Enzyme s and Vitamins (stu copy) PDF

CHY2026: General Biochemistry UNIT 5: ENZYMESAND VITAMINS Nature of Enzymes ❖ They are proteins ❖ Biological catalysts…Unlike inorganic catalysts (a) enzymes do not last indefinitely (b) They are biological products ❖ Enzymes can be denatured and precipitated with salts, solvents and ot...

CHY2026: General Biochemistry UNIT 5: ENZYMESAND VITAMINS Nature of Enzymes ❖ They are proteins ❖ Biological catalysts…Unlike inorganic catalysts (a) enzymes do not last indefinitely (b) They are biological products ❖ Enzymes can be denatured and precipitated with salts, solvents and other reagents. They have molecular weights ranging from 10,000 to 2,000,000 ❖ Enzymes, with a few exceptions, are water-soluble globular proteins. Nature of Enzymes  They speed up biochemical reactions by lowering the activation energy necessary for a reaction to take place or speeding up the rate determining step of the reaction Characteristics of Enzymes 1. Catalytic Efficiency  Catalyst increase the rate of a chemical reaction but are not used up in the reaction  Enzymes speed up biochemical reactions under mild pH and temperatures  Enzymes can accomplish in seconds reactions that would take weeks or months under lab conditions e.g. CO2 + H2O H2CO3 Carbon dioxide is moved out of the body by combing with water to form carbonic acid. The reaction is catalyzed by carbonic anhydrase. The process is able to take place at a rate of 36 million molecules per minute 2. Specificity  Unlike catalysts, enzymes are specific in the type of reaction they catalyze and the particular substance that will be involved in the reaction  E.g. acids will catalyze the hydrolysis of any amide, dehydration of alcohol etc., however the enzyme urease will only hydrolyze the amide urea  This is absolute specificity  Some enzymes display relative specificity by catalyzing the reaction of structurally related substances e.g. lipase  Stereo chemical specific enzyme is able to distinguish between stereo isomers e.g. D-amino oxidase will not catalyze the reaction of L- amino acids Type Reaction type Example Absolute Catalyze one type of Urease catalyses only the reaction for a single hydrolysis of urea substrate Group Catalyze one type of Hexokinase adds a reaction for a similar phosphate group to substrates hexoses Linkage Catalyze one type of Chymotrypsin catalyzes reaction for a specific the hydrolysis of peptide type of bond bonds  Enzyme Specificity  All enzymes are chiral (non superimposable mirror images)  Medications are also chiral as well  Chirals compounds can be classified as D, L and R, S  The chiral pairs are referred to as enantiomer  D- and L- are the two shape possibilities in amino acids and we use L- in our bodies  The enzymes are specific to the substrates, example is: only L-lactate is changed to pyruvate  D-lactate is not affected 8 Enzyme Specificity  The properties of many drugs depends on their stereochemistry: HN HN O O O O NHCH3 NHCH3NH CH CH3NH 3 Cl Cl Cl Cl (S)-ketamine (R)-ketamine anaesthetic hallucinogen  Two enantiomers have exactly the same chemical properties except for their reaction with chiral non-racemic reagents  Many drugs are chiral and often must react with a chiral receptor or chiral enzyme to be effective  One enantiomer of a drug may effectively treat a disease whereas its mirror image may be ineffective or toxic  Enzyme Specificity  Enzymes are capable of distinguishing between stereoisomers: 3. Regulation  The catalytic behaviour of enzymes can be regulated  Even though the cell contains thousands of different molecules only some reaction will take place due to the enzyme present  The cell controls the rate of reaction an the amount of product formed by regulating the action of the enzymes  Enzymes ❖ Their reactions may be anabolic - involved in synthesis or catabolic – involved in breakdown ❖ The name of the enzyme always end with the suffix –ase ❖ Substrtes bind to Enzymes ❖ The area on the enzyme that binds to the substrate is called the active site ❖ In the active site there are hydrophobic pockets and ionic or polar groups for binding to the substrate  Nomenclature and Classification (a) Substrate Acted Upon by the Enzyme The