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Consortium for Educational Communication Module on Discovery, Nomenclature and Characteristics of Enzymes By Ms Hadiya Shafi Research scholar Department of Botany University of Kashmir Srinagar Consortium for Educational Communication TEXT Enzymes Enzymes are the biological molecules that catalyze specific biochemical reactions. They are active under mild conditions of temperature and pressure found in the cell. They are usually single or multiple chain proteins that act as biological catalysts that catalyze intracellular and extracellular biochemical reactions. On the basis of their composition, enzymes are of two types, i.e., simple enzymes (Pepsin, trypsin, Urease and amylase) and conjugated enzymes. The simple enzymes are entirely made up of proteins while as the conjugated enzymes consist of a protein part and a non protein part. The protein part is called apoenzyme and the non protein part is called prosthetic group, coenzyme or cofactor. The complete enzyme including prosthetic group and or coenzyme or cofactor is called holoenzyme. Apopenzyme consists of one or more polypeptide chains of 10’s to 100’s of amino acids. The type of amino acid and the number of subunits determine the size and composition of the protein part. The Prosthetic group is tightly bound to the enzyme and is usually a much smaller inorganic portion of an enzyme. It is attached to the protein part of an enzyme by covalent bonds and is essential for its catalytic activity. The simple prosthetic groups are metal ions, such as, Cu, Zn, Mn and Mo. Some prosthetic groups may also be organic compounds which include cytochromes, Flavonoids, pyridoxe or biotinal phosphate, heme or biotin, etc. These groups are bound to active site of many enzymes that participate in catalysis. Coenzyme or cofactor is not tightly bound to the enzyme. The former is an organic molecule and the later is inorganic in nature. Coenzymes serve as carriers of various types of chemical groups. NAD+ (Nicotineamide adenine Consortium for Educational Communication dinucleotide) functions as carrier of electrons in oxidation reduction reactions. Several other coenzymes are involved in the transfer of a variety of additional chemical groups (e.g. carboxyl groups and acyl goups). Cofactors are essential for the catalytic activity of an enzyme. Some examples of cofactors are Clˉ, Cu2+, Fe2+, Zn2+, Co2+, K+, Ca2+, etc. There are also some special types of enzymes. These are isozymes and extremozymes. Isozymes are the multiple molecular forms of a single enzyme which are normally formed under normal conditions. They can differ in various physical and chemical properties and their intracellular localization may also differ in nature of cofactor and kinetic or regulatory properties. Extremoxymes are the enzymes which are active under extreme conditions of pH, temperature and pressure. These enzymes are well adapted to tolerate such harsh conditions. Enzymes have the ability to increase the reaction rate or biocatalysis without being destroyed or changed in the process. Because of this, one enzyme molecule can theoretically change an infinite amount of substrate, if given an infinite amount of time. Increasing the amount of enzyme, decreases the time required for completing the reaction. The enzymes enhance reaction rates by a factor of 105 to 10 17 times than the uncatalysed reaction and facilitate the attainment of chemical equilibrium fast. Enzymes function by lowering the free energy of the reactions transition state. They are effective in very small amounts. They are extremely unstable and are inactivated or denatured by high temperature or very alkaline or acidic conditions. For an enzyme to work, it must have access to its substrate, the material upon which it exerts its action. If no substrate is available to the enzyme, the enzyme performs no function. The substances on which an enzyme exerts its action are called reactants and the substances produced at the end of the reaction are called products. Consortium for Educational Communication Enzymes control all biochemical reactions in every living thing, from viruses to man. Breathing, digestion, heart action, formation of body tissues, movement of muscles and many more processes depend on enzymes. So without enzymes, life is not possible. More than 100,000 different types of enzymes have been identified, most of which are colorless solid, soluble