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CHAPTER 1: ENZYME KINETICS AND APPLICATIONS (Part 1a : Kinetics of Enzyme Catalyzed Reactions) ERT 317 Biochemical Engineering Sem 1, 2016/2017 [email protected] Role of Biochemical /Bioprocess Engineer • • • • exploit advances in biology to create new products design biochemical processes & operate plants develop energy resources Develop new, environmentally friendly, and safer processes to make the biochemical products that people depend on. • Work in research and development laboratories, creating polymeric materials with improved performance and durability. • Work in manufacturing, making vaccines and antibiotics. • Invent new ways to keep our food and water supplies safe. [email protected] Bioprocess Engineer’s Task – Minimize production of unwanted byproducts – Separate the good (product) from the bad (byproducts) – Recover the unused reactants – Maximize profit, minimize energy consumption – Minimize impact on the environment [email protected] OUTLINE • Introduction • Enzyme Structure • Enzyme Function [email protected] Enzymes • There are many chemical compounds in the living cell. • How they are manufactured and combined at sufficient reaction rates under relatively mild temperature and pressure? • How does the cell select exactly which reactants will be combined and which molecule will be decomposed? Catalysis by ENZYME [email protected] Enzymes •Enzymes are biological catalysts that are protein molecules in nature- react in mild condition •They are produced by living cells (animal, plant, and microorganism) and are absolutely essential as catalysts in biochemical reactions. •Almost every reaction in a cell requires the presence of a specific enzyme– related to its particular protein structure. •A major function of enzymes in a living system is to catalyze the making and breaking of chemical bonds. •Therefore, like any other catalysts, they increase the rate of reaction without themselves undergoing permanent chemical changes. [email protected] Over 2000 enzymes have been identified Often named by adding the - ‘ase’ to the name of substrate acted upon, or the reaction catalyzed such as urease, alcohol dehydrogenase The majority of cellular reactions are catalyzed by enzymes [email protected] Some protein enzyme required a non-protein group for their activity. Non protein group: Cofactors: metal ions, Mg, Zn, Mn, Fe. Coenzyme: complex organic molecule, NAD, FAD, CoA Vitamins Catalyze biochemical reactions breaking, forming and rearranging bonds. Catalytic function – very specific and effective (Specific because of conformational shape) Dictated by the enzyme active site. Some active sites allow for multiple substrates. [email protected] • Enzymes are catalysts – Catalyst: chemical that changes the rate of a reaction without being consumed – Recycled (used multiple times) • Enzymes reduce the activation energy of a reaction – Amount of energy that must be added to get a reaction to proceed [email protected] Catalysts • A catalyst is unaltered during the course of a reaction and functions in both the forward and reverse directions. • In a chemical reaction, a catalyst increases the rate at which the reaction reaches equilibrium. • For a reaction to proceed from starting material to product, the chemical transformations of bond-making and bond-breaking require a minimal threshold amount of energy, termed activation energy. • Generally, a catalyst serves to lower the activation energy of a particular reaction. [email protected] Enzyme Function Enzymes lower the activation energy of reaction catalyzed ( They do this by binding to the substrate of the reaction, and forming an enzyme-substrate (ES) complex) Substrate binds to a specific site on the enzyme called the active site Multi-substrate reactions possible ‘Lock and key’ model [email protected] The activation energy for the decomposition of hydrogen peroxide varies depending on the type of catalysis. Type of catalysis Activation energy Uncatalyzed reaction at 20°C 18 kcal/mol Enzymatically catalyzed (catalase) 7 kcal/mol Chemically catalysed (by collodial platinum) 13 kcal/mol Enzyme lower the activation energy of the reaction by binding the substrate and forming an enzymes-substrate complex. [email protected] Comparison of activation energies in the uncatalyzed and catalyzed decompositions of ozone. [email protected] Important Terms To Be Remember! • active site - a region of an enzyme comprised of different amino acids where catalysis occurs or a small portion of the surface of an enzyme which a specific chemical reaction is catalyzed • substrate - the molecule being utilized and/or modified by a particular enzyme