substance upon which enzymes act is called the SUBSTRATE Enzymes can be named by adding the suffix –ase to the name of the substrate catalyzed e.g. carbohydrates – carbohydrases lipids – lipases maltose - maltase b) Type of Reaction Catalyzed Enzymes are highly specific to the reaction they catalyze The suffix –ase is added to the reaction e.g. hydrolysis – hydrolases isomerization – isomerases transamination - transaminases (c) Substance that is Synthesized Substrate acted upon and type of reaction catalyzed can determine the name of the enzyme e.g. dehydration of succinic acid – succinic dehydrogenase Other classifications include: (d) Chemical composition of the Enzyme N.B. The International Union of Biochemistry and Molecular Biology (IUBMB) takes into consideration the overall chemical reaction when naming enzymes  There is 7 major classes of enzymes 1. Oxidoreductase: e.g. Dehydrogenase, oxidoreductase  This group is comprised of enzymes which were once categorized dehydrogenases  They catalyze electron transfer reactions and the transfer of hydrogen and oxygen atoms e.g. 2. Transferases: e.g. Transaminase, kinase  Enzymes that catalyze the transfer of a group (other than hydrogen) between a pair of substrate , e.g. glucose + ATP → glucose-6-phosphate + ADP kinase 3. Hydrolases: e.g. Estrases, digestive enzymes  Enzymes catalyze the hydrolysis of their substrates by adding constituents of water across the bond they split. e.g. sucrose → glucose + fructose sucrase 4. Lyases: e.g. Decarboxylase, aldolases  Splitting of a molecule into smaller unit without the molecule undergoing hydrolysis e.g. 5. Isomerase: e.g. Isomerase, epimerase  Catalyze the interconversion of optical, geometric or position isomers by intramolecular rearangement of atoms or groups e.g. Glucose -6-phosphate → fructose – 6- phosphate phosphoglucoisomerase 6. Ligases or Synthetases: They catalyze the linking together of two compounds utilizing the energy made available due to simultaneous breaking of the pyrophosphate bond in ATP or a similar compound e.g. 7. Translocase - Catalyze the movement of ions or molecules across membranes or their separation within membranes Enzymes Cofactors ❖ These are non protein components of an enzyme that is essential to carrying out their functions (cofactors) ❖ The enzyme-cofactor complex is called a holoenzyme ❖ Enzyme portion without the cofactor is called an apoenzyme http://academic.pgcc.edu/~kroberts/Lecture/Chapter%205/05-04_Holoenzyme_L.jpg Enzymes Cofactors ❖ There are three groups of cofactors 1. Activators/ Inorganic ions - e.g. Salivary amylase activity is increased in the presence of chloride ions (Cl-) 2. Prosthetic groups – eg. Flavin adenine dinucleotide (FAD), haem and biotin 3. Coenzymes – eg. Adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD [oxidised form], NADH [reduced form]) Activators/ Inorganic ions Metal ion Function Enzyme cofactor Fe2+/Fe3+ Oxidation-reduction Catalase Oxidation-reduction Cytochrome oxidase Zn2+ Used with NAD+ Alcohol dehydrogenase Carbonic anhydrase Carboxypeptidase A Mg2+ Hydrolyses phosphate esters Glucose-6-phosphatase Mn2+ Removes electrons Arginase Ni2+ Hydrolyses amides Urease  Coenzymes  Coenzymes are organic molecules that are required by certain enzymes to carry out catalysis  They bind to the active site of the enzyme and participate in catalysis but are not considered substrates of the reaction  It is a part of acetyl coenzyme A, the substance formed from all foods as they pass through stage II of catabolism  Coenzymes often function as intermediate carriers of electrons, specific atoms or functional groups that are transferred in the overall reaction. An example of this would be the role of NAD in the transfer of electrons in certain coupled REDOX reactions Chemical Structure of Coenzyme A  Coenzyme Abbreviation Entity transferred nicotine adenine dinucelotide NAD - partly composed of niacin electron (hydrogen atom) nicotine adenine dinucelotide NADP -Partly composed of electron (hydrogen atom) phosphate niacin FAD - Partly composed of flavine adenine dinucelotide electron (hydrogen atom) riboflavin (vit. B2) coenzyme A CoA Acyl groups coenzymeQ CoQ electrons (hydrogen atom) thiamine