in water or dilute solutions, but some are blue, green or greenish brown. Discovery of Enzymes The term ‘enzyme’ was introduced by Kuhne in 1878. It is taken from the Greek word Zume (Zyme) meaning ‘Leaven’. Enzymes are the principles found in Leaven. The first observation was carried out on vegetable and animal cell free extracts. Berzilius in 1836 coined the term catalysis which in Greek means ‘to discover’. After this, a certain number of enzymes called ‘diastases’ or ‘ferments’ were identified. Claude Bernard in 1848 discovered the role of Pancreatic juices and described the action of Pancreatin, an albuminous substance. In 1864, Bechamp observed that the enzyme Zymase is involved in the transformation of Saccharose into glucose. Raphael Dubois in 1886 discovered luciferase, the enzyme responsible for bioluminescence. Later in 1896, several oxidative enzymes were discovered by Gabriel Bertrand. He introduced the concept of Cofactor. In 1897, Hill discovered that maltase catalyzes a reversible reaction. Edward Buchner in 1897 showed that a yeast extract completely lacking cells could just as well stimulate alcoholic fermentation. He hypothesized that yeast cells ferment sugars not inside the cell but by secreting proteins into their environment, which is the actual mechanism. This posed challenge to vitalism by showing that the presence of living yeast cells was not necessary for fermentation. He was Consortium for Educational Communication awarded Nobel Prize in Chemistry in 1907 for this valuable work. Biocatalysis has been the basis of some of the oldest chemical transformations (e.g fermentation and brewing) and it involves enzyme catalysis in the living cells. The discovery of enzymes can be traced back to nineteenth century. In 1926, James Batcheller Sumner demonstrated that urease could be isolated and crystallized and showed by chemical tests that it was a protein. This was the first experimental proof that an enzyme is a protein. In 1937, he succeeded in isolating and crystallizing a second enzyme, catalase and devised a general crystallization method for enzymes, and also showed that all enzymes are proteins. By similar methods, John Howard Northrop also obtained other crystalline enzymes by this time, starting with pepsin in 1929. W. M. Stanley is also known for the preparation of enzymes and virus proteins in a pure form. In 1946, James Batcheller Sumner shared the Nobel Prize in Chemistry with John Howard Northrop and Wendell Meredith Stanley for the brilliant work on enzymes. Table 1: Enzymes and their discoverers. Enzyme Ptyalin Maltase Singrase Pepsin Discoverer Leuchs Payen and Persoz Faure Schwann Year of discovery 1831 1833 1835 1836 Nomenclature of enzymes Many enzymes have usually been named by adding the suffix “ase” to the name of their substrate or the catalytic reaction. Some enzymes have been named in an arbitrary way. Some of the examples are trypsin, pepsin, chymotrypsin, rennin and emulsion. The earlier system of nomenclature has been modified Consortium for Educational Communication in 1992 by International Union of Biochemistry and Molecular Biology (IUBMB) nomenclature system of enzymes, commonly known as Enzyme Commision (EC) numbers. It developed certain rules for nomenclature and classification of enzymes. This system divided enzymes into six classes, each with subclasses and subsubclasses on the basis of the reaction catalysed. According to standard nomenclature, each enzyme is assigned a four digit code number following the abbreviation EC (Enzyme Commission) and a systematic name, which identifies the reaction catalysed by it. Examples: ATP + D-glucose → ADP +D-glucose 6-phosphate Common name: Hexokinase Systematic name: ATP: glucose phosphotransferase Enzyme Commision number [EC no.] is EC 2.7.1.1 The first number 2 denotes that it belongs to class 2 of enzymes, i.e., transferases which catalyze group transfer reactions.The second number 7 denotes the subclass 7, i.e., phosphotransferases, which catalyse transfer of phosphoryl group.The third number 1 denotes the sub-subclass i.e., a phosphotransferase with a hydroxyl group as acceptor and the fourth number 1 indicates that the phosphoryl group acceptor is D- glucose. The six classes of enzymes are mentioned below:1- Oxidoreductases 2- Transferases 3- Hydrolases 4- Lyases 5- Isomerases Consortium for Educational Communication 6- Ligases 1-Oxidoreductases Oxidoreductases bring about redox