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CONTENTS 1. Introduction 2. Classification of enzymes 3. Properties: 3.1. Kinetics: Michaelis-Menton &Lineweaver-Burk Plots 3.2. Inhibition: Competitive, Uncompetitive & Non-competitive 3.3. Catalysis: Proximity, electrostatic, acid-base, covalent 4. Cofactors and coenzymes 5. Temperature & pH effects on reactions 6. Application in Industry and medicine INTRODUCTION How enzymes work ? 1. recognize very specific substrates, 2. perform specific chemical reactions high speeds. Enzymes make and break specific chemical bonds of the substrates at a faster rate without being consumed in the process. At the end of each catalytic cycle, the enzyme is free to begin again with a new substrate molecule HISTORY • As early as the late 1700s and early 1800s, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known. • However, the mechanism by which this occurred had not been identified. • In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms. • He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells.“ • In 1878 German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme, which comes from Greek ἔνζυμον "in leaven", to describe this process. • The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment used to refer to chemical activity produced by living organisms. • In 1897 Eduard Buchner began to study the ability of yeast extracts that lacked any living yeast cells to ferment sugar. • In a series of experiments at the University of Berlin, he found that the sugar was fermented even when there were no living yeast cells in the mixture. • He named the enzyme that brought about the fermentation of sucrose "zymase". • In 1907 he received the Nobel Prize in Chemistry for his biochemical research and his discovery of cell-free fermentation. • Following Buchner's example; enzymes are usually named according to the reaction they carry out. • Having shown that enzymes could function outside a living cell, the next step was to determine their biochemical nature. • Many early workers noted that enzymatic activity was associated with proteins, but several scientists (such as Nobel laureate Richard Willstätter) argued that proteins were merely carriers for the true enzymes and that proteins per se were incapable of catalysis. • However, in 1926, James B. Sumner showed that the enzyme urease was a pure protein and crystallized it; Sumner did likewise for the enzyme catalase in 1937. • The conclusion that pure proteins can be enzymes was definitively proved by Northrop and Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin. • This discovery that enzymes could be crystallized eventually allowed their structures to be solved by x-ray crystallography. • This was first done for lysozyme, an enzyme found in tears, saliva and egg whites that digests the coating of some bacteria; the structure was solved by a group led by David Chilton Phillips and published in 1965. • This high-resolution structure of lysozyme marked the beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail. PROPERTIES • Enzymes are generally globular proteins, some are simple proteins and others are conjugated proteins. • Enzymes catalyze reactions without bring destroyed or chemically changed. • Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. • Example: maltase only catalyses the hydrolysis of maltose and lipase catalyses the hydrolysis of oil. Properties of enzymes • A catalyst enhances the rate of reaction but is not permanently altered. • Catalysts work by decreasing the activation energy for a reaction by changing its pathway. • The structure of the active site of the enzyme (shape and charge distribution) is used to optimally orient the substrate for reaction. • The energy of the enzyme-substrate complex is then closer to the Transition Stage (Fig 6.1, p.162 of text) • An enzyme lowers the activation energy but it does not change the standard free energy change (DG) for the reaction nor the Keq. • Most enzymes are temperature/pH sensitive and will not work outside their normal temperature/pH range because the enzyme is denatured. LOCK AND KEY model • The correct substrate fits into the active site of the enzyme like a key into a lock. Only the right key fits. • The active site is a specialized region of the protein where the enzyme interacts with the substrate. Lock and Key Induced-fit Hypothesis CLASSIFICATION OF ENZYMES 1. Oxidoreductases catalyze redox reactions. Eg. Reductases or peroxidases 2. Transferases transfer a group from one molecule to