Biology 11 Enzymes - Complete Book PDF

# Chapter 3: Enzymes ## There is complete check and balance on the chemistry of cell, which is exhibited through various enzymatic reactions going on within a cell. The sum of all the chemical reactions going on in a cell is known as metabolism. These reactions have to be carried out very quickly s...

# Chapter 3: Enzymes ## There is complete check and balance on the chemistry of cell, which is exhibited through various enzymatic reactions going on within a cell. The sum of all the chemical reactions going on in a cell is known as metabolism. These reactions have to be carried out very quickly so that their products can be utilized in various life activities in the cells. Enzymes are biological catalysts and therefore they speed up the biochemical reaction without being consumed. The enzymes exist in the cell as colloids. ## We can define enzymes as 'biological polymers that catalyse biochemical reactions.' ### 3.1 Enzyme Structure All the enzymes are globular proteins which are made up of one or more polypeptides. Ribozymes are the only which consist of RNA and are found in ribosomes. For example, peptidyl transferase is a ribozyme which forms peptide bond during protein synthesis. The enzyme consists of linear chain of amino acids, which form the three dimensional structure of an enzyme. ### 3.1.1 Components of an Active Site of an Enzyme Majority of enzymes are protein in nature. The catalytic activity of an enzyme is located in its active site. It is a specific charge bearing, three-dimensional cavity. The substrate (the reactant which is to be converted into product) molecule is attached to the active site by non-covalent interactions like hydrogen bonding and hydrophobic interactions. Active site consists of 3-12 amino acids which may be scattered in the polypeptide but are brought together in a particular fashion due to secondary and tertiary folding of the protein molecule, e.g., the active site for aldolase consists of glycine, histidine, and alanine amino acids. An active site consists of two functional regions, i.e., binding site and catalytic site. Some amino acids have active site which makes bonds with substrate constitute the binding site while the other amino acids which cause conversion of substrate into product (catalysis) constitute the catalytic site. The shape of active site is designed according to the substrate therefore only a particular substrate can attach to the active site, however, sometime related substrate can also bind to the active site. ## Some enzymes also require a non-protein part, the cofactor which is not only responsible for the attachment of substrate to the active site but also participate in catalytic process. The final shape of active site is actually established after the attachment of cofactor. An enzyme which requires a cofactor becomes active only if the cofactor is combined with it. Such an active enzyme is called holoenzyme. If the cofactor is not available the remaining protein part of enzyme becomes catalytically inactive and is called apoenzyme. On the other hand, the enzymes which do not require cofactor can also show active and inactive states. Pepsin is an example of such enzyme. It is secreted by gastric gland from stomach wall in an inactive state, the pepsinogen. In this state, it has an additional polypeptide fragment attached to its active site which does not allow the binding of substrate, hence it remains inactive. When pepsinogen is exposed in HCl (as in stomach cavity) the additional polypeptide fragment is removed and as a result inactive (apoenzyme) pepsinogen is changed into its active (holoenzyme) form, the pepsin. ### 3.1.2 Types of Cofactors There are three types of cofactors: inorganic ions, organic molecules and prosthetic group. #### Inorganic ions The inorganic cofactors are different metallic ions such as Fe**, Mg++, Cu**, Zn**, etc. These are only attached to the enzymes when substrate gets bind i.e., they are detachable cofactors. Such cofactors are also called activators. #### Organic molecules The organic cofactors are either co-enzymes or prosthetic groups. The coenzymes are the derivatives of vitamins. For example ATP, NAD, FAD are common coenzymes. Like inorganic cofactors they are also attached to the enzymes when substrate gets bind i.e., they are also detachable cofactors. #### Prosthetic group On the other hand a prosthetic group is covalently bonded part of an enzyme which is permanently attached to enzyme and does not detach after the completion of reaction. ## 3.2 Mechanism of Enzyme Action The substrate first binds to the active site of the enzyme to form an enzyme-substrate (ES) complex. Then the substrate is converted into product while it is attached to the enzyme (EP complex). Finally the product is released, thus allowing the enzyme to start all over again. ### 3.2.1 Models of Enzyme Action The mechanism of enzyme action can be explained with the help of two different models. Emil Fischer proposed Lock and key model (in 1894). According to this model, the enzyme is a lock and the substrate is a key. As the key has a similar shape to that of the