reactions or oxidation-reduction reactions involving transfer of hydrogen or oxygen atoms, hydride ions or electrons between the molecules i.e., from one molecule (the reductant or the hydrogen or electron donar) to another molecule (the oxidant or the hydrogen or electron acceptor). Their systematic name is in the form of donar: acceptor oxidoreductase. If a hydrogen donating substrate is being oxidized, the enzyme is commonly called donar dehydrogenase. If oxygen is the acceptor, the term donar oxidase is used. The second number in the EC code of oxidoreductases denotes the sub-subclass of the enzyme and indicates the hydrogen acceptor. Some of the common examples of oxidoreductases are Alcohol dehydrogenase, Cytochrome oxidase and Heme oxygenase. The general reaction catalysed by oxidoreductases is mentioned below: Areduced + Boxidized → A oxidized + Breduced Table 2 : Classification of oxidoreductases upto the subclass level SubClass 1.1. 1.2 1.3 Oxidoreductases Example Acts on the CH–OH group of donors. Alcohol dehydrogenase Acts on the aldehyde or oxo group of F o r m a l d e h y d e donors. dehydrogenase Acts on the CH–CH group of donors. Fumarate reductase (NADH) Consortium for Educational Communication 1.4 1.5 1.6 1.7 1.8 1.9 1.10 Acts on the CH–NH2 group of donors. Glutamate synthase (NADPH) Acts on the CH–NH group of donors. Flavin reductase Acts on NADH2 or NADPH2. NAD(P)+ transhydrogenase Acts on other nitrogenous compounds as Nitrite reductase (NAD(P) donors. H) Acts on a sulfur group of donors. Cystine reductase Acts on a haem group of donors. Cytochrome oxidase 1.15. 1.16. 1.17. 1.18. Acts on diphenols and related substances as donors. Acts on a peroxide as acceptor (peroxidases). Acts on hydrogen as donor. Acts on single donors with incorporation of molecular oxygen. Acts on paired donors with incorporation of molecular oxygen. Acts on superoxide radicals as acceptor. Oxidizing metal ions. Acts on –CH2– groups. Acts on reduced ferredoxin as donor. 1.19. Acts on reduced flavodoxin as donor. Superoxide dismutase Mercury(II) reductase Ribonucleotide reductase Ferredoxin—NADP(+) reductase nitrogenase 1.97. Other oxidoreductases. Chlorate reductase 1.11 1.12 1.13 1.14 Coenzyme Q – cytochrome c reductase Catalase Hydrogen dehydrogenase Indole 2,3-dioxygenase Heme oxygenase 2. Transferases Transferases catalyze the transfer of functional groups from one substrate (donar) to another (acceptor). Thus, they are involved in group transfer reactions. The systematic name is donar: acceptor group transferase. The recommended name is acceptor grouptransferase or donar grouptransferase. Transferases catalyze the transfer of an atom (other than oxygen or hydrogen) Consortium for Educational Communication or a group of atoms like acyl-, alkyl-, glycosyl, etc. Some of the common examples of transferases are Hexokinase, Glutathione S-transferase and Methionine synthase. The general reaction of transferases is given below: A-C + B → A + B-C Table 3. Classification of transferases upto the subclass level. SubClass 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 Transferases Transfers one-carbon groups. Transfers aldehyde or ketone residues. Acyltransferases. Glycosyltransferases. Transfers alkyl or aryl groups, other than methyl groups. Transfers nitrogenous groups. Transfers phosphorus-containing groups. Transfers sulfur-containing groups. Transfers selenium-containing groups. Example Methionine synthase Transketolase Fatty acid synthase Glycogen synthase Glutathione s transferase Transaminase Hexokinase Biotin synthase L- seryl-tRNASec transferase selenium 3. Hydrolases Hydrolase catalyzes the hydrolytic cleavage of a chemical bond such as C–O, C–N, C–C and some other bonds, including phosphoric anhydride bonds. The systematic name is substrateXhydrolase, where X is the group removed by hydrolysis. In many cases, the recommended name is formed by the name of the substrate with the suffix “ase”. This suffix indicates that it is a hydrolytic enzyme. The main physiological function of the enzyme is the reaction with water as the acceptor. The first number in Consortium for Educational Communication the EC code indicates that it belongs to the class hydrolases. The second number indicates the nature of the hydrolysed bond, and the third number denotes the nature of the substrate. Some of the common examples of hydrolases are Alkaline phosphatase, Uracil DNA glycosylase and Exonuclease. For