pyrophosphate thiamine (vit. B1) aldehydes pyridoxal phosphate pyridoxine (vit B6) amino groups biotin biotin carbon dioxide carbamide coenzymes Cobalamin (vit. B12) alkyl groups  NADH and FADH2 are the major electron carriers in the oxidation of fuel molecules  Nicotinamide adenine dinucleotide (NAD+) is a major electron acceptor in the oxidation of fuel molecules. The reactive part of the NAD+ is the nicotinamide ring  In the oxidation of a substrate the nicotinamide ring NAD+ accepts a H+ and two electrons http://biology-forums.com/gallery/2056_04_05_12_9_36_12.jpeg  Flavin adenine dinucleotide (FAD) is another major electron carrier. The reactive part of FAD is the isoalloxazine ring  NADPH is the major electron donor in reductive biosynthesis. The extra phsophate group on the NADPH facilitates this  The reduced coenzymes (NADH and FADH2) are metabolized in the electron transport chain and oxidative phosphorylation process  They react with the oxygen we breathe in and are oxidized giving off energy used to make ATP from ADP 31  Active Site  The substrate molecules are much smaller than enzyme molecules  There exists sites on the enzyme on which a substrate will bind (active site)  Some common features of the active site includes: (a) The active occupies a relatively small portion of the enzyme molecule (b) The active site is a 3-D entity. It is made up of groups that come from different parts of the linear amino acid sequence. (c) The arrangement of atoms is the active site are well defined resulting in specificity of the enzyme. (d) The active site binds the substrate molecule by weak forces of attraction. (e) The active site are groves and crevices from which water is excluded. It is made up of amino acids. The side chain groups serve as catalytic groups in the active site  The enzyme-substrate complex is highly unstable and the complex decomposes to produce the end products of the reaction and to regenerate free enzyme.  The ES complex results in the release of energy  This energy raises the energy level of the substrate molecule, thus inducing an activated state  During this state the bonds in the substrate are susceptible to cleavage. Enzyme –Substrate Interaction ❖ The active site and the substrate have complementary shape enabling them to bind together (like pieces of a jig saw puzzle) ❖ The interaction is not rigid as would occur between a lock and a key (lock and key hypothesis – Fischer) http://hsc.csu.edu.au/biology/core/balance/9_2_1/image1.jpg  Enzyme –Substrate Interaction ❖ The enzyme has an internal flexibility and is able to undergo conformational changes, adjusting to the shape of the substrate (induced fit hypothesis – Koshland) http://upload.wikimedia.org/wikipedia/commons/thumb/2/24/Induced_fit_diagram.svg/450px- Induced_fit_diagram.svg.png Enzyme–Substrate Interaction  In the induced-fit model of enzyme action:  The active site is flexible, not rigid  The shapes of the enzyme, active site, and substrate adjust to maximum the fit, which improves catalysis  There is a greater range of substrate specificity Enzymes ❖ Enzymes are specific for a particular substrate ❖ When an enzyme-substrate is formed it is activated to form products ❖ The products formed cannot fit into the active site and escapes leaving the active site of the enzyme free N.B. When an enzyme is denatured, the active site no longer exists Enzyme Kinetics Enzyme Activity  This is the general catalytic ability of an enzyme to increase the rate of reaction. This rate is called the turn over number  Turn over number is the number of molecules of substrate acted on by one molecule of enzyme per minute Mechanism of Action  A simple enzymatic reaction  Michaelis-Menten Hypothesis  Leonor Michaelis and Maud Menten, while studying the hydrolysis of sucrose catalysed by invertase proposed this theory based on the following assumptions 1. Only a single substrate and a single product are involved 2. The process proceeds to completion 3. The concentration of the substrate is much greater than that of the enzyme 4. An intermediate enzyme-substrate complex is formed 5. The rate of decomposition of the substrate is proportional to the concentration of the enzyme substrate complex  