Enzymes 1 PDF - Biochemistry Notes

ENZYMES: Part 1 Kirstin Rhys S. Pueblos, MSc. Department of Chemistry 1st sem 2022-2023 College of Science and Mathematics MSU-Iligan Institute of Technology  Outline: Part 1 structure & classification Enzyme thermodynamics – transition-state stabilization...

ENZYMES: Part 1 Kirstin Rhys S. Pueblos, MSc. Department of Chemistry 1st sem 2022-2023 College of Science and Mathematics MSU-Iligan Institute of Technology  Outline: Part 1 structure & classification Enzyme thermodynamics – transition-state stabilization -- complementarity & binding energy catalytic strategies – general acid-base catalysis – covalent catalysis – metal ion catalysis BIOCHEMISTRY I | Page 2  Outline : Part 2 (continued) Regulatory enzymes – allostery --Proenzymes – feedback inhibition --Protein modification enzyme inhibition – competitive inhibition --Noncompetitive inhibition enzyme kinetics – Michaelis-Menten equation – kinetic parameters (Km, Vmax) BIOCHEMISTRY I | Page 3  Enzymes are catalytic proteins that increase the rate of biological reactions within the cells of the body. As catalysts, enzymes lower the activation energy for a chemical reaction (Figure 1). Less energy is required to convert reactant molecules to products, which increases the rate of a biochemical reaction compared to the rate of the uncatalyzed reaction. For example, carbonic anhydrase, an enzyme in the blood converts large amounts of carbon dioxide and water to carbonic acid. In the presence of the enzyme, this reaction occurs 10 million times, as rapidly as it does in the absence of the enzyme. One molecule of carbonic anhydrase can hydrate about 1 million molecules of CO2 a second. BIOCHEMISTRY I | Page 4  Figure 1. BIOCHEMISTRY I | Page 5  Figure 2. The enzyme carbonic anhydrase lowers the activation energy needed for the reaction of CO2 and H2O. BIOCHEMISTRY I | Page 6 ABA, Oct. 2021 | Page 7  Enzymes Enzymes are proteins capable of catalyzing covalent bond cleavage & formation – there are a few RNA molecules that have enzymatic properties Enzymes endow cells with the capacity to exert kinetic control over thermodynamic potentiality – enzymes alter the rate of product formation, but not the free energy change (ϪG) of a process (the chemical equilibrium is not changed) Enzymes are the agents of metabolic function – collections of enzymes work together in anabolic and catabolic pathways BIOCHEMISTRY I | Page 8  Enzymes key attributes of enzymes – catalytic power — enormous acceleration of rate of chemical transformations – specificity — can selectively bind and process substrates over closely- related molecular species -- controlled by structure: the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield – reactivity conditions — functional in aqueous environments at low temperatures (as found in cells) BIOCHEMISTRY I | Page 9  Types of Enzymes 1. Simple proteins – consist only of a polypeptide chain; it is the tertiary protein structure of the simple enzyme that makes it biologically active. 2. Conjugated proteins - protein portion is inactive without a cofactor; the cofactor is the nonprotein portion of an enzyme, such as a metal ion that is necessary for enzyme activity; if the cofactor is an organic compound, usually a vitamin, it is called a coenzyme. BIOCHEMISTRY I | Page 10  Conjugated Proteins apoenzyme - protein portion of the enzyme, inactive holoenzyme - apoenzyme + cofactor, active enzyme cofactors - organic compounds derived from B vitamins (coenzymes) --inorganic metal ions BIOCHEMISTRY I | Page 11  Figure 2. (a) The apoenzyme is unable to bind to its substrate. (b) When the required cofactor, in this case a copper ion, Cu2+, is available, it binds to the apoenzyme. Now the active site takes on the correct configuration, the enzyme-substrate complex forms, and the reaction occurs. BIOCHEMISTRY I | Page 12  Coenzymes A coenzyme is required by some enzymes An organic molecule bound to the enzyme by weak interactions / Hydrogen bonds Most coenzymes carry electrons or small groups Many have modified vitamins in their structure BIOCHEMISTRY I | Page 13  Water soluble vitamins. BIOCHEMISTRY I | Page 14  Structure of water soluble vitamins. BIOCHEMISTRY I | Page 15  Table 2. Fat soluble vitamins. BIOCHEMISTRY I | Page 16  Structure of fat soluble vitamins. BIOCHEMISTRY I | Page 17  Nicotinamide Adenine Dinucleotide BIOCHEMISTRY I | Page 18  NAD+ to NADH Mechanism The nicotinamide part of NAD+ accepts a hydride ion (H plus two electrons) from the alcohol to be oxidized The alcohol loses a proton ( H+ ) to the solvent Oxidized form Reduced form BIOCHEMISTRY I | Page 19  Two Other Adenine Dinucleotide Coenzymes BIOCHEMISTRY I | Page 20  Cofactors: Cofactors are important for the chemically reactive enzymes Cofactors:small organic molecules or Inorganic ions Organic molecule cofactors: also called as co-enzymes or co-substrates Co-enzymes/co-substrates are derived from dietary vitamins Inorganic ion cofactors Typical metal ion cofactors - Zn2+, Mg2+, Mn2+ & Fe2+ Nonmetallic ion cofactor - Cl- Inorganic ion cofactors derived from dietary minerals BIOCHEMISTRY I | Page 21  Metal Ions Required by Some Enzymes BIOCHEMISTRY I | Page 22  Names and Classification of Enzymes Enzymes are most commonly named using a system that provides information on the FUNCTION rather than the STRUCTURE of the enzyme. Important aspects in naming enzymes: 1. The suffix –ase identifies a substrate as an enzyme. ex. urease, sucrase, lipase – enzyme designation The suffix -in is still found in digestive enzymes (trypsin, pepsin, rennin, papain) 2. The type of reaction catalyzed by the enzyme is often noted with a prefix. ex. oxidase = catalyzes oxidation rxn, hydrolase = catalyzes hydrolysis rxn 3. The identity of the substrate is often noted in addition to the type of reaction. ex. glucose oxidase, pyruvate carboxylase, succinate dehydrogenase 4. Sometimes, the substrate but not the reaction type is given. ex. urease, lactase BIOCHEMISTRY I | Page 23  International Classification of Enzymes BIOCHEMISTRY I | Page 24  1. Oxidoreductases = catalyze redox reactions a. oxidases - oxidation of substrate b. reductases - reduction of substrate c. dehydrogenase - introduction of a double bond by removal of hydrogen from substrate BIOCHEMISTRY I | Page 25  2. Transferases = catalyze transfer of functional group a. kinases – transfer phosphate group b. transaminase – transfer amino group BIOCHEMISTRY I Page 26  3. Hydrolases = catalyze addition of water to a bond a. protease – hydrolysis of peptide linkages in protein BIOCHEMISTRYI I BIOCHEMISTRY | Page 27  Hydrolases b. carbohydrase – hydrolysis of glycosidic bonds in carbohydrates BIOCHEMISTRY I | Page 28  Hydrolases c. lipases – hydrolysis of ester linkages in lipids d. nucleases – hydrolysis of sugar-phosphate ester bonds in nucleic acids e. phosphatases – hydrolysis of phosphate ester bonds BIOCHEMISTRY I | Page 29  4. Lyases = catalyze the addition of a group to a double bond or removal of a group to create a double bond a. dehydratases/hydratase – removal/addition of water from substrate BIOCHEMISTRY I | Page 30  Lyases b. decarboxylases – removal of carbon dioxide c. deaminases – removal of ammonia BIOCHEMISTRY I | Page 31  5. Isomerases = catalyzes the conversion of a substrate into another compound that is isomeric with it. a. racemases – conversion of D to L or vice versa BIOCHEMISTRY I | Page 32  Isomerases b. mutases – transfer of functional grp w/in a molecule c. epimerases – conversion of a sugar epimer into another BIOCHEMISTRY I | Page 33  6. Ligases = catalyze bonding of 2 substrate w/ ATP a. synthetases – formation of new bond w/ ATP BIOCHEMISTRY I | Page 34  Ligases b. carboxylases – formatin of new bond w/ ATP & CO2 BIOCHEMISTRY I | Page 35 BIOCHEMISTRY I | Page 36 | Page 37 The Effect of Enzymes on the Activation Energy of a Reaction An enzyme speeds a reaction by lowering the activation energy, changing the reaction pathway – This provides a lower energy route for conversion of substrate to product Every chemical reaction is characterized by an equilibrium constant, Keq, which is a reflection of the difference in energy between reactants, aA, and products, bB | Page 38  Diagram of Energy Difference Between Reactants and Products The uncatalyzed reaction has a large activation energy, Ea, seen at left In the catalyzed reaction, the activation energy has been lowered significantly increasing the rate of the reaction BIOCHEMISTRY I | Page 39  Enzyme-Substrate Complex These reversible reaction steps represent the steps in an enzyme catalyzed reaction The first step involves formation of an enzyme-substrate complex, E-S E-S* is the transition state E-P is the enzyme-product complex BIOCHEMISTRY I | Page 40  Enzyme-Substrate Complex Details The part of the enzyme combining with the substrate is the active site Active sites characteristics include: – Pockets or clefts in the surface of the enzyme R groups at active site are called catalytic groups – Shape of active site is complimentary to the shape of the substrate – The enzyme attracts and holds the substrate using weak noncovalent interactions – Conformation of the active site determines the specificity of the enzyme BIOCHEMISTRY I | Page 41  Lock and Key Enzyme Model In the lock-and-key model, the enzyme is assumed to be the lock and the substrate the key The enzyme and substrate are made to fit exactly This model fails to take into account proteins conformational changes to accommodate a substrate molecule BIOCHEMISTRY I | Page 42  Induced Fit Enzyme Model The induced-fit model of enzyme action assumes that the enzyme active site is more a flexible pocket whose conformation