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ENZYMES Dr Nithin Kumar U Assistant professor Biochemistry Yenepoya medical college Enzymes: Basics Rate of a Reaction Reversible & Irreversible Reactions Reaction Equilibrium Catalysis Background: A reaction r1 A+B C+D Reaction rate: r1 α [A] [B] Therefore, r1 = k1 [A] [B] Reversible reaction r1 (k1) A+B C+D r2(k2) forward reaction (left to right) backward reaction (right to left) state of equilibrium – chemical equilibrium. At equilibrium, r1 = r 2 Background r1 = k1 [A] [B] & r2 = k2 [C] [D] At equilibrium, k1 [A] [B] = k2 [C] [D] [A] [B] k2 = Keq(equilibrium constant) = [C] [D] k1 Law of mass action - for reversible reactions (Effects of [S]and [P] on the direction in net reaction proceeds) Background [A] [B] K eq= [C] [D] Freely reversible reaction, K eq value is 1 There is no energy change (ΔG = 0); Irreversible reaction K eq value very high (endergonic reaction; ΔG = +ve ) or Negligible(exergonic reaction; ΔG =–ve). Catalysis Catalyst increases the rate of a chemical reaction , remains unchanged chemically at the end of the reaction Phenomenon is CATALYSIS neither cause chemical reactions to take place nor change the equilibrium constant of chemical reactions Catalysis Catalyze forward & backward reactions equally Only catalyze reaction in thermodynamically allowed direction Catalysts accelerate chemical reactions by lowering the activation energy or energy barrier Effect of catalyst on activation energy of a reaction Enzymes: Definition Biocatalysts synthesized by living tissues which increase the rate of reaction without getting consumed in the process Enzymes: Introduction Biocatalysts Neither consumed / permanently altered Colloidal organic compounds – proteins Formed by living organisms High specificity for their substrates and reaction types Enzymes: Introduction No formation of unnecessary by-products Function in dilute aqueous solutions under mild conditions of temperature and pH Physiological regulation Several enzymes can work together in a specific order creating - metabolic pathways Enzymes: Medical importance Accelerate 106 to 1012 times Regulatory enzymes sense metabolic signals Inherited genetic disorders(Phenylketonuria) Inhibitors of enzymes can be used as drug Ex: Lovastatin for HMG CoA reductase Clinical enzymology Enzymes: History En-zyme = in yeast In 1850s Louis Pasteur – “ferments” – fermentation of sugar into alcohol by yeast Urease – first enzyme to be isolated in crystalline form in 1926 Ribozymes ( made up of RNA) Active site Size of enzymes >substrates “small region at which the substrate binds and catalysis take place” Imparts efficiency: -Local concentration -shields substrate from solvent Situated in a pocket or cleft of the enzyme The active site contains -substrate binding site (substrate) -catalytic site (reaction) Active site Catalytic sites of enzymes contain sites for binding cofactors or coenzymes exists d/t tertiary structure of protein loss of native enzyme structure derangement of active site loss of function For catalysis to take place substrate/s should bind to the substrate binding site reversibly by weak non-covalent bonds (Hydrogen bond, Van der walls force, hydrophobic interactions) Active site Not rigid in structure and shape Flexible to promote the effective binding of substrate to the enzyme Responsible for substrate specificity If an enzyme is denatured or dissociated into subunits catalytic activity is lost Enzymes acts within the moderate pH and temperature Active site In active site 3–4 amino acids directly involved in catalysis- catalytic residues Amino acids far away in the primary structure contribute to the formation of active site amino acids at the active sites – *serine *aspartate *histidine *cysteine *Lysine *arginine *glutamate *tyrosine Active site Enzymes and Enzyme Catalyzed Reactions Substrate: Reactant/s on which the enzymes act to catalyze the reaction Enzymes are much larger than the substrates they act on Holoenzyme, Apoenzyme Some enzymes contain a non prosthetic group other than component protein protein Holoenzyme=Apoenzyme+cofactor (protein) (non protein) Cofactors Non-protein factors required for catalysis Bind to the catalytic site organic or inorganic Organic cofactors -prosthetic groups -- tightly bound -coenzymes – released from active site during reaction Inorganic cofactors – activators Cofactors coenzymes organic cofactors inorganic Prosthetic group activators Exceptions -- FMN ,FAD, and biotin are tightly bound to enzymes, but called as coenzymes Prosthetic group tightly bound to enzyme by covalent bond cannot be separated from enzyme by Dialysis Ex -Biotin in carboxylases Apoenzyme + Prosthetic group = Holoenzyme (active enzyme) Coenzymes derivatives of vitamins Bound reversibly by weak non-covalent bonds to active site and released during the reaction separated easily from enzyme by dialysis Affinity for the enzyme is similar to substrate chemically changed by catalysis considered as co-substrate Function: carriers of various groups Coenzymes Hydrolases (class 3) - not require coenzymes IUB classes: I,II,V and VI need coenzymes Coenzyme form Vitamin (derived from) Group transferred Thiamine pyrophosphate (TPP) Thiamine(Vit B1) NAD+ Niacin(Vit B2) FAD and FMN Riboflavin (Vit B3) Hydroxy ethyl Hydrogen/electron Hydrogen/electron Pyridoxal phosphate(PLP) Coenzyme A (CoA) Biotin Folate coenzymes Adenosine tri phosphate(ATP) Amino group Acyl group CO2 One carbon group Phosphate Pyridoxine(VitB6) Pantothenic acid Biotin Folic acid ------- Inorganic Cofactor/Activators Metals 2 types - Metal activated enzyme - Metallo enzymes metal activated enzymes metal is not tightly bound by the enzyme Ex *ATPase (Mg2+) *Enolase(Mg2+) *Chloride (Cl-) -salivary amylase *Ca2+ and Pancreatic lipase Inorganic Cofactor/Activators Metallo enzymes Metals are tightly bound with enzymes Ex - *alcohol dehydrogenase (zinc) *carbonic anhydrase (zinc) *DNA Polymerase (zinc) *Xanthine oxidase(molybdenum) *Catalase, peroxidase(Iron) *Cytochrome oxidase(iron)/(copper) *Hexokinase, pyruvate kinase(Mg2+) *Glutathione peroxidase( selenium) Mechanism of enzyme action Enzymes act by binding substrates & lowering activation energy (energy needed for reactants to undergo reaction) Higher the activation energy, lower the rate Transition state- Energy barrier has to be overcome Reaction coordinate diagram Catalyst for H2O2 decomposition Energy of activation (kcal / mol) None 18.0 Platinum 11.8 Catalase 4.2 Mechanism of enzyme action lower energy status of transition state is d/t – *Substrate strain *Proper orientation of substrates *Proximity of substrates *Change of electrostatic environment around the substrates Michaelis- Menten Theory Enzyme E combines with a single substrate S to form Enzyme-Substrate complex ES at the active site, which immediately dissociates to form free enzyme E and the product P S+ E ES E+P Models to explain mechanism of specificity and catalysis Formation of ES complex can be explained by 2 models: Fischer’s Lock and Key Model Koshland’s Induced Fit Model Fischer’s Lock and Key Model Fischer’s Lock and Key Model Theory active site of enzyme is pre-shaped & rigid Is complimentary to the substrate Fit exactly into one another like key in lock Explains only enzyme specificity Fails to explain the flexibility shown by allosteric enzymes Koshland’s Induced Fit Model Koshland’s Induced Fit Model Hand in glove Model -interaction of S & E induces a conformational change in E like glove when hand is introduced Model: *active site is not rigid *binding of substrate induces conformational changes in the enzyme – leads to precise orientation of the catalytic groups catalysis Explains both enzyme specificity and catalysis Nomenclature of enzymes Describe type of reaction & add suffix “-ase” Ex: Dehydrogenases proteases isomerases Modifiers: hormone sensitive lipase cysteine protease RNA polymerase III Nomenclature of enzymes International Union of Biochemists (IUB): unique name and 4 digit EC code number Enzyme name has 2 parts: -Names indicating substrate(s) & cofactor -Type of reaction catalyzed ( ends in ‘ase’) Additional information in parenthesis Classification of enzymes The International Union of Biochemistry and Molecular Biology (IUBMB) 6 major classes of enzymes Mnemonic: OTHLIL Oh Thank Heaven Learning Is Lively Classification of enzymes 1. 2. 3. 4. Oxidoreductases oxidation-reduction Transferases transfer of group of atoms Hydrolases hydrolysis Lyases cleavage of bonds without hydrolysis 5. Isomerases rearrangement of atoms 6. Ligases joining of molecules (using ATP) Oxidoreductases Catalyzes oxidation of one substrate with simultaneous reduction of another substrate or coenzyme Alcohol dehydrogenase Alcohol NAD+ Aldehyde NADH +H+ Malate dehydrogenase Malate NAD+ Oxaloacetate NADH +H+ Oxidoreductases AH2 + B A + BH2 Transferases These enzymes catalyze transfer of a group other than H such as- amino, phosphoryl, methyl, from one substrate to another A- X+ B A + B- X Transferases Transfer groups from one substrate to another Ex Hexokinase Hexose ATP Hexose-6-phosphate ADP Alanine transaminase Pyruvate Glutamate Alanine α-ketoglutarate Hydrolases These enzymes catalyze cleavage of a molecule by addition of water (hydrolysis) cleaves ester, peptide, glycosidic bonds A–B + H2O A-OH + B–H Hydrolases Ex Lactase Sucrase Lactase Lactose + H2O Glucose + Galactose Sucrase Sucrose + H2O Glucose + Fructose Lyases break bonds by other than hydrolysis Fructose 1, 6 bis phosphate Aldolase Dihydroxyacetone phosphate Glyceraldehyde-3phosphate Fumarase Malate Fumerate + H2O Isomerases These enzymes substrates. produce isomers of Include racemases, epimerases and cis-trans isomerases. A A' Isomerases Phosphohexose isomerase Glucose 6-P Fructose P Phosphotriose isomerase Glyceraldehyde 3-P Dihydroxyacetone 3-P Ligases These enzymes catalyze synthetic reactions two molecules joined by covalent bond at the expenses of ‘high energy phosphate bond’ of, usually ATP hydrolysis of ATP Synthetases A + B C ATP ADP + Pi Ligases Pyruvate carboxylase Pyruvate + CO2 ATP Biotin Oxaloacetate ADP + Pi Acetyl CoA carboxylase Acetyl CoA + CO2 ATP Malonyl CoA Biotin ADP + Pi Hexokinase ATP + D – Hexose ADP + Hexose 6 -P E.C.2.7.1.1 ATP:D-Hexose 6-phosphotransferase Class 2: Transferases Subclass 7: transfer of a phosphoryl group Sub-subclass 1: alcohol is phosphoryl acceptor 1: the alcohol phosphorylated is hexose Malic enzyme L-malate + NAD+ NADH + H+ + Pyruvate +CO2 EC.1.1.1.37 L-malate:NAD+oxidoreductase(decarboxylating) creatine kinase Creatine + ATP → Creatine-P + ADP EC 2.7.3.2 ATP: creatine phosphotransferase Class 2: Transferases Subclass 7: phosphotransferases Sub subclass 3: Phosphotransferases with a nitrogenous group as acceptor 2: designates creatine kinase Synthases They do not belong to ligases Eg: -Glycogen synthase (class II) -ALA synthase (class II) -ATP synthase (class III) Methods of enzyme assays Endpoint method readings are taken at the end of reaction Kinetic method readings are taken at different time intervals Enzyme units The rate of reaction catalyzed by the enzyme is proportionate to the quantity of enzyme present International unit (I.U) Katal Turnover number Enzyme units Katal The number of mol of substrate transformed per second per liter of sample. International unit One IU is amount of enzyme that converts 1µmol of substrate per minute per liter of sample (U/L) 1 I. U = 16.67 nKat 1 nKat = 0.06 U Enzyme units Turnover number The number of substrate molecules transformed per unit time by a single enzyme molecule Specific activity - a measure of purity The number of enzyme units present per milligram of protein Enzyme purification Salting out Gel filtration chromatography Ion exchange chromatography Electrophoresis Enzyme specificity Enzymes are reaction/substrate specific More specific than inorganic catalysts Specificity is because of d/t -complementary shape -charge characteristics of E & S Koshland’s induced fit model substrate specificity of enzymes explains Enzyme specificity : Types Absolute Specificity Certain enzymes act only on one substrate Ex: Glucose oxidase oxidize β D glucose only Glucokinase Glucose glucose-6-phosphate Glucokinase cannot act on galactose urease Urea ammonia +CO2 Enzyme specificity : Types Bond Specificity Act on substrate having specific bonds All proteolytic enzymes Ex: Trypsin & chymotrypsin- hydrolyze peptide bonds formed by carboxyl groups of Arg, Lys Glycosides on glycosidic bond of carbohydrate Lipases on ester links of lipids Enzyme specificity : Types Group Specificity Act on substrate having specific groups Act on a structurally related substances Ex: Hexokinase - several hexoses to form the respective hexose-6-phosphate Enzyme specificity : Types Stereo specificity Act on only one type of isomers (exceptionisomerase) Humans enzymes are specific for D– carbohydrates and L-aminoacids Ex: L amino acid oxidase D amino acid oxidase Enzyme kinetics Study of all the factors that affect the rates of enzyme catalyzed reactions The study of rate of enzyme catalyzed reactions & factors affecting these reaction rates Enzyme kinetics Enzyme concentration Substrate concentration Temperature pH Product concentration Presence of coenzymes, inhibitors activators or Importance of studying Enzyme kinetics understanding mechanism of enzyme action understanding inhibition mechanism of clinical diagnosis (measuring enzymes in disease conditions ) Understanding disease processes enzyme activity of Basics catalyst is required in much concentration than the substrates Similarly in an enzyme catalysed reaction [E] <<< [S] [E] nmoles/liter and [S] mmol/liter