keyhole of the lock, in the same manner, the substrate has a similar shape to the active site of the enzyme. The substrate binds tightly to the active site of the enzyme, just like the key into its lock. If the shape of the substrate and active site are not similar, the substrate will not be able to bind to the enzyme. An enzyme is a rigid structure and the shape of the active site will not change or modify during the binding process. In some books and diagrams you may find the reverse i.e. key as the enzyme and lock as the substrate. Actually, the notched portion of the key is equivalent to the active site on the enzyme. It reflects that enzymes are highly specific in their action and each enzyme can carry out only one particular reaction. The enzymes, which work according to this model, are called non-regulatory enzymes. However, this model is exercised by a very small number of enzymes, for example sucrase, maltase etc. The ability of enzyme to catalyze one specific reaction is perhaps its most significant property. Although, many enzymes show a broad range of specificity towards the substrate they catalyze. When one enzyme can catalyze only one substrate and essentially no others it is called absolute specificity e.g., urease. Koshland proposed Induced fit model (in 1959). According to this model the active site is flexible; therefore, it is modified as the substrate interacts with enzyme. The amino acids, which makeup the active site are molded into a precise shape which enables the enzyme to perform its catalytic function more effectively. The change which is induced in the shape of active site is responsible for the conversion of substrate into product. As the reaction is completed the active site regains its original shape. This is the flexibility of active site which allows more than one type of related substrates to be attached on active site and therefore, an enzyme can carry out more than one type of related reactions. The example is carbonic anhydrase which can add O₂ to haemoglobin as well as can control the formation of carbonic acid and bicarbonates in blood. Enzymes, which follow the induced fit mechanism, are called regulatory or allosteric enzymes for example hexokinase. ## 3.2.2 Energy of Activation Molecules do not react with one another unless they are activated in some way. The energy that must be added to cause molecules to react with one another is called the energy of activation. In non-living system, we use heat as energy of activation to increase the number of effective collision between molecules. In living systems large amount of heat cannot be used as energy of activation. Why? All living cells and organisms are mainly composed of temperature sensitive protein molecules. About 1,000 chemical reactions are being carried out in a cell at any time. Energy of activation required for such a large number of reactions cannot be provided by living system. The living system works in isothermal condition. The excited state of molecules or reactants is achieved by biochemical process. Enzyme (E) reacts with reactant (A) to form an AE transitional complex. The energy level of AE complex reaches to the energy level of reactant B. AE complex then reacts with reactant B to form AB and enzyme (E) is released. Enzyme does decrease the energy of activation by changing energy dependent process to energy independent process. Thus the energy of activation is "energy required to break the existing bonds and begin the reaction". An enzyme greatly reduces the activation energy necessary to initiate a chemical reaction. ## 3.3 Factors Affecting the Rate of Enzymatic Action The rate of enzymatic reaction is measured by the amount of substrate changed or amount of product formed, during a period of time. The external conditions which affect rate of enzyme reactions are: temperature, pH, concentration of enzyme and substrate concentration. ### 3.3.1 Temperature Heating increases molecular motion. Thus the molecules of the substrate and enzyme move more quickly, so probability of a reaction to occur is increased. Increasing temperature affect the rate of reaction in such a way that an increase of just 10°C in the existing temperature doubles the rate of reaction but this effect remains up to a certain limit. The temperature that promotes maximum activity is called an optimum temperature. If the temperature is increased above this level, then a decrease in the rate of the reaction occurs despite the increasing frequencies of collision. This is because the secondary and tertiary structures of the enzyme have been disrupted and the enzyme is said to be Denatured. The enzyme unfolds and the precise structure of the active site is gradually lost. This temperature which causes denaturation of enzyme is called maximum temperature. The bonds which are most sensitive to temperature change are hydrogen bonds. All human enzymes have a optimum temperature of about 37-38°C, but bacteria living in hot springs may have an optimum temperature of 70°C or higher. Such enzymes have been used in biological washing powders for high temperature washes. If temperature is reduced to near or below