UNIVERSITY OF TECHNOLOGY, HOCHIMINH CITY MICROBIOLOGY CODE: EN1015 ASSOC. DANG VU BICH HANH 1 Enzymes * Contents 1. Mechanism of Enzyme Action 4. Specificity *Chemistry Introduction ✓Biological catalysts → speed up the rate of the biochemical reaction.
✓Some special RNA species also act as enzymes and are called Ribozymes e. Hammerhead enzyme Structure of Enzymes *Active site : the region binds substrates *Active sites < 5% of the total surface area of enzyme. *Active site: specific shape due to tertiary structure of protein. Note: A change in the shape of protein affects the shape of active site and function of the enzyme.
Active site oActive site can be further divided into: Active Site Binding Site Catalytic Site It chooses the substrate It performs the catalytic and binds it to active site. action of enzyme. Co- factors oCo-factor: protein molecule; carries out chemical reactions that can not be performed by standard 20 amino acids. oCo-factors are of two types: ➢Organic co-factors ➢Inorganic cofactors Co- factors *Inorganic co- factors oThese are the inorganic molecules required for the proper activity of enzymes.
Examples: ➢ Enzyme carbonic anhydrase requires Zn++ for it’s activity. ➢ Hexokinase has co-factor Mg++ ORGANIC CO-FACTORS o These are the organic molecules required for the proper activity of enzymes. Example: ➢ Glycogen phosphorylase requires the small organic molecule pyridoxal phosphate. Types of Organic co-factors Prosthetic Group Coenzyme o A prosthetic group is a oA coenzyme is loosely tightly bound organic co- bound organic co-factor.
NAD groups and biotin. Types of co-factors Continued… *An enzyme with it’s co-factor removed is designated as apoenzyme. *The complete complex of a protein with all necessary small organic molecules, metal ions and other components is termed as holoenzyme of holoprotein. Types of co-factors Continued… * The reactant in biochemical reaction is termed as substrate.
* When a substrate binds to an enzyme it forms an enzyme- substrate complex. Substrate Joins Enzyme * substrate * Sites of enzyme synthesis oEnzymes are synthesized by ribosomes which are attached to the rough endoplasmic reticulum. oInformation for the synthesis of enzyme is carried by DNA. oAmino acids are bonded together to form specific enzyme according to the DNA’s codes.
*The reactant in biochemical reaction is termed as substrate. *When a substrate binds to an enzyme it forms an enzyme-substrate complex. * Sites of enzyme synthesis o Synthesized by ribosomes. oInformation for the synthesis of enzyme is carried by DNA.
* Intracellular and extracellular enzymes oIntracellular enzymes: are synthesized and retained in the cell for the use of cell itself. o They are found in the cytoplasm, nucleus, mitochondria and chloroplast. Example : Oxydoreductase catalyses biological oxidation. Or: enzymes involved in reduction in the mitochondria.
* Intracellular and extracellular enzymes oExtracellular enzymes are synthesized in the cell but secreted from the cell to work externally. Example : Digestive enzyme produced by the pancreas, are not used by the cells in the pancreas but are transported to the duodenum. Characteristics *Enzymes speed up the reaction by lowering the activation energy of the reaction. *Their presence does not effect the nature and properties of end product.
*They are highly specific in their action that is each enzyme can catalyze one kind of substrate. *Small amount of enzymes can accelerate chemical reactions. *Enzymes are sensitive to change in pH, temperature and substrate concentration. *Turnover number is defined as the number of substrate molecules transformed per minute by one enzyme molecule.
Catalase turnover number = 6 x106/min Nomenclature of enzymes oAn enzyme is named according to the name of the substrate it catalyses. oSome enzymes were named before a systematic way of naming enzyme was formed. Example: pepsin, trypsin and rennin oBy adding suffix -ase at the end of the name of the substrate, enzymes are named. oEnzyme for catalyzing the hydrolysis is termed as hydrolase.
Example : maltase maltose + water glucose +glucose * Examples substrate enzymes products lactose lactase glucose + galactose maltose maltase Glucose cellulose cellulase Glucose lipid lipase Glycerol + fatty acid starch amylase Maltose protein protease Peptides + polypeptide * Classification of enzymes *by International Enzyme Commission. *based on the type of reactions catalyzed by enzymes. *six major classes. *Each class → sub classes.
Classification of enzymes ENZYME CLASS REACTION TYPE EXAMPLES Oxidoreductases Reduction-oxidation (redox) Lactate dehydrogenase Transferases Move chemical group Hexokinase Hydrolases Hydrolysis; bond cleavage with Lysozyme transfer of functional group of water Lysases Non-hydrolytic bond cleavage Fumarase Isomerases Intramolecular group transfer Triose phosphate (isomerization) isomerase Ligases Synthesis of new covalent bond RNA polymerase between substrates, using ATP hydrolysis Mechanism of enzyme action *The catalytic efficiency of enzymes is explained by two perspectives: Thermodynamic Processes at the changes active site *Thermodynamic Activational barrier. changes *Thermodynamic changes *Only a few substances cross the activation barrier and change into products. *That is why rate of uncatalyzed reactions is much slow. * Enzymes provide an alternate pathway for conversion of substrate into products.
*Enzymes accelerate reaction rates by forming transitional state having low activational energy. *Hence, the reaction rate is increased many folds in the presence of enzymes. *The total energy of the system remains the same and equilibrium state is not disturbed. * Thermo-dynamic changes overview * Processes at the active site Covalent catalysis Acid base Catalysis catalysis by strain Catalysis by proximity oEnzymes form covalent linkages with substrate forming transient enzyme-substrate complex with very low activation energy.
oEnzyme is released unaltered after completion of reaction. *Covalent Acid-base catalysis *Mostly undertaken by oxido- reductases enzyme. *Mostly at the active site, histidine is present which act as both proton donor and proton acceptor. * Catalysis by proximity *In this catalysis molecules must come in bond forming distance.
*When enzyme binds: oA region of high substrate concentration is produced at active site. oThis will orient substrate molecules especially in a position ideal for them. *Catalysis by bond strain ❑Mostly undertaken by lyases. ❑The enzyme-substrate binding causes reorientation of the structure of site due to in a strain condition.
❑Thus transitional state is required and here bond is unstable and eventually broken. ❑In this way bond between substrate is broken and converted into products. *Lock and key model *Proposed by EMIL FISCHER in 1894. *Lock and key hypothesis assumes the active site of an enzymes are rigid in its shape.
*There is no change in the active site before and after a chemical reaction. *More recent studies have revealed that the process is much more likely to involve an induced fit model (proposed by DANIAL KOSH LAND in 1958). *According to this exposure of an enzyme to substrate cause a change in enzyme, which causes the active site to change it’s shape to allow enzyme and substrate to bind. *Induced fit model *Induced Fit Model 36 *Introduction “It is a branch of biochemistry in which we study the rate of enzyme catalyzed reactions.” *Kinetic analysis reveals the number and order of the individual steps by which enzymes transform substrate into products *Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of that enzyme, its role in metabolism, how its activity is controlled, and how a drug or an agonist might inhibit the enzyme * Rates of reaction and their dependence on activation energy * Activation Energy (Ea): “The least amount of energy needed for a chemical reaction to take place.” * Enzyme (as a catalyst) acts on substrate in such a way that they lower the activation energy by changing the route of the reaction.
* The reduction of activation energy (Ea) increases the amount of reactant molecules that achieve a sufficient level of energy, so that they reach the activation energy and form the product. Example: * Carbonic anhydrase catalyses the hydration of 10⁶ CO₂ molecules per second which is 10⁷x faster than spontaneous hydration. Enzymes lower the activation energy of a reaction Energy levels of molecules Activation energy Initial energy state Activation energy of uncatalysed of substrates of enzyme catalysed reactions reaction Final energy state of products Progress of reaction (time) * Kinetics of enzymes catalysis *Enzymes catalysis: “ It is an increase in the rate of reaction with the help of enzyme(as catalyst).” *Catalysis by enzymes that proceed via unique reaction mechanism, typically occurs when the transition state intermediate forms a covalent bond with the enzyme(covalent catalysis). *During the process of catalysis enzymes always emerge unchanged at the completion of the reaction.
* Factors affecting rate of enzyme catalyzed reactions 1. Hydrogen ion concentration(pH) 3. Substrate concentration Effect of Temperature *Raising the temperature increases the rate of enzyme catalyzed reaction by increasing kinetic energy of reacting molecules. *Enzymes work maximum over a particular temperature known as optimum temperature.
Enzymes for humans generally exhibit stability temperature up to 35-45 ᵒC. Effect of Temperature (cont.) *The temperature coefficient is a factor Q₁₀ by which the rate of biological processes increases for a 10 ᵒC increase in temperature. *For most biological processes Q₁₀ = 2. *However some times heat energy can also increase kinetic energy to a point that exceed the energy barrier which results in denaturing of enzymes.
5- 40oC Temperature Increase in Activity 40oC - denatures Rate of Reaction 0 10 20 30 40 50 60 <5oC - inactive * Effect of pH *Rate of almost all enzymes catalyzed reactions depends on pH *Most enzymes exhibit optimal activity at pH value between 5 and 9 *High or low pH value than optimum value will cause ionization of enzyme which result in denaturation of enzyme pH affects the formation of hydrogen bonds and sulphur bridges in proteins and so affects shape. trypsin arginase pepsin Rate of Reaction (M) Acidic 2 4 6 8 10 pH Basic * Enzymes kinetics * Michaelis-Menten model & effects of substrate * Michaelis-Mentenconcentration Model: “According to this model the enzyme reversibly combines with substrate to form an ES complex that subsequently yields product, regenerating the free enzyme.” k₁ k₂ E + S k₋₁ ES E + P where: ➢ S is the substrate ➢ E is the enzyme ➢ ES-is the enzyme substrate complex ➢ P is the product ➢ K1,K-1 and K2 are rate constants Michaelis-Menten Equation * Michaelis-Menten Equation: “It is an equation which describes how reaction velocity varies with substrate concentration.” Vmax [S] Vo= Km+[S] * Where ➢Vo is the initial reaction velocity. ➢Vmax is the maximum velocity. ➢Km is the Michaelis constant = (k₋₁+k₂)/k₁.
➢[S] is the substrate concentration.