lower Effect of Enzyme concentration Initial velocity of enzyme-catalyzed reaction is directly proportional to enzyme concentration As enzyme concentration [E] increases velocity (V) of enzyme reaction also increases progressively straight line is obtained when result is plotted on a graph with V on y axis and [E] on x axis Graph: Effect of Enzyme concentration on velocity y Velocity (V) x Enzyme concentration Effect of Substrate Concentration As substrate concentration [S] is increased, velocity also increases correspondingly in the initial phases; but the curve flattens afterwards If velocity(V) of an enzyme reaction is measured at various [S] & result is plotted on a graph with V on y axis and [S] on x axis, a rectangular hyperbolic curve is obtained Graph: Effect of substrate concentration on velocity y Vmax------------------------------------------------------------C ½ Vmax------------- B Velocity A x Km Substrate concentration Effect of Substrate Concentration Vmax A: Substrate concentration is very low –Voα [S] B: Substrate concentration is more – Vo is not directly proportional to [S] – smaller ↑ C: Substrate concentration is high –Vo is independent of [S] – vanishingly smaller ↑ Vmax – Maximal velocity Graph approaches, but never reaches a plateau Enzyme is “saturated” with its substrate Michaelis-Menten Equation Describes the relationship of substrate concentration [S] to velocity (V) for enzyme catalyzed reactions. V = initial velocity Vmax = maximum velocity [S] = substrate concentration Km = Michaelis constant Km value (Michaelis constant) Vi Initial velocity The velocity measured when very little substrate has reacted Vmax Maximal velocity The maximum reaction rate attainable in presence of excess substrate Km value (Michaelis constant) The substrate concentration at half the maximal velocity (½ Vmax) in an enzyme catalyzed reaction When V =½ Vmax , Km= [S] Denotes that 50% of enzyme molecules are saturated with substrate molecules at that particular substrate concentration Km value Km value is characteristic feature of a particular enzyme for a specific substrate Constant for an enzyme - signature Ranges from 10-5 to 10-2 moles/liter Km value : Significance A measure of affinity of enzyme for substrate -low Km value indicates a strong affinity -high Km reflects a weak affinity Helps to know natural substrate of an enzyme having more than one substrates Helps to study of mechanism of enzyme inhibition Km values of isoenzymes are different for the same substrate Hexokinase and Glucokinase Hexokinase is present in all cells except hepatocytes and β-cells of pancreas Glucokinase is present in hepatocytes and βcells Hexokinase has low Km for glucose (0.05mmol/L) – ensures supply even at low blood glucose Glucokinase – high Km value (10 mmol /L) – removes glucose after a meal from portal vein Hexokinase and Glucokinase Effect of temperature on enzyme activity Velocity (V) of an enzyme-catalyzed reaction increases when temperature of the medium is increased, reaches a maximum and then falls A graph with V on y axis and temperature on x axis, a bell-shaped curve is obtained ↑Temp: -increases kinetic energy of molecules -increases collision frequency -lowers energy barrier Graph: Effect of temperature on enzyme activity Optimum temperature Velocity Low 37oC Temperature High Effect of temperature on enzyme activity Temperature at which the velocity is maximum is - Optimum temperature (37oc for most enzymes in humans) If temperature is very high (>55oC)- heat denaturation activity of the enzyme decreases Exception: Thermus acquaticus enzymes are stable and active even in 1000C Used in PCR Effect of temperature on enzyme activity Q10 (temperature coefficient) The factor by which the rate of reaction increases for every 100 C rise in temperature. Q10 = 2 Effect of pH on enzyme activity Velocity V of an enzyme-catalyzed reaction is measured at various pH values and plotted ,a bell-shaped curve is obtained Enzyme have an optimum pH on both sides of which the velocity will be drastically reduced pH changes will cause alteration in charged state of enzyme /substrate or both Graph: Effect of pH on enzyme activity Optimum pH Velocity pH Effect of pH on enzyme activity The pH at which the velocity is maximum is called the optimum pH Usually 6 – 8 for most human enzymes Exceptions: -Pepsin (1-2) -Alkaline phosphatase (9-10) -Acid phosphatase (4 - 5) At extreme pH, enzyme gets denatured activity is drastically reduced Product concentration An increase in [P] decreases velocity of the enzyme-catalyzed reaction