. It has serial residues on both sides. When these residues aren’t phosphorylated, we call the enzyme phosphorylase b (the less active form of the enzyme), but when phosphorylation happens to both serine residues by glycogen phosphorylase kinase, it will become phosphorylase a (the active or the more active form of the enzyme). The reverse reaction occurs by phosphoprotein phosphatase, where there will be removal of phosphate group, and get the enzyme back to its original state (inactive state). 12 | P a g e àBoth phosphorylase b and phosphorylase a exist as equilibria between an active R state and less-active T state. àPhosphorylase b is usually inactive because the equilibrium favors the T state. àPhosphorylase a is usually active because the equilibrium favors the R state. Explanation: *phosphorylase b is converted to phosphorylase a by phosphorylation process in serine residues. Since both phosphorylases have more than one subunit (multimeric enzyme) as we mentioned earlier, they would exist in different conformations. *Phosphorylase b, the inactive form, would exist in T state more than R state àthat’s why the arrow converting R state to T state, is much bigger than (TàR) arrow. So if we have 100 molecule, and the ratio is 20:80 between R and T states, 20 molecule would be found in R state, and 80 molecule in T state. *while in phosphorylase a, the active form, would exist in R state more than T state. **to sum up: weather phosphorylase b, in T or R state, it would be phosphorylated and converted to the active form of phosphorylase a. but in its inactive form (phosphorylase b), there would be more molecules in T state than in R state. The transition of phosphorylase between the T and the R state is controlled by the energy charge of the muscle cell. 13 | P a g e Protein kinase (PKA)... In general it makes a phosphorylation . How.? Protein kinase A (PKA): refers to a family of enzymes whose activity is dependent on cellular levels of cyclic AMP (cAMP).cAMP: referred to a hormonal 2nd messenger. 🔴 Either dedicated or not. 🔴 Has several functions in the cell, including regulation of glycogen, sugar, & 🔴 lipid metabolism The structure of protein kinase is: two regulatory subunits and two catalytic subunits. (In this state it was inactive.) ➡ The regulatory subunits have 4 binding sites as you can see in this picture , so each 4 cAMP molecule will bind those places (maximum in saturation condition ) once they are bound or close to the regulatory subunits which will cause dissociation between the catalytic subunits from the regulatory subunits. Look bellow ⬇ ➡ Note: when the protein kinase A is activated, it loses its quaternary structure. So the catalytic subunits go and phosphorylate other proteins including 2 enzymes. 🌺 What is the main work that protein kinase does? The answer = it’s kinase so it does a phosphorylation for many enzymes and protein that is downstream for it, inhibits some of them and activates others. 💚 14 | P a g e So it has nothing to do with the fact that the phosphorylation did what it did, but the protein become phosphoprotein as in the last picture (A) This phosphate can bind to serine, tyrosine, threonine from the amino acids which found in phosphoprotein. Also one of the proteins that PKA makes them active is: phosphodiesterase enzyme (PDE) —> inactive by phosphorylation becomes active . Active PDE breaks down the cAMP to AMP so , So there will not be a source for the enzyme ( PKA ) to work (as a mode of regulation to itself : So it catalyzes the enzyme directly to stop its work Look to these sequence why adrenaline causes increase in the sugar level (phosphorylation cascade) So Adrenaline (epinephrine) bind to the receptors outside the cell causes Activation of that receptors( G-proteins are active)(adenylate Cyclase is active ) → increasing the concentration of cyclic ↑cAMP → activates protein kinase A which → phosphorylates & activates glycogen phosphorylase kinase is getting activating by phosphorylation → phosphorylates & activates glycogen phosphorylase ( it will cause break down of glycogen ) 15 | P a g e Are any other covalent modifiers other than the phosphate p that bind covalently then released from the enzyme, and controlled the enzymatic reaction? ⚫ Yes. The most common one is the phosphorylation process but the others are like: A) Adenylylati on process B) (addition of adenylyl group). AMP (from ATP) is transferred to a Tyr hydroxyl by a phosphodiester linkage. The addition of bulky AMP inhibits certain cytosolic enzymes. C) Uridylylatio n D) addition of uridylyl group ) look where the nitrogenous base here is adenine , the nitrogenous base here is uridine. It can cause activation or inhibition of a certain enzyme . 