another. Eg. Transaminases, transcarboxylases 3. Hydrolases cleave bonds by adding water. Eg. Phosphatases or peptidases • 4. Lyases catalyze removal of groups to form double bonds or the reverse. Eg. decarboxylasaes or synthases • 5. Isomerases catalyze intramolecular rearrangements. Eg. epimerases or mutases • 6. Ligases bond two molecules together. Many are called synthetases. Eg. carboxylases ENZYME KINETICS • Enzyme kinetics is the quantitative study of enzyme catalysis. • Kinetic studies measure reaction rates and the affinity of enzymes for substrates and inhibitors. • Kinetics provides insight into reaction mechanisms. Km is a constant that is characteristic of the enzyme and the substrate . The lower the value of Km, the greater the affinity of the enzyme for ES complex formation. When [S] << Km the velocity will be given by v = (Vmax/Km)[S]. The velocity depends linearly on [S]. Doubling [S] doubles the rate. When [S] >> Km, The equation reduces to v = Vmax, the velocity approaches Vmax , and the dependence of velocity on substrate concentration approaches a horizontal line If [S] = Km, the velocity will be one-half of Vmax. When there is no substrate present ([S] = 0), there is no velocity COMPETITIVE INHIBITORS Competitive inhibitors bind reversibly to free enzyme, not the ES complex, to form an enzyme-inhibitor (El) complex. At high substrate concentration, the effect of a competitive inhibitor can be overcome Lowering the k1, thus increasing the Km UNCOMPETITIVE INHIBITORS In uncompetitive inhibition, the inhibitor binds only to the enzyme-substrate complex, and not the free enzyme: NONCOMPETITIVE INHIBITORS In some enzyme-catalyzed reactions an inhibitor can bind to both the enzyme and the enzyme-substrate complex: A noncompetitive inhibitor binds at a site other than the active site of the enzyme and decreases its catalytic rate by causing a conformational change in the three-dimensional shape of the enzyme. The effect of a noncompetitive inhibitor cannot be overcome at high substrate concentrations. Effect : No change in Km IRREVERSIBLE INHIBITION • irreversible inhibitors form a reversible non-covalent complex with the enzyme (EI or ESI) and this then reacts to produce the covalently modified EI* called inactivation rate. • EI may compete with ES, binding of irreversible inhibitors can be prevented by competition either with substrate or with a second, reversible inhibitor Catalysis - Kinetic study is not enough to study how enzyme catalyze biochemical reactions. - Need other way : catalysis. - Factors contribute to enzyme catalysis : - proximity and strain effects electrostatics effects acid-base catalysis covalent catalysis Factors influencing catalysis (1) Proximity and Strain Effects • Substrate must closely approach the catalytic site with proper orientation. • The more tightly the active site can bind the substrate while it is in its transition state, the greater the rate of the reaction. Factors influence catalysis……. (2) Electrostatic Effects • the strength of electrostatic interactions is related to the capacity of surrounding solvent molecules to reduce the attractive forces between chemical groups. • the dielectric constant near the active site is often low, this may influence the chemical reactivity of the substrate. • weak electrostatic interactions in both the active site and the substrate, are believed to contribute to the catalysis. Factors influence catalysis (3)Acid-Base catalysis • Chemical groups can often be made more reactive by adding or removing a proton. • Enzyme active sites contain side chain groups that act as proton donors or acceptors. (4)Covalent Catalysis In covalent catalysis, a transient covalent bond is formed between the enzyme and the substrate. Consider the hydrolysis of a bond between groups A and B: In the presence of a covalent catalyst (an enzyme with a nucleophilic group X:) the reaction becomes FACTORS AFFECTING ENZYME REACTIONS • • • • TEMPERATURE Ph ENZYME CONCENTRATION SUBSTRATE CONCENTRATION TEMPERATURE The higher the temperature, the higher the reaction rate. The rates of enzyme-catalyzed reactions also increase with increasing temperature. pH Changes in [H] ion can affect the ionization of active site groups. If a substrate contains an ionizable group, a change in pH may alter its capacity to bind to the active site. Changes in ionizable groups may change the tertiary structure of the enzyme. REGULATION- refer from textbook • IMPORTANT for: - to maintain an orderly state - consevation of energy - responsivenes