at its active site • co-factor - organic or inorganic molecules that are required by some enzymes for activity. These include Mg2+, Fe2+, Zn2+ and larger molecules termed co-enzymes like nicotinamide adenine dinucleotide (NAD+), coenzyme A, and many vitamins. [email protected] Types of Enzymes • holoenzyme - a complete, catalytically active enzyme including all co-factors OR an enzyme containing a non protein group • apoenzyme - the protein portion of a holoenzyme minus the co-factors OR the protein part of holoenzyme • (holoenzyme = apoenzyme+cofactor) • isozyme - (or iso-enzyme) an enzyme that performs the same or similar function of another enzyme that occur in several different molecular forms. [email protected] Nomenclature of enzyme Originally enzymes were given non descriptive names such as: rennin : curding of milk to start cheese-making processor pepsin : hydrolyzes proteins at acidic pH trypsin : hydrolyzes proteins at mild alkaline pH The nomenclature was later improved by adding the suffix -ase to the name of the substrate with which the enzyme functions, or to the reaction that is catalyzed, for example: [email protected] Alcohol dehydrogenase Glucose isomerase Glucose oxidase Lactic acid dehydrogenase [email protected] Enzyme reactions are different from chemical reactions, as follows: 1. An enzyme catalyst is highly specific, and catalyzes only one or a small number of chemical reactions. A great variety of enzymes exist, which can catalyze a very wide range of reactions. 2. The rate of an enzyme-catalyzed reaction is usually much faster than that of the same reaction when directed by nonbiological catalysts at mild reaction condition. 3. A small amount of enzyme is required to produce a desired effect. 4. Enzymes are comparatively sensitive or unstable molecules and require care in their use. [email protected] Enzymatic Reaction Principles • Biochemically, enzymes are highly specific for their substrates and generally catalyze only one type of reaction at rates thousands and millions times higher than non-enzymatic reactions. • Two main principles to remember about enzymes are : a) they act as CATALYSTS (they are not consumed in a reaction and are regenerated to their starting state) a) they INCREASE the rate of a reaction towards equilibrium (ratio of substrate to product), but they do not determine the overall equilibrium of a reaction. [email protected] Reaction Rates The rate of the reaction is effected by several factors including the concentration of substrate, temperature and pH. For most standard physiological enzymatic reactions, pH and temperature are in a defined environment (eg; pH 6.97.4, 37oC). This enzymatic rate relationship has been described mathematically by combining the equilibrium constant, the free energy change and first or second-order rate theory. Keq = e−∆Go/RT The net result for enzymatic reactions is that the lower the activation energy, the faster the reaction rate, and vice versa. [email protected] Specificity • Most synthetic catalyst are not specific i.e., they will catalyze similar reactions involving many different kinds of reactants. • While enzymes are specific. They will catalyze only one reaction involving only certain substances. [email protected] Binding Energy • The interaction between enzyme and its substrate is usually by weak forces. • In most cases, Van der Waals forces and hydrogen bonding are responsible for the formation of ES complexes. • The substrate binds to a specific site on the enzyme known as the active site. [email protected] Classification of Enzyme Enzymes fall into 6 classes based on function 1. Oxidoreductases: which are involved in oxidation, reduction, and electron or proton transfer reactions 2. Transferases : transfer of functional group 3. Hydrolases : which cleave various covalent bonds by hydrolysis 4. Lyases : catalyse reactions forming or breaking double bonds 5. Isomerases : catalyse isomerisation reactions 6. Ligases : join substituents together covalently. [email protected] [email protected] References • Shuler, M. L. and Kargi, F. (2014). Bioprocess Engineering: Basic Concept. 2nd Ed. Upper Saddle River, NJ: Prentice Hall PTR • Rao, D.G. (2010). Introduction to Biochemical engineering. 2nd Ed. Tata McGraw Hill Education Private Limited • Dutta, R. (2008). Fundamentals of Biochemical Engineering. New Delhi: Ane Books India • Katoh, S. and Yoshida, F. (2009). Biochemical Engineering: A Textbook for Engineers Chemists and Biologists. Weinheim: Wiley-VCH Verlag GmbH & Co • Nielsen, J., Villadsen, J. and Gunner L. (2011). Bioreaction Engineering Principles. 3rd Ed. New York: Springer Science+Bussiness Media [email protected] [email protected]