example,the hydrolase catalyzes the following reaction:A–B + H2O → A–OH + B–H Table 4: Classification of hydrolases upto the sub class level. Subclass 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 4. Lyases Hydrolases Acts on ester bonds. Glycosidases. Acts on ether bonds. Acts on peptide bonds (peptidase). Acts on carbon–nitrogen bonds, other than peptide bonds. Acts on acid anhydrides. Acts on carbon–carbon bonds. Acts on halide bonds. Acts on phosphorus–nitrogen bonds. Example Alkaline phosphatase Urasil DNA glycosylase Adenosylmethionine hydrolase carboxypeptidase A Aminohydrolase Acylphosphatase Acetylpyruvate hydrolase Alkylhalidase phosphoamidase Acts on sulfur–nitrogen bonds. Cyclamate sulfohydrolase Acts on carbon–phosphorus Phosphonopyruvate hydrolase bonds. Acts on sulfur–sulfur bonds. Trithionate hydrolase Consortium for Educational Communication Lyases catalyze the cleavage of various chemical bonds such as C–N, C–O, C–C, C-S and others by reactions other than hydrolysis and oxidation. Thus, they remove a group of atoms from the substrate and lead to the formation of a new double bond or a new ring structure. Lyases require only one substrate for the unidirectional reaction, but two substrates for the reverse reaction. The systematic name is substrate grouplyase. If the reverse reaction is much more important, or is the only one demonstrated, the name synthase may be used rather than synthetase. Some of the common examples of lyases are Fumarase, Isocitrate lyase and pyruvate decarboxylase. One of the reactions catalyzed by lyases is given below: ATP → cAMP + PPi Table 5. Classification of Lyases. SubClass 4. 1. 4. 2. 4. 3. 4. 4. 4. 5. 4. 6. 4.99 Lyases Carbon–carbon lyases Carbon–oxygen lyases Carbon–nitrogen lyases Carbon–sulfur lyases Carbon–halide lyases Phosphorus-oxygen lyases Other lyases Example Isocitrate lyase Fumarase Aspartate ammonia-lyase Cysteine lyase DDT-dehydrochlorinase Adenylate cyclase Aliphatic aldoxime dehydratase 5.Isomerases Isomerase catalyzes the interconversion or structural rearrangement of optical, positional or geometric isomers. These enzymes catalyze structural or geometric changes within one molecule. The first number indicates that they are isomerases. Consortium for Educational Communication The second number denotes the type of isomerism involved, and the third number shows the type of substrate. In certain cases, the intermolecular oxidoreduction reaction is involved. They are classified as isomerases and not oxidoreductases because the donor and the acceptor groups are present in the same molecule. They may also contain firmly bound NAD or NADP. Some of the common examples of isomerases are Inositol-3-phosphate synthase, Phosphoglucomutase and Type II topoisomerase. Thus, isomerases catalyze reactions of the form: A→C where C is an isomer of A. Table 6. Classification of isomerases. Subclass 5.1. 5.2. 5.3. 5.4. 5.5. 5.99. Isomerases Racemases and epimerases cis–trans-Isomerases Intramolecular oxidoreductases Intramolecular transferases Intramolecular lyases Other isomerases Example Alanine racemase Retinal isomerase Ribose-5-phosphate isomerase Phosphoglucomutase Inositol-3-phosphate synthase DNA gyrase 6. Ligases The word ligase has been taken from the Latin verb ligare which means “to bind” or “to glue together”. It catalyzes the joining of two large molecules to form a new covalent bond coupled with the hydrolysis of a small dependent chemical group. For example, a pyrophosphate bond in ATP, GTP or a similar compound is hydrolyzed by ligase. The enzyme links together two compounds and catalyzes the joining of C-N, C-S, C-O, C-C bonds etc. The systematic name of enzyme is A: B ligase. The recommended name is A–B ligase. To emphasize the synthetic nature of the reaction, sometimes, the name synthase is used Consortium for Educational Communication for the recommended name. The name synthetase can also be sometimes used instead of synthase in the names of enzymes in this class. Some of the common examples of ligases are DNA Ligase, Pyruvate carboxylase and Succinyl co A synthetase. The general reaction catalysed by Ligase is shown below:Ab + cD → A–D + b + c Here, the lower case letters indicate the small dependent groups. Table 7: Classification of ligases upto the subclass level SubClass 6. 1. 6. 2. 6. 3. 6. 4. 6. 