Enzymes PDF

# 4 Enzymes ## Learning Objectives After completing this chapter, you will be able to understand the following: - Role of enzymes and their structure. - Mechanism of enzyme action. - Shape and properties of enzymes. - Factors affecting enzymatic activity. - Various classes and nomenclature of en...

# 4 Enzymes ## Learning Objectives After completing this chapter, you will be able to understand the following: - Role of enzymes and their structure. - Mechanism of enzyme action. - Shape and properties of enzymes. - Factors affecting enzymatic activity. - Various classes and nomenclature of enzymes. - Role of co-factors. - Significance of enzymes. ## 4.1 Enzymes In living cells, most catalysts are protein molecules called enzymes which catalyze numerous biochemical reactions occurring in a cell. Some can also be nucleic acids that behave like enzymes such as ribozymes. They are species of RNA molecules having enzymatic activity. They have a high degree of specificity, that is, they catalyze only one type of reaction, and most act on only one particular substrate. Enzymes of commercial importance are available from plant and animal sources. However, they have certain limitations. The production of enzymes from plants and animals is in short supply and cannot meet the increased demands of the industries. Besides, the industries require a continuous supply of these enzymes. The microbial enzymes faced initial hiccups in their acceptance as the people feared of the enzymes being contaminated by the microbes. However, the microbial enzymes have certain advantages: 1. They can be produced on a large scale by the process of fermentation. 2. If the enzymes are excreted into the medium, they can be isolated easily.. 3. A large variety of enzymes can be produced using microbes. 4. The microbes can be genetically engineered to give high yield of enzymes. The majority of enzymes are now derived from microorganisms followed by plants and animals. Enzymes are especially useful in industrial processes because of their specificity. They act on a certain substrate and yield a certain product. This minimizes the problems of product purification. ## Enzymology The study of enzymes is known as enzymology. Louis Pasteur and others believed that living systems had a "vital force" that allowed them to bypass the laws of nature governing non-living matter as they were unable to reproduce most biochemical reactions in the laboratory. Justus von Liebig and others were of the opinion that biological processes were caused by the action of chemical substances known as ferments. The term enzyme (en = in; zyme = yeast) was coined in 1878 by Wilhelm Kühne. It indicated that something present in the yeast, as opposed to the yeast itself, catalyzes the fermentation reactions. Eduard Buchner reported that a cell-free yeast extract was able to carry out the synthesis of ethanol from glucose by the following reaction in which 12 enzyme-catalyzed steps are involved: ``` C6H12O6 → 2CH3CH2OH + 2CO2 ``` Since enzymes are proteins, they get destroyed if the temperature increases beyond 40°C. This is the main point of difference between an enzyme and an inorganic catalyst which can function at high temperatures for example, the hydrothermal vents, a place with high pressure and temperature. However, there are certain enzymes present in hot sulphur springs and hydrothermal vents like the DNA polymerase used in polymerase chain reaction. They are functional at 80-90°C and exhibit thermal stability. ## Mode of action of Enzymes Most of the enzymes are globular proteins. Few are RNA enzymes like ribozyme and ribonuclease P. They are generally hydrophilic in nature. Enzymes act as catalysts by increasing the rates of reactions. Like proteins, most of them have secondary and tertiary structures. In their tertiary structure, the crevices, clefts or pockets are present that act as active sites (portion of the enzyme molecule which is active and wherein substrate usually binds for efficient biocatalysis). It is the region of the enzyme where catalysis occurs. Catalysis depends on the arrangement of functional groups in the enzyme's active sites which helps in increasing the rates of reactions. Substrates like amino acids bind to the enzyme at this active site. The amino acids present in the active site are usually situated at a distance from each other along the length of the polypeptide chain, but they are brought into close proximity to one