Enzymes PDF

28 Sewar Habahbh Dental student Nafez Abutarboush ENZYMES ❖ In the previous lecture we talked about the active sites of enzymes, and we said that these active sites make enzymes special because of their special properties. ✓ Active sites ➢ Active sites of enzymes are specific three-dimensional s...

28 Sewar Habahbh Dental student Nafez Abutarboush ENZYMES ❖ In the previous lecture we talked about the active sites of enzymes, and we said that these active sites make enzymes special because of their special properties. ✓ Active sites ➢ Active sites of enzymes are specific three-dimensional shapes which include a region where the biochemical reaction takes place.they contain a specialized amino acid sequence that facilitates the reaction. ➢ If you visualize the 3D shape of the enzyme, you will find that the active site is within the structure of the enzyme and it can’t be on its surface, and when you visualize this site as a 3D shape inside the enzyme then you have to know the lining of that space. ➢ This lining is made of the backbones of the amino acids which create this space, these amino acids are from different parts of the protein itself. Accordingly, they are not in sequence, and you have to know that the side chains are projecting toward the space. They are not in sequence means that: the first aa in the active site may have number 1 in the polypeptide chain and the second aa have the number 300, while the third is number 500, etc… (amino acids that create the active site come from different secondary structures in the protein) Note: There should be polar aa in the active site to make different types of bonds. ➢ Active sites are different between different enzymes (small-large). When the active site has enough space to be divided into sub-sites, one of these sub-sites will be responsible for binding the substrate and it doesn’t participate in the catalytic process and the other is responsible for the catalysis ➢ Binding site: binds substrate through ionic, H-bonding or other electrostatic forces, or hydrophobic interactions ➢ Catalytic site: contains the catalytic groups (where the reaction takes place) ➢ . If there is no space for these two sub-sites, the same site that binds the substrate acts as a catalytic site.. ✓ Features of active sites ➢ Enzymes are globular proteins. ➢ active sites structures can look like: a) canals. b) clefts c) pockets. d) invaginations. e) crevices. ➢ Enzymes are found in an aqueous medium , so when the active site is not binding to a substrate, you will find water filling the space (because water molecules are small). ➢ When it binds to a substrate, water will be excluded unless it participates in the reaction. ➢ Binding occurs at least at three points (chirality), the question is why? Ans:1) having more binding points prevent tipping 2)as well as to differentiate between isomers. (If you look at 2 points you will not be able to differentiate between L & D isomers such as L amino acids that are digested by our body because it can be recognized by our enzymes) ➢ The substrates are bound to enzymes by multiple weak attractions (electrostatic, hydrogen, van der waals , hydrophobic) because covalent bonds are a very high energy bonds, so if a substrate binds to the active site covalently, it will stop processing, so the substrate won’t be converted to its products. ▪ There are two important points here: 1) there are some materials that can bind to the active site initially covalently (drugs, toxins) 2) However, these materials are not found physiologically (in normal conditions). ➢ The active site is small in size (if we compare the size of the enzyme to the size of the active site, we will find that the active site is small, like your hand with respect to your body) ➢ Forms by groups from different parts of the amino acid sequence usually forming a domain made of multiple secondary structures ➢ Takes up a relatively small part of the total volume ➢ The “extra” amino acids help create the three-dimensional active site& in many enzymes, may create regulatory sites. ➢ Enzymes can change their shape easily and this property makes them special, but each active site is capable of binding to a specific substrate, because active sites are supported by different parts of the protein (these parts maintain the proper 3-D structure of the active site) as well as for regulation purposes (as we will discuss now). ➢ If a material binds to an enzyme in a place other than the active site (regulatory site), it will cause a conformational change in the enzyme. This conformational change can lead to a change in the active site’s shape. ➢ So, if this material increases the strength of binding to the substrate, we called it activator. In the other hand, if it decreases the strength of binding, we called it inhibitor. ✓ How Do Enzymes work?? We can answer this question from different perspectives (model, energy, mechanism of actions) ➢ At the beginning we will talk about the models. There are two models ,the first one is lock and key, this model was emplaced to explain how enzymes work, but actually it does not explain how enzymes function because of two reasons: 1)Some enzymes can bind to more than one substrate. 2)Proteins are dynamic, so they have many conformations, one of these conformations at least has full activity and it is called native conformation, others can be partially active or inactive. ➢ The second model is (the induced-fit model) ,and as the name implies the hundred percent complementary between the substrate and the active site is induced , and the inducer is the substrate itself. ❖ This picture is a real representation of an enzyme (glucokinase), it adds a phosphate group to the glucose converting it to glucose 6 phosphate. ❖ The active site was open, after the glucose (inducer)enters the active site ,the active site changes its shape so the substrate can fit better, (both the active site and substrate change their shape to fit each other and this occurs due to the binding of substrate ) Note : glucose appears in red. ➢ ➢ ➢ ➢ ➢ **HOW DO ENZYME WORK?