ENZYMES Notes PDF

ENZYMES Learning Outcomes: At the end of the three hours lecture –discussion on enzymes, the students will be able to: 1. identify the functions of enzymes, 2. familiarize the nomenclature of enzymes, 3. classify the different types of enzymes,...

ENZYMES Learning Outcomes: At the end of the three hours lecture –discussion on enzymes, the students will be able to: 1. identify the functions of enzymes, 2. familiarize the nomenclature of enzymes, 3. classify the different types of enzymes, 4. give the roles of coenzyme, 5. differentiate the two models of enzyme activity, 6. trace the enzymatic cycle, 7. describe the specificity of enzyme –substrate complex, 8. enumerate the environmental factors that regulate enzyme activity, 9. determine the different types of inhibition. INTRODUCTION Enzymes are proteins that are considered as biological catalyst. They speed up the biological reactions in our body. In the absence of enzymatic catalysis, most biochemical reactions are so slow that they would not occur under the mild conditions of temperature and pressure that are compatible with life. Enzymes accelerate the rates of such reactions by well over a million-fold, so reactions that would take years in the absence of catalysis can occur in fractions of seconds if catalyzed by the appropriate enzyme. Cells contain thousands of different enzymes, and their activities determine which of the many possible chemical reactions actually take place within the cell. Catalysts are substances that increase product formation by (1) lowering the energy barrier (activation energy) for the product to form and (2) increase the favorable orientation of colliding reactant molecules for product formation to be successful. Below is an illustration of how activation energy of a reaction is lowered by a catalyst;  Enzymes lower activation energy and speed up reactions by several mechanisms: 1. Active site can hold two or more reactants in the proper position so they may react. 2. Induced fit of the enzyme's active site may distort the substrate's chemical bonds, so less thermal energy (activation energy) is needed to break them during the reaction. 3. Active site might provide a micro-environment conducive to a particular type of reaction (e.g., localized regions of low pH caused by acidic side chains on amino acids at the active site). 4. Side chains of amino acids in the active site may participate directly in the reaction. 5.1 NOMENCLATURE OF ENZYMES An enzyme's name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase. Examples are lactase, alcohol dehydrogenase and DNA polymerase. The International Union of Biochemistry and Molecular Biology have developed another nomenclature for enzymes, the EC numbers; each enzyme is described by a sequence of four numbers preceded by "EC". The first number broadly classifies the enzyme based on its mechanism: Number Classification Biochemical Properties Act on many chemical groupings to add or remove hydrogen EC1. Oxidoreductases atoms. Transfer functional groups between donor and acceptor molecules. Kinases are specialized transferases that regulate EC2. Transferases metabolism by transferring phosphate from ATP to other molecules. EC3. Hydrolases Add water across a bond, hydrolyzing it. Add water, ammonia or carbon dioxide across double bonds, EC4. Lyases or remove these elements to produce double bonds. Carry out many kinds of isomerization: L to D isomerizations, EC5. Isomerases mutase reactions (shifts of chemical groups) and others. Catalyze reactions in which two chemical groups are joined (or EC6. Ligases ligated) with the use of energy from ATP. Examples of these six major classifications of enzymes that catalyzed a certain type of reactions are as follows: 1. OXIDOREDUCTASES - enzymes that catalyze oxidation-reduction reaction. These enzymes are named as oxidoreductase. 2. TRANSFERASES - enzymes that catalyze the transfer of functional groups from one molecule to another. They are named as transaminase, kinase, transmethylase. 3. HYDROLASES - enzymes that encourage the addition of water molecule to a compound resulting in a bond breakage. 4. LYASES - speed up the attachment of a group to a double bond or the removal of a functional group to form a double bond. 5. ISOMERASES - enzymes that rearrange the position of a functional group in a molecule and converts one isomer to another. 6. LIGASES - enzymes that break or make a C-C, C-S, C-O or C-N bond. 5.2 CLASSIFICATION OF ENZYMES BASED ON THEIR COMPOSITION Enzymes are also classified on the basis of their composition. Enzymes composed wholly of protein are known as simple enzymes in contrast to complex enzymes, which are composed of protein plus a relatively small organic molecule. Complex enzymes are also known as holoenzymes. In this terminology the protein component is known as the apoenzyme, while the non-protein component is known as the coenzyme or prosthetic group where prosthetic group describes a complex in which the small organic molecule is bound to the apoenzyme by covalent bonds; when the binding between the apoenzyme and non-protein components is non-covalent, the small organic molecule is called a coenzyme. Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins. It should be noted that the main clinical symptoms of dietary vitamin insufficiency generally arise from the malfunction of enzymes, which lack sufficient cofactors derived from vitamins to maintain homeostasis. The non-protein component of an enzyme may be as simple as a metal ion or as complex as a small non-protein organic molecule. Enzymes that require a metal in their composition are known as metalloenzymes if they bind and retain their metal atom(s) under all conditions that is with very high affinity. Those which have a lower affinity for metal ion, but still require the metal ion for activity, are known as metal-activated enzymes 5.3 ROLE OF COENZYMES The functional role of coenzymes is to act as transporters of chemical groups from one reactant to another. The chemical groups carried can be as simple as the hydride ion (H + + 2e-) carried by nicotinamide adenine dinucleutide (NAD) or the mole of hydrogen carried by flavine adenine dinucleutide (FAD); or they can be even more complex than the amine (-NH2) carried by pyridoxal phosphate. Since coenzymes are chemically changed as a consequence of enzyme action, it is often useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different holoenzymes. In all cases, the coenzymes donate the carried chemical grouping to an acceptor molecule and are thus regenerated to their original form. This regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme as a chemical catalyst, since (unlike the usual substrates, which are used up during the course of a reaction) coenzymes are generally regenerated. 5.4 MODELS OF ENZYME ACTIVITY 1. Lock and Key Model According to this model, each enzyme molecule may have as few as one active site on the surface of the enzyme molecule itself. An active site is an indentation or cavity whereby a reactant molecule (substrate) is attracted to. This is called the enzyme-substrate complex. The polar and non-polar groups of the active site attract compatible groups on the substrate molecule so that the substrate molecule can effectively lock into the cavity and position itself for the necessary collisions and bond breaks and formations that must take place for successful conversion to a product molecule. Once the product molecule has been formed the electrical attractions that made the substrate molecule adhere to the active site no longer are present, and the product molecule can disengage itself from the active site thus freeing the site for another incoming substrate molecule. This process occurs in a highly efficient manner hundreds or even thousands of times in a short time span. This model assumes that molecules that lock into the active site must form a perfect fit. Also the assumption is that the active site conformation is ridged. Enzyme Substrate Enzyme – Substrate Complex 2. Induced Fit (Hand and Glove) Model Modification of the lock and key model assumes that the active site has a certain amount of elasticity whereby the active site can expand or contract in a limited way in order to accommodate the substrate molecule. The analogy is like a hand fitting into a glove. The glove adjusts in shape and size to fit various sized hands within a certain range. This tolerance would explain why bogus molecules of slightly different size compared to the true substrate molecule can still be accommodated by the elastic active site. Small changes in temperature would distort the active site conformation but not so much that the active site could not still accommodate the substrate molecular size. pH changes which would also change the active site conformation but not so much that the active site could not flexibly accommodate the substrate molecule. The Induced Fit Model seems to explain why there is some flexibility in the ability of the active site to accommodate other molecules and at limited temperature and pH ranges. 5.5 THE ENZYMATIC CYLCE Below is an illustration of the entire enzymatic cycle which is quite rapid. The enzymatic cycle has the following steps: a. An enzyme, depending on its degree of specificity reacts with the substrate. b. The enzyme-substrate complex is formed either in the lock and key method or the induced key method. Either way, it can be seen that there now starts a reaction between the two. c. The continued interaction between the two (enzyme-substrate) change the position and shape of the substrate, thus, they are energetically unstable. The enzymes and substrate is held by a covalent bonding. The unstable energy might be derived form: during the continued interaction, the enzyme might have added stress to the substrate in order to break the bond An enzyme might bring the substrate to its close proximity so that the reaction would hasten.  