16 | P a g e E) ADB ribosylatio n, F) methylation . G) acetylation 17 | P a g e F) We will discuss in the next semester in shaa allah , in the process of glycogen synthesis ADP ribose and ADP glucose is there in the process to cause activation or inhibition and we call it the high energy intermediate in the pathways . ● adding a methyl group . ● Methyl group has a positive charge. So this process masks the negative charges accordingly to increase the hydrophobicity on carboxylate side chains ● adding acetyl groups. ● The acetyl group has a negative charge. F and D is how they can change the enzymatic action by masking the negative, and positive charge consedring both of these modifiers. 3. C.Conformational changes from protein - protein interactions G protein: a family of trans-membrane proteins causing changes inside the cell. They communicate signals from hormones, neurotransmitters, and other signaling factors 🔴 An Example where a hormone binds to a receptor, we have talked about it before (Adrenaline), this receptor is bound to G-protein ((which is composed of a 3 subunits α,β,¥(trimer)) The hormone causes a conformational changes to the receptor which will cause activation of Alfa, then the activation of alfa causes exchanging of GDP to a GTP molecule so GDP get out and GTP would come instead of it .. Once GTP is bound it causes dissociation of Alpha from Beta-Gama dimer which is associated with, Alfa will go to the adenylate cyclase enzyme, which is one of its targets, because of activation of adenylate cyclase causing conversion AMP to cAMP. Now Why we discuss this topic about G protein in enzyme lectures? The answer is: G-protein is an enzyme in itself, Alpha subunit is an enzyme by itself, it has an internal hydrolytic activity, it can hydrolyze GTP into GDP GDP gets out and GTP joins. But How GTP turns to GDP because there will not be an exchange there so the GDP will be hydrolyzed by alpha subunit, so alpha works as an enzyme. α-Subunit can be stimulatory or inhibitory 18 | P a g e monomeric G-protein: There are a lot of them Same as Gα . Hydrolysis vs. exchange. Activation or inhibition. Example: RAS 🔴 E.g RAS protein, you can understand it exactly as a G-alpha, it works as GAlpha which causes exchange in between GTP and GDP , then it slowly hydrolysis the GTP to become GDP again , and alpha then becomes inactive . The difference between the RAS and the last one is that it’s a monomer while the alpha G-protein is trimer, it’s free in the solution in the cytoplasm, inside the nucleolus and so on . 🔹 The last way in the conformational changes is 3-D proteolytic cleavage: 3.D.Proteolytic Cleavage Zymogens (Pro- or -gen) Cleavage means cutting the protein itself. As you can see in the picture that the active site of the enzyme is blocked, why? Because there is a certain piece of the enzymes itself blocking the active site , now how these enzymes are getting active ?....by cutting this piece and the active site is open There are a lot of enzymes which can be regulated in their activity in this manner, now this cut (the cleavage that will occur (the hydrolysis) to this peptide bond is reversible, once it’s active it cannot come back and binds again. Irreversible cut usually at the N-terminus 19 | P a g e chymotrypsin chymotrypsinogen Q; why do a lot of enzymes are produced in this inactive manner and then become active? You need the enzyme to be ready all the time in the area so as after that there will be a fast activation of the enzyme and then the enzyme can work , or it is synthesized in a place and you need to transfer it to another area , you do not need it to work in the area where the production is . This is related to many enzymes such as: 1- digestive enzymes : trypsin, chymotrypsin, pepsin. 