changes to accommodate the substrate molecule BIOCHEMISTRY I | Page 43  Specificity of the Enzyme – Substrate Complex Enzyme specificity is the ability of an enzyme to bind only one, or a very few, substrates and thus catalyze only a single reaction. BIOCHEMISTRY I | Page 44  Classes of Enzyme Specificity 1. Absolute: - catalyze a particular reaction for one particular substrate only and will have no catalytic effect on substrates which are closely related eg. urease acts only on urea H2N – CO – NH2 not for methylureaH2N – CO – NHCH3 nor biuret H2N – CO – NH – CO – NH2 2. Group: - enzyme catalyzes reaction involving any molecules with the same functional group - less selective and will act upon structurally similar molecules - eg, carboxypeptidase catalyzes the hydrolysis of C-terminal groups regardless of what amino acid BIOCHEMISTRY I | Page 45  Classes of Enzyme Specificity 3. Linkage: - enzyme catalyzes the formation or break up of only certain category or type of bond -the least specific; attack a particular kind of chemical bond, irrespective of the structural features in the vicinity of the linkage -eg, lipase catalyzes the hydrolysis of any kind of ester 4. Stereochemical: enzyme recognizes only one of two enantiomers - catalyze a specific stereochemical representation - eg. acid dehydrogenase catalyzes the oxidation of L-lactic acid (in muscle tissues) but not D-lactic acid (in microorganisms) BIOCHEMISTRY I | Page 46  Factors Affecting Enzyme Activity Temperature Figure 4.The effect of temperature on (a) uncatalyzed reactions and (b) enzyme-catalyzed reactions. BIOCHEMISTRY I | Page 47  The rate of the uncatalyzed reaction steadily increases with increasing temperature because more collisions occur with sufficient energy to overcome the energy barrier for the reaction. At the temperature optimum, the enzyme is functioning optimally and the rate of the reaction is maximal. Above the temperature optimum, increasing temperature begins to increase the vibrational energy of the bonds within the enzyme. Eventually, so many bonds and weak interactions are disrupted that the enzyme becomes denatured, and the reaction stops. BIOCHEMISTRY I | Page 48  pH Most enzymes are active and function best pH of 7. A plot of reaction rate versus pH for a typical enzyme is shown in Figure 5. The pH at which an enzyme functions optimally is called the pH optimum. Figure 5. Effect of pH on the rate of enzyme-catalyzed reactions. This enzyme functions most efficiently at pH 7. The rate of the reaction falls rapidly as the solution is made either more acidic or more basic. BIOCHEMISTRY I | Page 49  Substrate Concentration For a given amount of enzyme, a reaction will go faster as the amount of substrate increases. However, when all of the enzyme molecules are combined with substrate, a maximum reaction rate is reached. At this point, the enzyme molecules are saturated, and there can be no further increase in rate even if more substrate is added. Uncatalyzed Enzyme-Catalyzed Reaction Reaction BIOCHEMISTRY I | Page 50  Enzyme Concentration In general, the concentration of substrate in a reaction is much higher than that of the enzyme. As the enzyme concentration is increased, the reaction rate increases, because more substrate molecules can undergo reaction. BIOCHEMISTRY I | Page 51  Figure 6. (a) Increasing substrate concentration increases the rate of reaction until the enzyme molecules are saturated. (b) Increasing enzyme concentration increases the rate of the reaction. BIOCHEMISTRY I | Page 52  The Transition State and Product Formation How does the enzyme promote a faster chemical rxn? As the substrate interacts with the enzyme, its shape changes and this new shape is less energetically stable. This transition state has features of both substrate and product and falls apart to yield product, which dissociates from the enzyme. BIOCHEMISTRY I | Page 53  Possible Types of Transition State Changes 1. The enzyme might put “stress” on a bond facilitating bond breakage BIOCHEMISTRY I | Page 54  2. The enzyme might bring two reactants into close proximity and maintain proper orientation 3. The enzyme might modify the pH of the microenvironment, donating or accepting a H+ BIOCHEMISTRY I | Page 55  Enzyme reactions and energy changes Activation energy -determines forward reaction rate S P determines distribution of S and P at equilibrium BIOCHEMISTRY I | Page 56 BIOCHEMISTRY I | Page 57 BIOCHEMISTRY I | Page 58  Some Rate Enhancement Produced Enzymes BIOCHEMISTRY I | Page 59 BIOCHEMISTRY I | Page 60  Recall: noncovalent ‘weak’ molecular forces BIOCHEMISTRY I | Page 61  Binding energy in enzyme catalysis BIOCHEMISTRY I | Page 62  Three general enzymatic strategies: 1) BIOCHEMISTRY I | Page 63 BIOCHEMISTRY I | Page 64  2) BIOCHEMISTRY I | Page 65  3) BIOCHEMISTRY I | Page 66 BIOCHEMISTRY I | Page 67