Lets define the number of moles of product (P) formed per time as V. The variable, V, is also referred to as the rate of catalysis of an enzyme. For different enzymes, V varies with the concentration of the substrate, S  At low S, V is linearly proportional to S, but when S is high relative to the amount of total enzyme, V is independent of S  Michaelis-Menten Equation:  When [S] = Km, then V = Vmax/2. Thus Km is equal to the substrate concentration at which the reaction rate is half the maximal value  The reciprocal of the Michaelis-Menten Equation = Lineweaver- Burke equation  Vmax (maximum reaction rate) represents the turnover number of an enzyme V0 = initial velocity (reaction rate) [S] = concentration of substrate  Km = amount of substrate needed for enzyme to obtain half of its maximum rate of reaction (Michaelis-Menten constant)  Low Km = high affinity for substrate  High Km = low affinity for substrate  Ka = association constant of the enzyme-substrate complex  Low Ka = low affinity for substrate (enzyme-substrate complex is less stable)  High Ka = high affinity for substrate (enzyme-substrate complex is more stable)  Kd = dissociation constant of the enzyme-substrate complex  Low Kd = high affinity for substrate (enzyme-substrate complex is more stable)  High Kd = low affinity for substrate (enzyme-substrate complex is less stable)  kcat = number of substrate molecules each enzyme converts to product per unit time (turnover number)  Low kcat = enzyme-substrate complex converts less of substrate it binds into product  High kcat = enzyme-substrate complex converts more of substrate it binds into product  Factors Affecting Enzyme Kinetics ❖ The activity of an enzyme is affected by its environmental conditions ❖ Changing these alter the rate of reaction caused by the enzyme ❖ In nature, organisms adjust the conditions of their enzymes to produce an Optimum rate of reaction, where necessary, or they may have enzymes which are adapted to function well in extreme conditions where they live  Factors affecting Enzyme Kinetics: Effect of Temperature ❖ Little activity at low temperature ❖ Rate increases with temperature ❖ Enzymes are most active at optimum temperatures (usually 37°C in humans) ❖ Increasing temperature increases the Kinetic Energy that molecules possess ❖ Since enzymes catalyse reactions involves enzymes randomly colliding with Substrate molecules, increasing temperature increases the rate of reaction, forming more products ❖ However, increasing temperature also increases the movement of enzyme molecules. This puts strain on the bonds that hold them together ❖ As temperature increases, more bonds, especially the Hydrogen and Ionic bonds, will break as a result of this strain ❖ Breaking bonds within the enzyme will cause the active site to change shape ❖ This change in shape means that the active site is less complementary to the shape of the substrate, so that it is less likely to catalyse the reaction. Eventually, the enzyme will become denatured and will no longer function Factors affecting Enzyme Kinetics: Effect of pH ❖ Enzyme works best within a narrow pH range ❖ Each enzyme works best at particular pH, known as its optimum pH level ❖ At extreme pH levels, enzymes lose their shape and function and become denatured ❖ H+ and OH- ions are charged and therefore interfere with Hydrogen and Ionic bonds that hold the enzyme, since they will be attracted or repelled by the charges created by the bonds. This interference causes a change in shape of the enzyme, and importantly, its active site ❖ Any change in pH above or below the optimum will quickly cause a decrease in the rate of reaction, since more of the enzyme molecules will have active sites whose shapes are not, or at least less, complementary to the shape of their substrate ❖ Small changes in pH above or below the optimum do not cause a permanent change to the enzyme, since the bonds can be reformed. However, extreme changes in pH can cause enzymes to denature and permanently lose their function Optimum pH Levels  Most enzymes of the body have an optimum pH of about 7.4  In certain organs, enzymes operate at lower and higher optimum pH values Enzyme Location Substrate Optimum pH