An enzyme might change the pH of the environment of the substrate by donating or accepting H+. d. The substrate is converted to a product, but it is still attached to the enzyme. This is otherwise known as the enzyme-product complex. e. Finally, the newly converted product detaches itself from the enzyme. The enzyme is now ready to take another substrate for product conversion. 5.6 FACTORS AFFECTING ENZYME ACTIVITY Each enzyme has optimal environmental conditions that favor the most active enzyme conformation. The following are environmental factors that will affect enzymatic activity; A. Temperature Every enzyme has a temperature range of optimum activity. Outside that temperature range the enzyme is rendered inactive and is said to be totally inhibited. This occurs because as the temperature changes these supplies enough energy to break some of the intramolecular attractions between polar groups (Hydrogen bonding, dipole-dipole attractions). Most enzymes (and there are hundreds within the human organism) within the human cells will shut down at a body temperature below a certain value which varies according to each individual. This can happen if body temperature gets too low (hypothermia) or too high (hyperthermia). Optimal temperature allows the greatest number of molecular collisions without denaturing the enzyme. Enzyme reaction rate increases with increasing temperature. Kinetic energy of reactant molecules increases with rising temperature, which increases substrate collisions with active sites. Beyond the optimal temperature, reaction rate slows. The enzyme denatures when increased thermal agitation of molecules disrupts weak bonds that stabilize the active conformation B. pH Changes in the pH or acidity of the environment can take place that would alter or totally inhibit the enzyme from catalyzing a reaction. This change in the pH will affect the polar and non-polar intramolecular attractive and repulsive forces and alter the shape of the enzyme and the active site as well to the point where the substrate molecule could no longer fit, and the chemical change would be inhibited from taking place as efficiently or not. Optimal pH range for most enzymes is pH 6- 8. Some enzymes operate best at more extremes of pH. For example, the digestive enzyme, pepsin, found in the acid environment of the stomach has an optimal pH of 2. In an acid solution any basic groups such as the Nitrogen groups in the protein would be protonated. If the environment was too basic the acid groups would be deprotonated. This would alter the electrical attractions between polar groups. Every enzyme has an optimum pH range outside of which the enzyme is inhibited. Some enzymes like many of the hydrolytic enzymes in the stomach such as Pepsin and Chymotrypsin effective operate at a very low acidic pH. Other enzymes like alpha amylase found in the saliva of the mouth operate most effectively at near neutrality. Still other enzymes like the lipases will function most effectively at basic pH values.  If the pH drops in the blood called acidosis then enzymes in the blood will be inhibited outside their optimal pH range. If the pH climbs to an unacceptably high value called alkalosis then enzymes ceases to function effectively. Normally, these conditions do not take place because of the highly efficient buffers found in the blood that restrict the pH of the blood to a very narrow range. Buffers are a substance or mixtures of substances that resist any change in the pH. There are many buffer systems found in the body to adjust the pH so that enzymes might continue to catalyze their reactions. 5.7 REGULATION OF ENZYME ACTIVITY One of the major ways in which enzymes differ from nonbiological catalysts is that the activity of the enzyme is often regulated by the cell. The production of enzyme depends on the availability of the substrate and the need in body’s processes. The body’s cells are the ones that regulate the enzyme activity. They send messages to the different parts of the body which is interpreted as a command. When the cell can sense that the body lacks an enzyme for the substrate to be converted to products, then it sends signal such that enzymes would be produced by the cells. On the other hand, if there are more converted products present than what is needed for the body, the cell again sends signal to stop the production. There are five main ways that enzyme activity is controlled in the cell: 1. Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished by a cell in response to changes in the cell's environment. This form of gene regulation is called enzyme induction and inhibition. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam ring within the penicillin molecule. Another example are the enzymes in the liver called cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of these enzymes can cause drug interactions. 2. Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular compartments. For example, fatty acids are synthesized by one set of enzymes in the cytosol, endoplasmic reticulum and the Golgi apparatus and used by a different set of enzymes as a source of energy in the mitochondrion, through β-oxidation.[ 3. Enzymes can be regulated by inhibitors and activators. For example, the end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. 4. Enzymes can be regulated through post-translational modification. This can include phosphorylation, myristoylation and glycosylation. For example, in the response to insulin, the phosphorylation of multiple enzymes, including glycogen synthase, helps control the synthesis or degradation of glycogen and allows the cell to respond to changes in blood sugar. Another example of post-translational modification is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut. This type of inactive precursor to an enzyme is known as a zymogen. 5. Some enzymes may become activated when localized to a different environment (eg. from a reducing (cytoplasm) to an oxidising (periplasm) environment, high pH to low pH  etc). For example, hemagglutinin in the influenza virus is activated by a conformational change caused by the acidic conditions; these occur when it is taken up inside its host cell and enters the lysosome. 5.8 REASONS FOR REGULATING THE ENZYME ACTIVITY 1. For energy consideration – conservation of cellular activity. If the cell runs out of chemical energy, it will die. Example, It is a great waste of energy to produce an enzyme if the substrate is not available. Similarly, if the product of an enzyme – catalyzed reaction is present in excess, it is a waste of energy for the enzyme to continue to catalyze the reaction, thereby producing more of the unwanted product. 2. Use of allosteric enzymes Allosteric enzymes are enzymes that have two binding sites. The activity of this enzyme is regulated by a small molecule. Allosterism is divided into two: 1. Positive allosterism - when the effector molecule converts the active site to the active configuration. An example of this is when the body experiences a strenuous activity. More demand of energy is needed; therefore, the effector molecule enhances the conversion of the substrate into products. 2. Negative allosterism - when the effector molecule converts the active site in an inactive configuration. An example to this is when the body is at rest or not needing so much energy, then the effector molecule binds to the enzyme to deactivate its binding site. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps allocate materials and energy economically, and prevents the manufacture of excess end products. Like other homeostatic devices, the control of enzymatic action helps to maintain a stable internal environment in living organisms. Allosteric regulation Allosteric site = Specific receptor site on some part of the enzyme molecule other than the active site. Most enzymes with allosteric sites have two or more polypeptide chains, each with its own active site. Allosteric sites are often located where the subunits join. Allosteric enzymes have two conformations, one catalytically active and the other inactive Binding of an activator to an allosteric site stabilizes the active conformation. Binding of an inhibitor (noncompetitive inhibitor) to an allosteric site stabilizes the inactive conformation. Enzyme activity changes continually in response to changes in the relative proportions of activators and inhibitors (e.g., ATP/ADP). ´ Subunits may interact so that a single activator or inhibitor at one allosteric site will affect the active sites of the other subunits. 3. Feedback inhibition Feedback inhibition = Regulation of a metabolic pathway by its end product, which inhibits an enzyme within the pathway. Feedback inhibition is also a process of regulating the enzyme activity by using the product as an effector molecule in one of the series of catalytic process. Consider this example; In the process of converting the substrate A to a final product C Substrate A binds to E 1 to be converted to a sub-product B. Sub-product B binds to E2 to be converted to a final product C. During the cellular activity, if product C is not needed, then it would be used to stop its own production. Product C would act as an effector molecule of E 1, such that when substrate A attaches to E1, sub-product B would not be produced. Non-production of sub- product B would mean that there will be no binding in E 2, thus, A is stopped from being produced. From this process, it is the product itself that causes its own production. 