Anas Kishawi. Zaid Emad. Nafez abu tarboush

Transcription

1 24 Anas Kishawi Zaid Emad Nafez abu tarboush

2 Hello everybody, this sheet is done according to Dr. Nafith s lecture so try to use his slides for the best understanding, and good luck. WAYS OF CHANGING THE ENZYME CONFORMATION 1) THE ALLOSTERIC BEHAVIOUR OF SOME ENZYMES: *Not all enzymes are simple in their behavior. *There are some allosteric enzymes which adopts sigmoidal plot such as: Aspartate carbamoyltransferase (also known as aspartatetranscarbamoylase or ATCase). *Those allosteric enzymes have a Vmax value and they have the same concept as Km, but we don t call it Km (where m refers to Michaelis), we call it (K0.5) as Michaelis-Menten model can`t explain the kinetics of these enzymes. *They re allosteric because materials can bind to certain subunits and affect the behavior of other subunits. *Those materials which are bound to certain subunits (regulatory subunits) in the enzyme affecting the activity of the catalytic subunit are called (allosteric modifiers)>> they modify the allosteric behavior of their protein>> they re either activators or inhibitors. *Those activators or inhibitors can be the substrate itself so we call them a Homotropic effectors or they can be a totally different material so we call them heterotropic effectors. *If u add an allosteric inhibitor to an allosteric enzyme, you re decreasing the velocity per unit of time. SO, in order to keep up with the same velocity, you need to increase the substrate concentration. As a result, the reaction plot will shift to the right and at the end we ll reach Vmax. (Look at the graph below) *Be aware that it s not an irreversible inhibitor, so there will be a shift to the right and the plot will become more sigmoidal and less hyperbolic. *If you add an allosteric activator, with the same substrate concentration, we ll get a higher velocity, so, the plot will shift to the left and it will become less sigmoidal and more hyperbolic. *You have to know that for allosteric enzymes the plot will NEVER be hyperbolic. *There must be a certain lag at the beginning. 1 P a g e

3 *A previous question regarding to the previous plot: You ve done this experiment under the NORMAL CONDITIONS and under ALLOSTERIC INHIBITION only, which line represents the normal behavior of the enzyme?? Ans: Notice that you ve done it under normal conditions and inhibition only THERE IS NO ACTIVATION(NO SHIFT TO THE LEFT ). So the answer will be the one to the left. *Allosteric enzymes, as in hemoglobin and myoglobin, can exist in two states: the T(tight) state and the R(Relaxed) state. *(T) state means that it has LOW affinity for the substrate, while the (R) state means it has HIGH affinity for the substrate. *Sometimes, the allosteric enzymes exist in the active and inactive state. *The inactive state can also exist between the (T & R) state, same as the active state. *Always the ACTIVE state favors the R state, while the INACTIVE state favors the T state. *If we have 100 molecules of the enzyme, and the environment is good for it to function, you ll see that 80 molecules are active, 20 are inactive. *If bad environement then 20 active and 80 inactive. *What determines if the enzyme is active or not is the ratio (T/R) which is called L ratio. *It represents how much do you have T state over the R state of each enzyme and this ratio is usually kept HIGH the inactive state is more than the active state(the DEFAULT SITUATION). *The inactive state should be more Once you need it, you ll activate it and it ll get back to its original situation which is the inactive state. *L ratio is increasing T state is becoming more and more>> The plot will shift to the right the shape becomes more sigmoidal. 2 P a g e

4 *Ex: ATCase catalyzes the conversion of aspartate amino acid through a long metabolic pathway into (UTP &CTP), Which means : it participates in DNA & RNA synthesis. *The behaviour of an uninhibited enzyme: *The final product is CTP CTP comes back and inhibits the enzyme in a feedback manner. *Once you have a high concentraition of CTP, it will come back and inhibit the enzyme. *So, under the high concentration of CTP, the plot will shift to the RIGHT. *ATP as a molecule is considered an allosteric activator for this enzyme. *ATP has a major role in our body, which is making nucleotides (adinine) used in DNA & RNA synthesis, but adinine alone can t make DNA &RNA as we need also CTP &UTP. So we need ATP to activate the pathway for their synthesis to make balance in nucleic acids components. * Once you have a high concentration of ATP, the plot will shift to the LEFT (less sigmoidal and more hyperbolic). ATPCase structure : ( look at the previous picture ) *It has (12) subunits: 6 are regulatory & 6 are catalytic. *Scientists have done an experiment when they detached the REGULATORY subunit from the CATALYTIC subunit & they added aspartate. *The result: the enzyme became hyperbolic as normal, which means, what causes the allosteric behavior of the enzymes are the REGULATORY subunits P a g e