5. Ligases Forming carbon–oxygen bonds Forming carbon–sulfur bonds Forming carbon–nitrogen bonds Forming carbon–carbon bonds Forming phosphoric ester bonds Example Aminoacyl tRNA synthetase Succinyl co A synthetase Glutamine synthetase Pyruvate caboxylase DNA ligase Characteristics of enzymes Enzymes can easily be identified by characteristics possessed by them. The various characteristic features of enzymes are specificity, reversibility of action, temperature sensitivity, pH sensitivity, high catalytic rate, colloidal properties, molecular weight, chemical nature, inhibition by poisons. All these properties are explained below: 1. Specificity Enzymes are highly specific in the type of reaction they catalyze and the choice of reactants they act upon. A particular enzyme can catalyze only a particular type of reaction. Each enzyme acts on Consortium for Educational Communication a single substrate or small group of closely related substrates. The presence of specific functional groups adjacent to the bond to be cleaved determines the specificity of the enzyme. The active sites of enzymes are highly specific centre’s composed of varying number and sequence of amino acids and a particular binding site which complexes only with a specific substrate (in a lock and key process). 2. Reversibility of action The enzyme catalyzed reactions are reversible as well as irreversible. In the presence of an appropriate enzyme, the conversion of substrate to products is accelerated without altering the equilibrium between the reactants and the products. Although, the rate at which the chemical equilibrium is established is enhanced. The enzymes affect only the rate and not the direction. They can accelerate the reaction in either direction. Many enzymes which are required for the synthesis of starch and sucrose are responsible for their breakdown also. However, by removing the products of enzymatic reaction as quickly as they are formed, the reversion can be checked. The enzyme must accelerate both the forward and backward reactions equally. The reversible reaction can be written as: S⇌P Example: Alcohol hydrogenase act in both directions. CH3CH2OH + NAD+ (Alcohol) ⇌ CH3CHO +NADH + H+ (Aldehyde) On the other hand, some enzymes carry out irreversible reactions Consortium for Educational Communication only in one direction, e.g., hydrolytic enzymes catalyzing cleavage of bonds between two atoms. In fact large molecules such as fats, proteins, starch and nucleic acids are synthesized by one enzyme and degraded by the other. Synthetic and degradative enzymes are often kept separate from each other by membranes or are formed at different stages, so that competition between them is minimized. 3. Temperature sensitivity The enzymes are sensitive to heat and hence are thermo labile. They function best at optimum temperature range of 250C to 400C. Both low as well as high temperatures are unfavorable for enzymatic activity. Their activity decreases with decrease as well as increase in temperature. Enzymatic reactions occur slowly at 00C because of the lower level of the molecular kinetic energy which limits substrate enzyme collision and attainment of the transition state. With increase in temperature, both these events are favoured. With every 100 rise in temperature, the reaction rate shows enhancement. Most enzymes are inactivated or denatured by temperature about 600C. It is because majority of the enzymes are proteinaceous in nature. Early denaturation by heat is observed in enzymes present in moist conditions. However the enzymes in drier or dehydrated conditions are quite stable. Denaturation disrupts the secondary structure of an enzyme and it looses its active site and hence the catalytic power. However, some enzymes are resistant to high temperature and continue to work at high temperature conditions. 4.pH sensitivity Consortium for Educational Communication In the catalytic site of an enzyme, the state of ionization of amino acid residues is pH dependent. Since, the specific states of ionization of these residues determine the enzyme’s catalytic activity, enzymes activity is also pH dependent. Different enzymes function at different pH ranges. There is a restrictive pH range which remains constant for each enzyme and is its characteristic property. The activity of an enzyme decreases with increase or decrease in optimum pH. pH changes also modify the ionic substrates. For example, if a COO- group of an aspartate is used to bind a positively charged substrate, protonation would reduce the force of