another as the polypeptide folds to form the tertiary structure. Amino acid residues lining active site facilitate the bond breakage/formation, group transfer, rearrangement, acceptance/donation of protons, thus facilitate the product formation and accomplish the reaction. Some species of RNA molecules have enzymatic activity, they are known as ribozymes. They were discovered in the early 1980s. RNA functioned both as a catalyst for some biochemical reactions instead of protein enzymes as well as genetic material. RNA was involved in many important life processes such as metabolism, translation, splicing, etc. A few reactions, such as the cleavage and ligation of phosphodiester bonds required for RNA splicing and the formation of peptide bonds during protein synthesis, are catalyzed by naturally occurring RNAs. Ribosomal RNA that catalyzes the formation of peptide bonds between amino acids is an example of ribozyme. ## Properties of Enzymes Enzymes do not initiate a chemical reaction, but increase the rate of reaction without getting used up themselves. All the chemical reactions that are controlled by enzymes are reversible. The number of substrate molecules transformed per minute by an enzyme is called turn over number of the enzyme. Enzymes catalyze specific reactions. They do so with great efficiency and with many built-in controls. Three important properties of enzymes are as follows: 1. **Enzymes are highly specific:** Each particular enzyme binds only to specific substrates (the reactant molecules on which the enzyme acts). Of the more than 1000 known enzymes in one's body, each has a characteristic three-dimensional shape with a specific surface configuration, which allows it to recognize and bind to certain substrates. The enzyme specificity is supported by two theories: * **Lock and Key hypothesis (by Emil Fischer in 1894) (Figure 1):** Substrate gets fit into the active site of the enzyme as a key fit in a lock. The enzyme specificity is due to the complementary shapes of enzyme and the substrate. * **Induced-fit model (by Daniel Koshland, 1958) (Figure 2):** The active site changes its shape to fit around the substrate once the substrate enters the active site. The substrate induces a change in the enzyme shape to fit the shape of the substrate. 2. **Enzymes are very efficient:** Under optimal conditions; enzymes can catalyze reactions at rates 100 million to 10 billion times more rapid than those of similar reactions occurring without enzymes. 3. **Enzymes are subject to a variety of cellular controls:** Their rate of synthesis and their concentration at any given time are under the control of a cell's genes. Substances within the cell may either enhance or inhibit the activity of a given enzyme. ## Chemical Reactions In a chemical reaction, the bonds are broken and new bonds are formed. A chemical reaction can be inorganic or organic. The amount of product formed in a given time determines the rate of reaction of physical and chemical processes. It is given by the following equation: ``` Rate=d[P]/dt ``` where, [P] = product formed and t = time. If a catalyst is added to a reaction, it increases the rate many times higher than those that are not catalyzed. For example, when carbon dioxide reacts with water to form carbonic acid without catalyst, 200 molecules of carbonic acid are formed per hour. However, if the enzyme carbonic anhydrase is present, 600,000 molecules are present per second and the rate of reaction increases by ten million times. Its reaction rate is one of the fastest of all the enzymes. ## High Rates of Chemical Conversions and Nature of Enzyme Action Enzymes help bring the substrates together in the proper orientation so that the reaction can occur. Figure 3 depicts how an enzyme works: ## Importance of Enzymes 1. They act as biocatalysts synthesized by living cells and take part in various biochemical reactions. 2. Due to their unique properties of specificity and flexibility, they act as dynamic catalysts and affect the reaction based on the conditions. 3. Cofactors act as co-substrates and bring about prosthetic groups, coenzymes and metal ions bind to the active sites of enzymes to maintain the shape of the site so that a stable enzyme-substrate complex is formed. 4. Inhibitors are used to study the properties of enzymes and are used in the form of drugs, antibiotics, or pesticides. ## Key Terms - Activation energy - Enzymes - Active site - Holoenzyme - Apoenzyme - Prosthetic group - Chemical kinetics - Coenzyme - Turn over number ## Multiple Choice Questions 1. Enzyme often have additional parts in their structures that are made up of molecules other than proteins. When this additional chemical part is an organic molecule, it is called * (a) cofactor. * (b) coenzyme. * (c) substrates. * (d) Both (a) and (b). 2. Enzyme inhibition caused by a substance resembling substrate molecule is * (a) competitive inhibition. * (b) non-competitive inhibition. * (c) feedback inhibition. * (d) allosteric inhibition. 3. Holoenzyme is produced by * (a) combined coenzyme and apoenzyme. * (b) only prosthetic group. * (c) only protein. * (d) only cofactor. 4. Feedback inhibition of enzyme is influenced by * (a) enzyme. * (b) external factors. * (c) end product. * (d) substrate. 5. Combination of apoenzyme and coenzyme produces * (a) prosthetic group. * (b) holoenzyme. * (c) enzyme-substrate complex. * (d) enzyme-product complex. 6. Blocking enzyme action through blocking it active sites is * (a) allosteric inhibition. * (b) feedback inhibition. * (c) competitive inhibition. * (d) non-competitive inhibition. ## Fill in the blanks 1. Combination of apoenzyme and coenzyme produces _______. 2. Cellulose eaten by grazing animals is broken down by _______. 3. Feedback Inhibition of enzyme is influenced by _______. 4. Enzyme amylase belongs to _______. ## 4.2 Classification and Nomenclature of Enzymes A substance that binds to a site other than the active site and influences the active site either negatively or positively is called a modulator or effector molecule. The effector molecules that enhance the enzymes activity are called allosteric activators. When it causes a decrease in enzyme activity, the effector molecule is called allosteric inhibitors. Feedback inhibition takes place by a product molecule which inhibits an early step in a reaction sequence in its own biosynthesis. This happens when a product accumulates beyond in optimal amount, it binds to the allosteric site of the enzyme catalysis its synthesis, thus altering the conformation of the enzyme such that it no longer binds to the substrate. Enzymes are commonly named by appending the suffix -ase to the name of the enzyme's substrate (urease catalyzes the hydrolysis of urea) or to the word describing the enzyme action (alcohol dehydrogenase catalyzes the oxidation of primary and secondary alcohols to their corresponding aldehydes and ketones by removing hydrogen). A number of enzymes are known today. In the beginning, there were no systematic rules for naming enzymes which resulted in using two different names for the same enzyme, thereby creating confusion. To avoid these problems, the International Union of Biochemistry and Molecular Biology (IUBMB) adopted the systematic functional classification and nomenclature of enzymes. On the basis of nature of the reactions that the enzymes catalyze, they are divided into 6 classes (Table 1). | Classification Type | Type of Reaction Catalyzed | Examples | |---|---|---| | Oxidoreductases/ dehydrogenases | Oxidation-reduction reactions | Nitrate reductase, succinate dehydrogenase | | Transferases | Transfer of functional groups, example, amino group from glutamate to pyruvate in alanine synthesis | CoA transferase, N-acetyl transferase | | Hydrolases | Hydrolysis reactions of ether, ester, C-C, C-X, peptide, glucosidic bonds | Amylase, lactase, maltase | | Lyases | Group elimination to form double bonds | Histidine decarboxylase, aldolase | |Isomerases | Isomerization to form optical, positional or geometrical isomers | Alanine racemase, tartrate epimerase | | Ligases | Bond formation of C-O, P-O, C-S, C-N coupled with ATP hydrolysis | Pyruvate carboxylase | Few examples of enzyme and their functions are: 1. Lactate dehydrogenase: It belongs to the class of dehydrogenase. It facilitates the interconversion between lactate and pyruvic acid with the reduction of nicotinamide adenine dinucleotide (NAD to NADH) which is essential for cellular respiration. 2. Nucleoside monophosphate kinase: It belongs to the class of transferase enzyme as it facilitates the transfer of the phosphoryl group from ATP to nucleoside monophosphate. It catalyzes the following chemical reaction: ``` ATP + Nucleoside phsopshate → ADP + Nucleoside diphosphate ``` This enzyme plays an important role in nucleotide metabolism and is used in the synthesis of deoxyribonucleotides, which are the building blocks of DNA.