5 2 ) THE REVERSIBLE COVELANT MODIFICATION : *It changes the conformation of the enzyme due to the binding of small molecules or atoms (like PHOSPHATE) covelantly, soon it will be broken down and changes the activity again. *phosphorylation: it changes the conformation and the activity of enzymes, because it s a BULKY molecule with TWO NEGATIVE CHARGES. *As a result, it ll make conformational changes by binding to the protiens in less than a second or it could take a long time. *This is why phosphorylation is common and linked to amplification of signals in the signaling process. *Notice that phosphorylation is REVERSIBLE, although it s covalently bonded to the protien. So it could dissociate without disrupting the structure of the enzyme. *Phosphorylation adds a PHOSPHATE GROUP to a HYDROXYL GROUP of an aminoacid (Ser, Thr, & Tyr). *The addition of this phosphate is catalyzed by an enzyme called (KINASE) and the removal of this phosphate is catalyzed by another enzyme called (PHOSPHATASE). *ENZYMES can be regulated by ANOTHER ENZYMES. For example, (Kinases & phosphatases) add or remove a phosphate group affecting the activity of another enzyme. **PHOSPHORYLATION DOESN T ALWAYS LEADS TO ACTIVATION** *IS IT LOGICAL THAT PHOSPHORYLATION LEADS TO INACTIVATION?! Human body metabolisms is contradictory to each other whether you are in a feed or fasting state (degarding or building up energy). So, when you are doing phosphorylation it will activate certain enzymes, on the other hand, it should inactivate another enzymes which go in the reverse metabolic pathways of these processes. Eg. Glycogen phosphorylase reaction: *Glycogen phosphorylase phosphorylates glycogen to break it down. *you add phosphate to the terminal glucose, then it breaks down, resulting in another free terminal glucose each time you add phosphate. *Glycogen phosphorylase exists between two states(active or inative). *The inactive state is called PHOSPHORYLAZE B. *The active state is called PHOSPHORYLAZE A. *Both the active and inactive forms exist between two states (T&R). *PHOSPHORYLAZE B (inactive)>> the equilibrium favours the (T STATE). *PHOSPHORYLAZE A (active)>> the equilibrium is favours the (R STATE). 4 P a g e

6 **The transition of phosphorylase B between the T and the R state is controlled by the energy charge of the muscle cell.** *Protein kinase A (PKA): *once the hormone binds to the receptor, the receptor will change its conformation affecting the G protien the G protien affects the Adenylcyclase which produces high concentration of (camp) which will affect Protien kinase A. *Protien kinase A is a TETROMER which is copmosed of two regulatory subunits and two catalytic subunits. *There are 4 binding sites for camp at the regulatory subunits * 1 camp is produced by Adenylcyclase it will bind to the regulatory subunits causing dissociation of the regulatory subunits from the catalytic subunits the catalytic subunits are active now beginning of phosphorylation of an enzyme called glycogen phosphorylase kinase which phosphorylates another enzyme called glycogen phosphorolase which has the ability to phosphorylate glycogen. THIS IS WHAT IS CALLED A PHOSPHORYLATION CASCADE. (FOR MORE EXPLANATION LOOK AT THE NEXT PICTURE ) 5 P a g e

7 *Other reversible covalent modifiers: *1)Adenylylation (addition of AMP) which is bulky and can change the activity of certain cytosolic enzymes. *2)Uridylylation (addition of UMP) which inactivates certain groups on the enzyme. *3)ADP-ribosylation: inactivates key cellular enzymes. (Ribose attached to ADP) *4)Methylation: masks a negative charge & adds hydrophobicity on carboxylate side chains of Aspartate & Glutamate and thus changing the enzyme activity. *5)Acetylation: masks positive charges when added to lysine residues. (Acetate group has a ve charge, so it s added to the +ve charges which are founded in protiens such as Lysine, and as a result, it masks it leading to changes in its activity). 3) Conformational changes due to protein-protein interactions: *Eg: G protien *G protien is a TRIMERIC protien (Alpha, Beta & Gamma). *Alpha, beta & Gamma have a fatty acid which is connected to the membrane to be close to the receptor, so, when a conformational change affects the receptor, it will affect the activity of the G protein. *G protien(alpha is monomer)(beta & Gamma are dimers) they have high affinity to each other, once the receptor changes its conformation, the alpha subunit will have a conformational change leading to decrease in its affinity towards Beta and Gamma subunits. *When the G protien is inactive, alpha subunit is attached to GDP molecule. The reason for calling them G protiens. (attached to guanine molecule) *Once the conformational changes occurs, the affinity towards GDP becomes less, increasing the affinity towards GTP. (It s a REPLACEMENT process). *When GTP is bound, alpha subunit will dissociate leading to another confomational change, and it will bind to Adenylcyclase enzyme mainly causing another conformational change to initiate the activity of camp. 6 P a g e