Pepsin Stomach Peptide bonds 2 Urease Liver Urea 5 Sucrase Small intestine Sucrose 6.2 Pancreatic amylase Pancreas Amylase 7 Trypsin Small intestine Peptide bonds 8 Arginase Liver Arginine 9.7 Factors Affecting Enzyme Kinetics: Effect of concentration ❖ Changing the Enzyme and Substrate concentrations affect the rate of reaction of an enzyme catalysed reaction ❖ Controlling these factors in a cell is one way that an organism regulates its enzyme activity and so its metabolism ❖ Changing the concentration of a substance only affects the rate of reaction if it is the limiting factor: that is, it the factor that is stopping a reaction from preceding at a higher rate ❖ (a) Substrate Concentration ❖ Increasing substrate concentration increases the rate of reaction. This is because more substrate molecules will be colliding with enzyme molecules, so more product will be formed ❖ However, after a certain concentration, any increase will have no effect on the rate of reaction, since substrate concentration will no longer be the limiting factor ❖ The enzymes will effectively become saturated, and will be working at their maximum possible rate  (b) Enzyme Concentration ❖ Increasing Enzyme Concentration will increase the rate of reaction, as more enzymes will be colliding with substrate molecules ❖ However there will be no effect when the enzyme concentration is now the limiting factor Enzyme Inhibition Competitive Inhibitors  The inhibitor has a similar shape to the usual substrate for the enzyme, and competes with it for the active site  However, once it is attached to the active site, no reaction takes place Normally With inhibitor Competitive Inhibitors Competitive Inhibitors Competitive Inhibitors ❖ The complex does not react any further to form products - but its formation is still reversible. It breaks up again to form the enzyme and the inhibitor molecule ❖ That is if you increase the concentration of the substrate, then the substrate can out- compete the inhibitor, and so the normal reaction can take place at a reasonable rate ❖ Methanol poisoning occurs because methanol is oxidized to formaldehyde and formic acid which attack the optic nerve causing blindness. Ethanol is given as an antidote for methanol poisoning because ethanol competitively inhibits the oxidation of methanol Ethanol is oxidized in preference to methanol and consequently, the oxidation of methanol is slowed down so that the toxic by-products do not have a chance to accumulate Competitive Inhibitors ❖ The complex does not react any further to form products - but its formation is still reversible. It breaks up again to form the enzyme and the inhibitor molecule ❖ That is if you increase the concentration of the substrate, then the substrate can out- compete the inhibitor, and so the normal reaction can take place at a reasonable rate ❖ Methanol poisoning occurs because methanol is oxidized to formaldehyde and formic acid which attack the optic nerve causing blindness. Ethanol is given as an antidote for methanol poisoning because ethanol competitively inhibits the oxidation of methanol. Ethanol is oxidized in preference to methanol and consequently, the oxidation of methanol is slowed down so that the toxic by-products do not have a chance to accumulate. Competitive Inhibition  A competitive inhibitor competes with the substrate for the active site of the enzyme  This means that increasing the concentration of substrate will decrease the chance of inhibitor binding to the enzyme. Hence, if the substrate concentration is high enough the enzyme will reach the same Vmax as without the inhibitor  Because it will require a higher concentration of substrate to achieve this, the K m of the enzyme will also be higher http://www.ucl.ac.uk/~ucbcdab/enzass/images/compinh.png http://www.ucl.ac.uk/~ucbcdab/enzass/images/complb.png Non-competitive Inhibitors/ Allosteric Inhibitors ❖ A non-competitive inhibitor does not attach itself to the active site, but attaches somewhere else on the enzyme ❖ By attaching somewhere else it affects the structure of the enzyme (conformational change at the active site) and so the way the enzyme works ❖ Because there is no competition involved between the inhibitor and