4. Cooperativity The phenomenon where substrate binding to the active site of one subunit induces a conformational change that enhances substrate binding at the active sites ofthe other subunit Substrate molecules themselves may enhance enzyme activity. 5. Production of Zymogens The production of enzyme in an inactive form called zymogen or proenzyme. It is then converted, usually by proteolysis (hydrolysis of protein), to the active form when it has reached the site of activity. This can often be seen in enzymes containing toxic materials or enzymes living in a pH lower than what is normal and standard. Enzymes in the stomach, having a pH optimum of 2 – 3 can destroy or kill the cell that produces then if they are released in active form. Protein enzymes secreted in the stomach are produced in an inactive form. They only become active when they are in contact with an acid. Thus, to buffer the acidity of the stomach, a zymogen is released. ZYYMOGEN OF THE DIGESTIVE TRACT ZYMOGEN ACTIVATOR ENZYME Proelastase Trypsin elastase Trypsinogen Trypsin trypsin Chymotrypsinogen A Trypsin + chymotrypsin chymotrypsin Pepsinogen Acid pH + pepin pepsin Procarboxypeptidase trypsin Carboxypepetidase A or B 6. Protein Modifications An enzyme modification that either turns off or activates it. This is done by adding or cleaving a chemical group from the cleaving process. 5.9 TYPES OF INHIBITION OF ENZYME ACTIVITY A. Competitive Inhibition Competitive Inhibition occurs when a bogus molecule that is close enough to the shape of the true substrate will fit into the active site. Once locked into position, the blocker molecule prevents the true substrate molecule from getting into position. This effectively blocks the active site. The bogus molecule competes for the active site with the true substrate molecule. Many toxic substances owe their toxic properties to their ability to act as inhibitors to important enzymes responsible for catalyzing important biochemical processes. Once the enzyme is inhibited the process cannot take place, and a toxicological symptom occurs that often leads to paralysis, coma or even death of the organism. For example, cyanide poisoning is due to the cyanide ion competitively inhibiting the active site of the cytochromases enzymes responsible for catalyzing the Oxidation and Reduction processes of the Electron Transport System which is responsible for cellular respiration. B. Non-Competitive Inhibition Other inhibitors attached themselves not to the active site itself but to some portion of the enzyme molecule close to the active site which results in the changing of the shape of the active site. This is referred to as non-competitive inhibition. Many heavy metals like Lead, Mercury,and Chromium will function as non-competitive inhibitors. Toxicology is the study of how toxicological substances can interfere with life sustaining enzymes via inhibition. The pesticide and herbicide industries make use of competitive and Non-Competitive Inhibitors Biological warfare owes its success to enzyme inhibition but so does the life giving chemotherapeutic treatment of cancerous tumor growths with agents that inhibit important cancel cell enzymes. All in all the use of inhibitors can be used for the benefit of mankind or its destruction. 5.10 TYPES OF ENZYME INHIBITORS Enzyme inhibitors are classified into: A. IRREVERSIBLE INHIBITOR These are chemicals that hinders the formation of an enzyme-substrate complex by blocking the active site of it; thus, effectively eliminating catalysis. This kind of inhibitor affects many enzymes. B. REVERSIBLE COMPETITIVE INHIBITOR An inhibitor in which the substrate competes for the molecule that resemble its structure and charge distribution. These molecules are referred to as structural analogs. The inhibitor of product formation depends on the concentration of the substrate and structural analogs. If there are more substrates than the structural analogs, inhibition will fail because more product will be converted. Consequently, if more analogs are present, then inhibition is successful. The term competitive comes from the fact that enzymes and structural analogs will compete in binding to the active binding site. C. REVERSIBLE NON-COMPETITIVE INHIBITORS A temporary state of inhibition. The inhibitor acts or binds temporarily to the enzyme making the enzyme unproductive. But when the inhibitor detaches from the enzyme, it makes the enzyme active and ready for a reaction with the substrate. It should be noted that this inhibitor does not bind to the active site of the enzyme. Illustrations: