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Enzymes BIOCATALYSTS VS

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Enzymes BIOCATALYSTS VS ENZYMESENZYMOLOGY BIOCATALYSTS 2009/2010 INGRID ŽITŇANOVÁ HISTORY 17th -18th century – digestion of meat caused by stomach secretions - conversion of starch to glucose by saliva Louis Pasteur 19th century – L. Pasteur – fermentation of sugar to alcohol by yeasts - vital force in yeasts required for this fermentation 1897 – Eduard Buchner – ability of yeast extracts that lacked any living yeast cells to ferment sugar – in 1907 – Nobel Prize for chemistry - discovery of cell free fermentation 1926 – James B. Sumner isolated the first enzyme – urease and prooved its protein character Eduard Buchner en zyme – in yeasts • Enzymes are biocatalysts ✓ Increase the rate of a reaction ✓ Not consumed by the reaction ✓ Enzymes are often very “specific” – promote only 1 particular reaction ✓ In the single cell - more than 3000 enzymes BIOCATALYSTS VS. INORGANIC CATALYSTS Enzymes (biocatalysts): 1) More efficient - higher reaction rate 2) Milder reaction conditions (20 - 40°C, pressure 0.1 MPa, pH = 7) 3) Higher specificity of the reaction 4) Ability to be regulated at different levels (inhibitors, activators) 5) They are non-toxic 6) Enzymes – organic compounds, chemic. catalysts – inorg. compounds Catalyst rate enhancement Inorganic catalysts 102 -104 fold Enzymes up to1020 fold How much is 1020 fold? Catalyst time for reaction With enzyme 1 second Without enzyme 3 x 1012 years ENZYME STRUCTURE • Enzymes are proteins (chain of amino acids) • Enzyme will twist and fold into a specific shape due to how the amino acids are attracted to each other • Enzyme shape attracts specific molecules - substrates – molecules that bind to the enzyme ➢ Enzymes DO NOT change the equilibrium constant of a reaction (accelerate the rate of the forward and reverse reactions equally) Carbonic anhydrase CO2 + H2O H2CO3 Carbonic anhydrase in tissues: CO + H O 2 2 H2CO3 Carbonic anhydrase in lungs: H2CO3 CO2 + H2O ENZYMES SIMPLE COMPLEX (HOLOENZYME) APOENZYMES COFACTOR (Nonprotein) (Protein) ORGANIC INORGANIC COENZYME PROSTHETIC GROUP (loosely bound) (tightly bound) HOLOENZYME Inorganic elements serving as enzyme cofactors Cytochrome oxidase Cytochrome oxidase, catalase, peroxidase Pyruvate kinase Hexokinase, pyruvate kinase Arginase, Dinitrogenase Urease Glutathione peroxidase Carboanhydrase, alcohol dehydrogenase Cu2+, Zn2+, Mn2+ Superoxide dismutase Cofactors Role of organic cofactors: transport of chem. groups from 1 reactant to another ◼ cofactors can serve several apoenzymes: NAD+ (nicotinamide adenine dinucleotide) - a cofactor for a great number of dehydrogenases: alcohol dehydrogenase, malate dehydrogenase lactate dehydrogenase reactions Classification of cofactors according to the type of a transferred molecule 1) Transfer of H atoms NAD+ (nicotine amide adenine dinucleotide) - transport of H- FAD (flavine adenine dinucleotide) – transport of 2H FMN (flavine mononucleotide), lipoic acid - transport of 2H 2) Transfer of electrons coenzyme Q, porfyrin derivatives 3) Transfer of groups of atoms adenosine phosphates (ATP, ADP) - phosphate group coenzyme A – acyl groups thiamine diphosphate - aldehydes pyridoxal phosphate – amine groups biocytin – CO2 tetrahydrofolate (coenzyme F) – one-carbon groups Vitamins are often converted to coenzymes Vitamin Coenzyme Function Thiamin diphosphate decarboxylation B1 Flavin mononucleotide (FMN) carries hydrogen B2 Nicotinamide adenine dinucleotide carries hydrogen H- B3 (NAD+), (NADH) B 5 acyl group carrier pantothenic acid Coenzyme A H (B7) Biocytin CO2 fixation B9-folic acid Tetrahydrofolate carries one carbon units B12-cobalamine Methylcobalamine, adenosylcobalamine ACTIVE SITE Active site Substrate ACTIVE SITE BINDING SITE CATALYTIC SITE (where a substrate binds) (where the reaction proceeds) ▪ ACTIVE SITE = pocket in the enzyme where substrates bind and catalytic reaction occur ▪ Some enzymes contain more active sites (2 - 4), they can bind more substrate molecules ▪ Aminoacids of the active site can be located at different regions of a polypeptide chain Aminoacids of the active site can be located at different regions of a polypeptide chain ➢ Substrates bind in active site by following interactions: ➢ hydrogen bonds ➢ hydrophobic interactions ➢ ionic interactions ➢ covalent bonds (occasionally) ➢ The interactions hold the substrate in the proper orientation for most effective catalysis ➢ The ENERGY derived from these interactions = “Binding energy“ binding pocket hydrophobic ionic interaction hydrogen interaction bonding ionic interaction 2. non-covalent interactions between substrate and Interactionsthe active site:between enzyme and substrate - hydrogen bonding - ionic interactions - hydrophobic interactions Stages of enzyme reaction 1/ E + S E-S Formation of E-S complex 2/ E-S E-S* Activation of the complex 3/ E-S* E-P Conversion of substrate to a product 4/ E-P E + P Separation of product from enzyme ES* = enzyme/transition state complex First step of enzyme catalysis FORMATION OF THE ENZYME-SUBSTRATE COMPLEX E + S ES Second step FORMATION OF THE TRANSITION STATE COMPLEX Note change ES transition state complex Transition State: a. Old bonds break and new ones form. b. Substance is neither substrate nor product c. Unstable short lived species with an equal probability of going forward or backward. Third step FORMATION OF THE ENZYME-PRODUCT COMPLEX ES* EP Fourth step RELEASE OF THE PRODUCT EP E + P Mechanisms of substrate conversion • Enzyme binds • Charges in the • Deformation of S 2 substrates, active site facilitates its that they are in induce changes conversion to a close vicinity in the charges in product S molecule MECHANISM OF ENZYME ACTION • Enzymes decrease the activation energy of a reaction by formation of the active enzyme - substrate complex • Activation energy is the energy required to start a reaction. Transition state Uncatalyzed reaction Catalyzed reaction Substrate Energy Product • The lower the free energy of activation, the more molecules have sufficient energy to pass through the transition state, and, thus, the faster the rate of the reaction. Enzyme activity Enzyme Substrate Product The katal (symbol: kat) - the SI unit of catalytic activity One katal is the catalytic activity that changes one mole of substrate per second at optimal pH. 1 kat = mol . s-1 ◼ SPECIFIC ACTIVITY – katal/kg (μkat/mg) protein ◼ MOLAR ACTIVITY – katal/mol protein 1 U = μmol . min-1 1 kat = mol/s = 60 mol/min= 60.106 μmol.min-1 = 6.107 U 1 U = μmol.min-1 = 10-6 mol/60 s = 16.7 . 10-9 kat ENZYME SPECIFICITY Enzyme has SPECIFICITY – it can discriminate among possible substrate molecules: S S S Substrate Enzyme Enzyme Enzymes are very specific and only work with certain substrates SUBSTRATE SPECIFICITY (apoenzyme responsible) 1) Strictly specific enzymes - only react with a single substrate (DNA polymerase, urease) 2) Less specific enzymes a. Group specific - recognize a functional group (-OH, -NH2...) (alcoholdehydrogenase - converts methanol, ethanol, ethylene glycol) b. Linkage specific – particular type of chemical bond regardless . of the rest of the molecular structure (peptidase, esterase) SPECIFICITY OF EFFECT (cofactor responsible) OXIDOREDUCTASES – oxidation/reduction reactions - transfer of H and O atoms or electrons from one substance to another (alcoholdehydrogenase) TRANSFERASES – transfer of a functional group - methyl-, acyl-, amino- or phosphate group (hexokinase) HYDROLASES – catalyze hydrolysis of various bonds (carboxypeptidase A) LYASES – cleave bonds by means other than hydrolysis and oxidation (pyruvate decarboxylase) ISOMERASES – intramolecular changes of „S“ (maleate isomerase) LIGASES – join two molecules with covalent bonds with the use of energy from ATP (pyruvate carboxylase) MODELS FOR ENZYME/SUBSTRATE INTERACTIONS 1) Lock and Key Model (Emil Fischer 1894) Substrate Active site ES complex Enzyme ➢ This model assumed that only a substrate of a proper shape could fit with the enzyme 1) Lock and Key Model (Emil Fischer 1894) Substrate Enzyme A. Substrate (key) fits into a perfectly shaped space in the enzyme (lock) B. Highly stereospecific C. Site is preformed and rigid 2) Induced Fit Model (Daniel Koshland 1958) Substrate Active site Enzyme ES complex ➢ This model assumes continuous changes in the active site structure as a substrate binds Induced fit model ◼ Takes into account the flexibility of proteins ◼ A substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation) ◼ Change in protein configuration leads to a near perfect fit of substrate with enzyme Principles of Catalysis • Uncatalyzed reactions often are extremely slow. • They are slow because of the heigh activation energy • Enzymes lower the activation energy by creating an ES (enzyme-substrate) complex which reduces bond strength in the substrate and makes the substrate easier to convert to the product. Enzyme Nomenclature 1. Trivial names 2. Systematic nomenclature Enzyme Nomenclature 1. Trivial names ◼ everyday use (pepsin, trypsin) Usually named by suffix –ase to: - the name of a substrate (urease) - the catalytic reaction (glucose oxidase) Some examples: Alcohol dehydrogenase - oxidation of alcohols DNA polymerase - polymerization of nucleotides Protease - hydrolysis of proteins Methyltransferase - methyl group transfer 2. Systematic names ◼ Introduced in 1961 (enzyme commision of IUB) ◼ Systematic names: a) characterizing catalytic reaction b) recommended – commonly used c) international – code number 2. Systematic names L-lactate + NAD+ pyruvate + NADH + H+ name of substrates
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