the substrate, increasing the substrate concentration will not help ❖ Some non-competitive inhibitors attach irreversibly to the enzyme, and therefore stop the enzyme from working permanently. Others attach reversibly Non-competitive Inhibitors  Non-competitive inhibitors ❖ Since many enzymes contain sulfurhydrl (-SH), alcohol, or acid groups as part of their active sites, any chemical which can react with them acts as an irreversible inhibitor ❖ Heavy metals such as Ag+, Hg2+, Pb2+ have strong affinities for -SH groups  Non Competitive Inhibition  Noncompetitive inhibitor binds to free enzyme (E) or the enzyme-substrate complex (ES) forming an unreactive enzyme-inhibitor complex (EI) or enzyme-inhibitor-substrate complex (EIS), respectively  Can bind whether or not the substrate has bound  The inhibition is not reduced or reversible with increasing [S] - must remove inhibitor  Decrease Vmax and have no effect on Km http://www.ucl.ac.uk/~ucbcdab/enzass/images/nci.png http://www.ucl.ac.uk/~ucbcdab/enzass/images/ncilb.png Competitive vs Non Competitive Inhibition Uncompetitive Inhibition  Bind to the enzyme-substrate complex (ES) only forming an unreactive enzyme- inhibitor-substrate complex (EIS)  Can only bind if the substrate has bound  Are reversible with decreasing [S]  Decrease Km and decrease Vmax  Uncompetitive Inhibition https://d2jmvrsizmvf4x.cloudfront.net/KzYd27yWTGn3SD2yILfQ_Uncompetitive_inhibitor_graph.jpg Mixed Inhibition  Bind to the enzyme at a site other than the active site  Bind to free enzyme (E) or the enzyme-substrate complex (ES) forming an unreactive enzyme-inhibitor complex (EI) or enzyme-inhibitor-substrate complex (EIS), respectively  Can bind whether or not the substrate has bound  Produce a conformational change in the enzyme - binding of the inhibitor reduces the substrate’s affinity for the active site  Are reduced, but not reversible with increasing [S]  Increase or decrease Km and decrease Vmax Noncompetitive inhibition is sometimes considered a special case of mixed inhibition. Noncompetitive inhibitors have the same affinity for free enzyme (E) and the enzyme-substrate complex (ES) whereas mixed inhibitors tend to have a higher affinity for either free enzyme (E) or the enzyme-substrate complex (ES). Mixed Inhibition Enzyme Inhibition  Ki = binding affinity of the inhibitor  Ki > 1 - inhibitor has higher affinity for enzyme than substrate  Ki < 1 - inhibitor has lower affinity for enzyme than substrate  IC50 = half-maximal inhibitory concentration  Tells how much of a drug is needed to inhibit a biological process by 50% Effect of Enzyme Inhibition Inhibitor Binds Reversible? Effect Competitive Free enzyme (E) Yes with ↑[S] Increases Km Uncompetitive Enzyme-substrate Yes with ↓[S] Decreases Km and complex (ES) Vmax Mixed Free enzyme (E) Reduced with Increases or or enzyme- ↑[S] decreases Km and substrate complex decreases Vmax (ES) Noncompetitive Free enzyme (E) Yes with removal Decreases Vmax or enzyme- of inhibitor The Use of Inhibition in Drug Therapy  If the requirement is to increase the intracellular concentration of the substrate, then either a competitive or non-competitive inhibitor can be used since both will inhibit the utilisation of substrate, so that it accumulates  However, if the requirement is to decrease the intracellular concentration of the product, then a non competitive inhibitor must be used...As unused substrate accumulates it will compete with a competitive inhibitor, and the final result will be a more or less normal rate of formation of product  Increasing the concentration of substrate does not affect a non-competitive inhibitor Vitamins  Vitamins are organic micronutrients that the body cannot produce in amounts needed for good health ❖ Differences between enzymes and vitamins (a) All enzymes are proteinaceous (b) Vitamins are not synthesized by animal cells  Water soluble -Vitamins B and C  Soluble in aqueous solutions  Used as cofactors by many enzymes  Not stored in the body  Fat Soluble -Vitamins A, D, E and K  Soluble in lipids, but not in aqueous solutions.  Important in vision, bone formation, antioxidants, and blood clotting.  Stored in the body.  Antioxidants -Vitamins A, C and E