attraction and decreases the affinity of the enzyme for the substrate. Most enzymes show maximal activity around neutral pH, but there are many exceptions. Table 8.Optimum pH of different enzymes Enzyme Pepsin Egg white lysozyme Acid phosphatase Pancreatic alpha amylase yeast hexokinase Alpha-chymotrypsin Alkaline phosphatase Fumarase Ribonuclease Optimum pH 1.5-2.0 4.5-5.0 5.0 6.9 7.4 8-8.5 9.5-10 7.0 7.0 3. Catalytic properties Enzymes increase reaction rate by a factor of 105-1017 than that of an unanalyzed reaction. Enzymes are active in extremely small amounts, e.g., one molecule of catalase can effectively catalyze conversion of 50, 00,000 molecules of H202 into H20 and H2.The Consortium for Educational Communication enzyme remains unaffected at the end of the reaction. It also doesn’t alter the position of chemical equilibrium of a reversible reaction but helps in its quick establishment. Weak interactions (hydrogen bonds and hydrophobic and ionic interactions) between substrate and enzyme provide a significant part of the energy used for reaction rate enhancement. Carbonic anhydrase is one of the fastest enzymes known. It catalyzes the hydration of CO2 and transfers it from tissues into the blood and then to the alveolar air. Each enzyme molecule can hydrate 105 molecules of CO2 per second. 5. Colloidal properties of enzymes Enzymes are large proteinaceous substances of very high molecular weight. They have the size in the range of colloidal particles (0.001µm-0.1µm in diameter). Their colloidal nature provides large surface area for reactions to take place. Enzymes display all the colloidal properties. They don’t pass through a collodion (or parchment membrane) but can pass through filter paper. This helps in their separation from true solution. The enzymes, being colloidal particles, have positive or negative charges. 7. Molecular weight Catalase has a molecular weight of 250,000 Daltons and is one of the largest enzymes. On the other side, peroxidase, one of the smaller enzymes has a molecular weight of 40,000 Daltons. Thus enzymes are larger than usual simple organic molecules. Consortium for Educational Communication 8. Chemical nature of enzymes Enzymes are mostly proteinaceous in nature except a small group of catalytic RNA and DNA molecules. There catalytic activity depends on the integrity of their native protein conformation. If an enzyme is dissociated into subunits or is denatured, it looses its catalytic activity. Breaking down an enzyme into its component amino acids always destroys its activity completely. Thus, the primary, secondary, tertiary and quaternary structures of proteinaceous enzymes are absolutely important for their catalytic activity. 9. Inhibition by poison The activity of an enzyme is altered by many foreign substances that combine with it in a way that influences its chemical substrate binding property and turnover number. Substances or agents that reduce the activity of an enzyme or inactivate it in this way are called as inhibitors or enzyme poisons. Inhibitor binds to enzyme either reversibly or irreversibly. Thus, the inhibitors are of two types:- A. Reversible inhibitors Reversible inhibitors can easily be removed from the enzyme because they bind reversibly to the enzyme by noncovalent interactions. There are three main types of reversible inhibition of enzymes. These are mentioned below. a. competitive reversible inhibition A competitive inhibitor binds at and blocks the binding site of Consortium for Educational Communication substrate on the enzyme. Most of them resemble the substrate and combine with the enzyme to form an EI complex but don’t lead to the catalysis. By adding more substrate, its combination with enzyme is favoured. b. Uncompetitive reversible inhibition An uncompetitive inhibitor binds only to site distinct from substrate active site and binds only to ES complex. Ultimately, it leads to no reaction. c. Mixed reversible inhibition A mixed reversible inhibitor binds either to ES complex or directly to the enzyme. It also binds to a site which is distinct from substrate binding site. B. Irreversible inhibitors These are the inhibitor which bind covalently with an enzyme and destroys a functional group on it that is essential for its catalytic activity. Some inhibitors form a particular stable non covalent association with the enzyme but the formation of a covalent bond between a irreversible inhibitor and an enzyme is common.