freezing point, enzymes are inactivated, not denatured. They will regain their catalytic influence when higher temperatures are restored. This temperature where an inactive enzyme becomes active again is called minimum temperature. ### 3.3.2 pH Every enzyme functions most effectively over a particular pH range. This narrow range of pH at which the maximum rate of reaction is achieved is called optimum pH. Enzyme conformation is sensitive to pH changes because pH influences the charges on the amino acid side chains that are involved in maintaining tertiary and quaternary structure of enzyme. Slight change in optimum pH of an enzyme causes ionization of amino acid of the enzyme therefore, they become inactive temporarily. On the other hand, extreme changes in optimum pH alter the ionic charge of the acidic and basic groups of enzyme and therefore disrupts the ionic bonding (denaturation) that helps to maintain the specific shape of the enzyme. The optimum pH values for most enzymes fall in the range of pH 6-8, but there are exceptions. Protein digesting enzyme pepsin is active in acidic medium at pH 2 and trypsin is inactive at this pH but shows maximum activity in alkaline medium at pH 8. Some enzymes like papain from green papaya act both in acidic and alkaline media. Papain is a cysteine protease acquired from the latex of the papaya plant. It has been used for protecting plants against insects. The enzyme has a high optimal temperature (65°C) and a wide pH range (5-8) for its activity. ### 3.3.3 Enzyme Concentration Provided that the substrate concentration is maintained at a high level (unlimited availability), and other conditions such as pH and temperature are kept constant, the rate of reaction becomes directly proportional to the enzyme concentration. If there is only one enzyme in the system it can convert hundreds of substrates into products but it takes more time. By increasing concentration of enzyme, numbers of active sites become more available and the rate of conversion of substrate into product becomes fast. Such effect persists till the equilibrium state (when concentration of enzyme and substrate becomes equal), after that further increase in enzyme concentration will have no effect upon rate of reaction. ## 3.4 Enzyme Inhibition The phenomenon in which an enzyme fails to catalyze a reaction is called enzyme inhibition and the molecules which react with enzyme but are not converted into desired products are called enzyme inhibitors. In general, the enzyme inhibition is a normal part of the regulation of enzyme activity within cells but sometimes when external factors cause enzyme inhibition; it may become dangerous for life. The molecules which act as inhibitors include poisons, cyanides, antibodies, anti-metabolites, penicillin, sulpha drugs etc. Inhibition may be competitive or noncompetitive. ### 3.4.1 Competitive Inhibition A type of enzyme inhibition in which enzyme activity is blocked by the presence of a chemical that compete with the substrate for binding to the active site is called competitive inhibition. Usually, a competitive inhibitor is structurally similar to the normal substrate and so fits into the active site of the enzyme. However, it is not similar enough to substitute fully for the normal substrate in the chemical reaction and the enzyme cannot catalyze it to form reaction products. Competitive inhibition is usually temporary, and the inhibitor eventually leaves the enzyme hence it is also called reversible inhibition. This means that the level of inhibition depends on the relative concentrations of substrate and inhibitor, since they are competing for places in enzyme active sites. Therefore, if the concentration of the substrate is increased relative to the concentration of the inhibitor, the active site will usually be occupied by the substrate. An example of inhibitor is malonate. Succinate dehydrogenase that catalyzes the formation of fumarate from succinate is competitively inhibited by malonate. The importance of competitive inhibitors is: (a) It supports lock and key hypothesis. (b) It shows that substances which are similar to substrate are not acted upon by enzymes. (c) Competitive inhibitors are used as drugs in the control of bacterial pathogens. Antibiotics known as sulphonamides are used to combat bacterial infection. ### 3.4.2 Non-Competitive Inhibitors In non-competitive inhibition the inhibitor molecule binds to an enzyme other than active site. The other binding site of enzyme is called allosteric site. The non-competitive inhibitors inactivate the enzyme temporarily (reversible inhibition) or they denature the enzyme permanently (irreversible inhibition). Reversible non-competitive enzyme inhibitors work not by preventing the formation of enzyme-substrate complexes, but by preventing the formation of enzyme-product complexes. So they prevent the substrate to be converted into product. Feedback inhibition is an example of reversible non-competitive enzyme inhibition On the other hand, an irreversible non-competitive enzyme inhibitor destroys enzyme by altering its shape so that the substrate cannot bind to the active site. The