to envirinmental change • ACCOMPLISHED by: - genetic control - covalent modification - allosteric regulation - compartment Cofactors & Coenzymes • Many enzymes require the presence of small, nonprotein units or cofactors to carry out their particular reaction. • Cofactors can be : – inorganic ions, exp: Fe2+ and Cu2+ and the alkali and alkaline earth metals (e.g., Na+, K+, Mg2+, and Ca2+). – a complex organic molecule called a coenzyme. Exp : • Nicotinamide adenine dinucleotide (NAD) • Nicotinamide adeninedinucleotide phosphate (NADP) • Flavin adenine dinucleotide (FAD) • flavin mononucleotide (FMN) Enzyme Application : In Industry Some examples : 1. In Textile industry : Amylase enzyme as a sizing agent 2. In Sugar industry : Amylase to reduce the starch content of sugar cane syrup 3. In energy related Ind : Lipase to speed up the esterification process of Biodiesel 4. In Beverages : Pectinases for juice clarification 5. In Detergent ind : amylase & protease to enhance the cleaning power of detergent 6. Enzyme cellulase breaks down cellulose to beta-glucose in the ruminating chambers of herbivores; it is also used in processing coffee and fermentation of biomass into biofuels; it is a fading agent in textile industry. Enzyme technology currently plays a role in two aspects of medical practice: diagnosis and therapy. A. Diagnosis : 1. Enzyme activity as an indicator of diseases. Exp : blood levels of acid phosphatase (enzyme) for diagnosing prostatic carcinoma (a urinary tract tumor that occurs in males). 2. Enzymes as reagents. Exp : enzyme urate oxidase is used to measure blood levels of uric acid (which is usually high in patients with gout. ) B. Therapeutic The use of enzymes in medical therapy has been limited because when administered to patients, enzymes are often rapidly inactivated or degraded. Exp : The enzyme asparaginase is used to treat of cancer. It catalyzes the conversion of Asparagine to Aspartate .Infusing asparaginase reduces the blood's concentration of asparagine and often causes tumor regression. Antioxidant co-enzymes Antioxidant co-enzymes: – glutathione peroxidase – superoxide dismutase (SOD), works with catalase, scavenge and neutralize cell-damaging free radicals by turning them into stable oxygen and H2O2, and then into oxygen and water. Fibrinolysis A process that dissolves blood clots • Enzymes – Trypsin – chymotrypsin • One use of enzymes, is in the case of thrombosis, this is when blot clots form in damaged blood vessels, if these clots are carried to an small artery and may become blocked a heart attack or stroke can be caused. Sports injuries • Sports injuries are often treated with protease enzymes because of their ability to reduce inflammation and speed the healing of bruises, swelling, and other injuries • A study conducted by Dr M.W. Kliene and his coworkers at the Sports Medicine Investigation Center in Grunwald, Germany researched the effectiveness of proteolytic enzymes on 100 athletes. • The results favored the enzyme treated subjects. Seventy-six percent evaluated the success rate as ‘good’ with protease enzymes. Surgery Several studies showed that when enzymes were taken before surgery, the swelling from the injury left around seven days quicker, on average, than those taking a placebo; post surgery recovery was also much more rapid. Digestive aid • A concentrated form of pancreatic amylase was available as Diastase Vera. • Amylase obtained from malt—produced from the action of Aspergillus oryzae on sterilized rice hulls or on wheat bran—was also marketed, • Diastase was mainly indicated for use by people who had overindulged in starchy food such as pasta Summary 1. Enzymes are biological catalysts which enhance reaction rates. Enzymes are specific to the types of reactions they catalyze. 2. There are six major enzyme categories: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. 3. Enzyme kinetics is the quantitative study of enzyme catalysis. Ref : MichaelisMenten, Km and Vmax 4. The most common types of reversible inhibition are competitive, uncompetitive, and noncompetitive. 5. Allosteric enzymes are enzymes containing multisubunit proteins. The kinetic properties of allosteric enzymes are not explained by the Michaelis-Menten model. 6. Several factors contribute to enzyme catalysis: proximity and strain effects, electrostatic effects, acid-base catalysis, and covalent catalysis. 7. Enzymes are sensitive to environmental factors such as temperature and pH. Each enzyme has an optimum temperature and an optimum pH.