8 *Are all G protiens a multi-subunit protiens?! No, there are some monomeric G protiens. *Alpha subunit has enzymatic activity. It slowly hydrolyzes the GTP to GDP and thus increasing its affinity toward the Beta-Gamma dimer. *Monomeric G protiens work in the same manner as trimeric G protiens. *They have one subunit that works exactly as alpha subunit in the trimeric G protiens. *The mechanism is the same as the one mentioned above. *Eg: RAS protien. *** G protiens, whether monomeric or trimeric, don t always lead to activation (same concept as phosphorylation) so, Gα could be devided into stimulatory (S) or inhibitory (I) by their nature. 4) PROTEOLYTIC CLEAVAGE: (Zymogens) *The active site of the enzyme is blocked by a piece of the same enzyme, so to activate the enzyme you need to do proteolytic cleavage. **Usually cleavage happens irrevesibly at the N-terminus. *you cleave this piece of peptide, leading to the exposure of the active site. *We need the enzyme in the inactive form due to two reasons: 1)When you produce it at a place and you need it to be used in another place. *Eg. Digestive enzymes. They re made in pancreas in the inactive form and they re being transformed to the active form in the intestine. 7 P a g e

9 2) When u produce it but you don t want it to function right now. Eg. Pepsin which is found in stomach. *Once the food is ingested, HCL is sectreted leading to the activity of the pepsin. *Zymogens either start with (pro-) or end with (-gen) Eg: Trypsin, chymotrypsin, pepsin and Thrombin all were trypsinogen, chymotrypsinogen, pepsinogen, prothrombin. you have to be familiar with these names NON-SPECIFIC REGULATION: *All what we discussed before can be specific for a certain enzyme. But the following regulatory mechanisms always apply for all enzymes : A. Regulating Enzyme Synthesis: Regulated by increasing or decreasing the rate of gene transcription. Usually slow in humans (hours to days) Sometimes through stabilization of the messenger RNA. B. Regulating Protein Degradation: Can be degraded with a characteristic half-life within lysosomes. 1) EFFECTS OF TEMPERATURE: *Temperature affects all the enzymes at the same way regardless of their nature. *As long as u are increasing the temperature, the activity of the enzyme should increase, WHY? Ans: because you are increasing the KINETIC ENERGY, so you are increasing the probability of binding per the same unit of time.(velocity) *So as the temperature is being increased, the enzyme activity should increase and the rate should increase until it reaches a maximum between 40 and 50 degrees ( Denaturation temperature ). *Most of enzymes are protiens, so after reaching these high temperatures they will denaturate. *Some enzymes tolerate higher temperatures. *CLINICAL CASE: *In cardiac surgery, there is no problem when you remove the heart and connect the body with a machine that pumps blood. 8 P a g e

10 *The problem is that when you do a surgery on a MAJOR ARTERY, as it distributes the blood to all of the body, but you need to prevent the blood from being transported through it, so, HOW WILL THE BLOOD REACH THE TISSUES? Ans: The metabolisms of the human body are controlled by enzymes, so, when we lower the temperature, all the enzymatic activity will become less. The same as the concept of the freezer which stops the metabolism of microrganisms to preserve food. *Some organisms, when you remove them from the low temperautre, they live again, but it s too risky in the human body. *They put ice around the patient to lower the temperature decreasing the metabolic activity performing the operation more efficiently and at the end of the surgery they must heat the body again. 2)EFFECTS OF ph: *ph is different than temperature as it depends on the amount of +ve or ve amino acids which perform an important function to the enzyme and the amount of ionic and electrostatic interactions within the enzyme. *When you change the ph, you change the PROTONATION STATE, so if a change in ph occurs, the ionic or electrostatic interactions between amino acids will be broken resulting in denaturation of the enzyme. *If the enzyme has high number of those ionic interactions and the ph was changed, the effect in the activity of the enzyme will be remarkable, but if u have a very low number of those interactions, it won t be affected verry much. **This is why the effect of ph on enzymes is called ENZYME-DEPENDANT. *Most enzymes have their maximum activity at ph between (5-9) *Enzymes are affected in different ways: Some enzymes don t change their bahviour when changing the ph because they don t have ionic interactions to preserve the 3D structure of the protien. **MOST DENATURATIONS ARE IRREVERSIBLE** 9 P a g e

11 3)EXTREMOZYMES: *Extremozymes are enzymes which can tolerate risky temperatures or ph values. *They re found in certain bacteria which lives in a very hot fountains or in a very cold water. *Scientists extracted the enzymes of those bacteria and used them in cleaning materials, paper production..etc. *Xylanases can tolerate very high ph values. *Taq polymerase tolerate very high temperatures. *Note that: Thermophiles means heat lovers. Psychrophiles means cold lovers. *ABZYMES cutting edge technology: *The mechanism: *The substrate is injected to an animal, so this animal will make antibodies which are specific for this substrate the binding site of the antibody looks like an active site, so these antibodies have catalytic activities, then they extracted and used in industry. *Ribozymes: *RNA molecules that have a catalytic activity. *Examples: telomerase & RNase P. *The catalytic efficiency of catalytic RNAs is less than that of proteinaceous enzymes, but can greatly be enhanced by the presence of proteinaceous subunit. **Ribozymes are very weak. Their strength will increase when they bind to a protein, so enzymes are the best biological catalysts in the world. MA Thank you all. Love you all have a good time and sorry for any mistake NEVER GIVE UP!! TODAY IS HARD!! BUT TOMORROW WILL BE SUNSHINE!! 10 P a g e