examples of irreversible non-competitive inhibitors include cyanides and salts of heavy metals. Cyanides are potent poisons of living organism because they can kill an organism by inhibiting cytochrome oxidase essential for cellular respiration. They block the action of these enzymes by combining with iron which may be present in the prosthetic group. lons of heavy metals such as mercury, silver and copper (Hg**, Ag, and Cu**) combine with thiol (-SH) groups in the enzyme breaking the disulphide bridges. These bridges are important in maintaining tertiary structure. When these bridges are broken, the enzyme becomes denatured and inactive. ### 3.4.3 Feedback Inhibition The activity of almost every enzyme in a cell can be regulated by its product. When the activity of an enzyme is inhibited by its own product, it is called feedback inhibition. This is a type of reversible non-competitive inhibition. This phenomenon is a part of normal regulatory mechanism and usually happens during the regulation of metabolic pathways. For example, the amino acid aspartate becomes the amino acid threonine by a sequence of five enzymatic reactions. When threonine, the end product of this pathway, is present in excess, it binds to an allosteric site on enzyme 1 on this pathway and then the active site is no longer able to bind aspartate. When all the threonine is consumed in cellular events, the threonine molecule which is attached to the allosteric site is also removed; the pathway resumes its activity once again. ## 3.5 Classification of Enzymes Enzymes can be classified either on the basis of reaction types that they catalyze or on the basis of substrate which are acted upon by the enzyme. ### 3.5.1 Classification based upon reaction type A systematic nomenclature and classification of enzymes based on reaction types and reaction mechanism was given by International Union of Biochemistry (in 1961). On that basis all the enzymes have been classified into six groups: 1. Oxidoreductases 4. Lyases 2. Transferases 5. Isomerases 3. Hydrolases 6. Ligases - **Oxidoreductases** These enzymes catalyze oxidation/reduction of their substrate and act by removing or adding electron or H+ ions from or to the substrate. For example cytochrome oxidase oxidizes cytochrome. - **Transferases** These enzymes catalyze the transfer of specific functional group other than hydrogen from one substrate to another. The chemical group transferred in the process is not in a free state, for example hexokinase transfers a phosphate group from ATP to glucose. - **Hydrolases** These enzymes bring about the breakdown of large complex organic molecules into smaller ones by adding water (hydrolysis) and breaking the specific covalent bonds. Examples are proteolytic enzymes which breakdown proteins into peptones and peptides such as pepsin, renin and trypsin. Other digestive enzymes that work in digestive tract are also the examples of hydrolases. - **Lyases** These enzymes catalyze the breakdown of specific covalent bonds and removal of groups without hydrolysis. For example histidine decarboxylase breaks the covalent bonds between carbon atoms in histidine forming carbon dioxide and histamine. - **Isomerases** These enzymes bring about intra-molecular rearrangement of atoms in the molecules and thus forming one isomer from another. For example phosphohexose isomerase changes glucose 6-phosphate to fructose 6-phosphate. - **Ligases (Synthetases)** These enzymes bring about joining together of two molecules. The energy is derived by hydrolysis of ATP. For example polymerases are responsible for linking monomers into a polymer such as DNA or RNA. ### 3.5.2 Classification based upon substrate Enzymes can be classified on the basis of substrates they use. Some of the examples are: proteases, lipases, carbohydrases and nucleases. - **Proteases** These enzymes act upon proteins. Examples are: pepsin and trypsin (both digest large polypeptides into small polypeptides or peptones), aminopeptidases and carboxypeptidases (both digest peptones into dipeptides) and erypsin (digest dipeptides into amino acids) - **Lipases** These enzymes hydrolyze lipids into fatty acids and glycerols. Examples are pancreatic lipases. - **Carbohydrases** These enzymes cause breakdown of carbohydrates. Examples are: * amylase (digest starch or glycogen into maltose) * cellulase (digest cellulose into cellubiose, a disaccharide) * maltase (digest maltose into glucoses) * sucrase (digest sucrose into glucose and fructose) * lactase (digest lactose into galactose and glucose) - **Nucleases** These are involved in the breakdown of DNA and RNA. Examples are: * RNAases (digest RNA into ribonucleotides) * DNAases (digest DNA into deoxyribo nucleotides). * ATPases (cause hydrolysis of ATP in muscles etc.) **Diagnostic uses of enzymes.** * Aldolase: progressive muscular dystrophy, viral hepatitis and advanced cancer of the prostate * Creatine Phosphokinase: damage to muscle cells. * Gamma-glutamyl Transpeptidase: in assessing liver function. * Lactic Dehydrogenase: in differentiating heart attack, anemia, lung injury, or liver disease. * Lipase: Damage to the pancreas.