2-Enzymes related to blood clotting: thrombin ,fibrinogen ,fibrin. How do we distinguish between them by the names? Either there is a suffix (gen) or prefix (pro), E.g= Pepsinogen = it will become converted to pepsin. Prothrombin will become thrombin Chymotrypsinogen: single polypeptide chain (245 residues), 5 (S—S) bonds Blood clotting The soluble protein fibrinogen is converted to the insoluble protein fibrin 20 | P a g e Non -specific regulation We Are controlling all enzymes non -specifically , when we have discussed the regulation of the enzymes before , we discussed the specific mechanism that affects specific enzymes for example this enzyme (glycogen phosphorylases) control throw the phosphorylation reaction process , and all the previous techniques affects enzymes fast in milli second . (the specific pathway is fast) The non-specific regulations affect enzymes, non-specificlly for a certain enzyme. The first thing is controlling the amount of enzyme, this regulates the synthesis or degradation of enzymes, you will need in it more of the enzyme or less of the enzyme , you will activate the catalytic activity more or you will decrease it depending on how much enzyme do you have , it takes more time to synthesis more enzymes or to degrade the enzyme that you have , not as the other processes that occurs instantly. (the non-specific pathway is slow) This process is adaptive : it adapts your body according to the situation you are in, according to your age, environment, gender, living place … etc A. Regulation enzymes Regulated by increasing or decreasing the rate of gene transcription (induction & repression) § Usually slow in humans (hours to days) ¡ Sometimes through stabilization of the messenger RNA B. Regulated Protein Degradation ¡ Can be degraded with a characteristic half-life within lysosomes § During fasting or infective stress: gluconeogenesis increase & synthesis of antibodies (protein degradation increases) § Increased synthesis of ubiquitin The effect of temperature: Increase in T° increases the rate until reaches a max (≈ 50°): the optimal temperature of each enzyme is its’ denaturation Autoclave steam heating Hypothermia, metabolic reactions, cardiac surgery. In general the increase in temperature will increase the enzymatic activity, because enzymes are a proteins ,it increases the kinetic energy in the substrate and the enzyme itself, Collisions increase. Is there is a limit? Yes it has a limit, it’s denaturation of the enzymes. Around 40 degree Celsius many enzymes denaturant ( remember when our body temperature reach this degree what happen ) , also at 50 most enzymes will be denatured . 🔴 21 | P a g e This is why the elevation of human being temperature over 40 is very risky, rather than the fat that will melt . The effect of temperature, open the lecture at 1h 16m: 24 sec. E.g : In the heat it’s easy to connect the blood vessels to a pump, which will pump the blood to the whole body , to prevent the lack of oxygen that causes tissue death, it’s easy to work with the heart , it’s a problem to work on a high vessel , like the aorta . If we want you to work with the aorta, what we can do, how blood will spread every where from the aorta? We decrease the temperature of the patient, if we do that the rate of reaction will be decreased of course , so metabolism will be decreased , so you don’t need oxygen, you need ATP for the metabolism,( and you will have a certain window to work on that ,like 40or 30 minutes ) . The effect of the pH: The effect of pH is different , changing of pH cause denaturation of enzymes , because it affect the ionic interaction inside the protein , and enzymes are proteins , and depending on how much ionic interaction are there inside the proteins will get affected by changing the PH . If this enzyme or protein does not have an ionic interaction the three dimensional structures is not preserved throw ionic interaction, will it get affected by changing the PH ? No and this is the case in the papain enzyme, a hydrolytic enzyme that does not get changed if you change the PH . 🖤 22 | P a g e In the case of pepsin which works in the stomach once the PH is getting elevated the activity decreased dramatically. 🧡 🧡 Cholinesterase , works best for example Around PH 7 and does not get changed after , but if you decrease the PH the activity Will decrease 💜 dramatically . The chymotrypsin, which works in the intestine around pH 8 , decreasing or increasing it will cause drastic changes on the enzyme . 💚 23 | P a g e