(slides) Binding leads to formation of transition-state. Usually, substrate binds by non-covalent interactions to the active site. The catalyzed reaction takes place at the active site, usually in several steps. Two models, lock-and-key vs. induced-fit model. Glucose and hexokinase, phosphorylation ➢ Improving the binding site for ATP & excluding water (might interfere with the reaction ✓ Energy and biochemical reactions ❖ Now we will talk about the energy point of view, so we can understand how do enzymes function from this perspective. ➢ Any reaction happens to achieve stability. There are two theories to study the reaction, the first one is to study the initial and the final states without caring about what is happening in between , and we call it thermodynamic science.(will be discussed in the next semester) ➢ The second theory is to study the pathway between reactants and products.(kinetic science) ➢ kinetic science deals with rates of biochemical reaction.(we will study it this semester) Transition (point) state Activation energy (line) ➢ ΔG++ is the -free- energy that is required to convert the stable condition (reactants) into the non-stable condition (transition state). ➢ ΔG is the free energy that describes the difference between the free energy values of the products and reactants. ➢ ΔG does not change in the same reaction even if there is an enzyme (enzymes just change the activation energy EA) ➢ Transition state: the intermediate state between the reactants and products, which is very unstable with a high level of energy. slides: ➢ ΔG = ΔH - TΔS ➢ Spontaneous vs. non-spontaneous, favorable vs non-favorable, exergonic vs. endergonic, exothermic vs. endothermic, switch of signs ➢ ΔG, ΔG° ➢ Biochemical pathways; storage (endergonic) &release (exergonic) ➢ Kinetics (rate) vs. Thermodynamics (favorability) ➢ Enzymes speed up reactions, but have no relation to equilibrium or favorability. ‡ ➢ The activation energy (ΔG° ): It’s the minimum energy required to cause a process to occur ➢ Specificity varies (stereoisomers), however, there is none non- specific ➢ Spontaneous vs. rate! ➢ According to the random collision theory )‫ (نظرية التصادم العشوائي‬molecules are moving randomly, so they will collide together and produce energy. However, most of these collisions will not make any chemical changes because the energy didn’t make the reactants reach the transitional state. ➢ In the presence of enzymes, they will reduce the activation energy and then, the energy of collisions will reach the transition state. Transition-state complex binds more tightly to the enzyme substrate compared to ✓ Alternative pathways: ➢ Substrates of enzymatic reactions often undergo several transformations when associated with the enzyme and each form has its own free energy value. ➢ Which one is the activation energy in the two pictures ? The wave with the highest peak ➢ Example:Adenosine Deaminase ➢ The reaction of adenosine deaminase ,as we notice, it has three intermediates and the third one represents the activation energy (the highest one). ✓ How do enzymes works? (Mechanical wise) ➢ Proximity effect: Bring substrate(s) and catalytic sites together (because catalytic sites have high affinity to there substrates ) ➢ Orientation effect: Hold substrate(s) at the exact distance and in the exact orientation necessary for reaction (induced-fit theory ) ➢ Catalytic effect: Provide acidic, basic, or other types of groups required for catalysis ➢ Energy effect: Lower the energy barrier by inducing strain in ➢ bonds in the substrate molecule 1)catalysis by proximity & orientation ➢ Enzyme-substrate interactions orient reactive groups and bring them into proximity with one another favoring their participation in catalysis ➢ Such arrangements have been termed nearestattack conformations (NACs). ➢ NACs are precursors to reaction transition states. ❖ IMP note:All the enzymes have these mechanism (proximity , orientation, catalytic and energy effects ) ,but there are some mechanisms that are specific to some enzymes such as: 1)catalysis by bond strain. 2)catalysis involving proton donors (acids) &acceptors (bases). 3) covalent catalysis. 2)catalysis by bond strain ➢ In this form of catalysis, the induced structural rearrangements produce strained substrate bonds reducing the activation energy. ‫رن‬ ‫رن‬ ‫يصي؟‬ ‫ماسكي ايدين بعض‬ ‫شخصي‬ ‫(تخيل‬ ‫وييج شخص ثالث يشد ايد الشخص األول شو رح ر‬ ‫ي‬ ‫ن‬ ‫أكيد رح تضعف الرابطة ر ن‬ ‫الشخص الثالث هو‬.............‫بصي فعل ًيا‬ ‫بي الشخص األول‬ ‫يل ر‬ ‫ي‬ ‫والثان وهاد ي‬ ) ‫االنزيم‬ ➢ Example:lysozyme ➢ The substrate, on binding, is distorted from the typical 'chair' hexose ring into the 'sofa' conformation, which is similar in shape to the transition state. 3)catalysis involving proton donors (acids) &acceptors (bases): ➢ The R groups act as donors or acceptors of protons. • Histidine is an excellent proton donor/acceptor at physiological pH. • Example: serine proteases. 4)covalent catalysis: ➢ A covalent intermediate forms between the enzyme or coenzyme and the substrate. ➢ Examples of this mechanism is proteolysis by serine proteases, which include digestive enzymes (trypsin, chymotrypsin, and elastase). ❖ Note: we talked that substates cannot bind initially to the active site covalently because covalent bond is a very high energy bond …. But what we mean by covalent catalysis is during the reaction ( after the substrate has bind ) ➢ serine proteases is an example of catalysis involving proton donors (acids) &acceptors (bases) and covalent catalysis so the enzyme can use more than mode of catalysis You must be the change you wish to see in the world End of sheet 28