Introduction to Enzyme 2022 Lecture 1 PDF

STB3613 ENZYMOLOGY INTRODUCTION  Enzymology is the study of enzymes which relevant to both biological and physical sciences.  The study of enzymes may include;  enzyme activity  enzyme properties & structure  enzyme kinetics  catalysis and mechanisms  production (extractio...

STB3613 ENZYMOLOGY INTRODUCTION  Enzymology is the study of enzymes which relevant to both biological and physical sciences.  The study of enzymes may include;  enzyme activity  enzyme properties & structure  enzyme kinetics  catalysis and mechanisms  production (extraction & purification) to know their functions  Its application is diverse; industries, clinical diagnosis, medicine, fermentation processes Overview: The Energy of Life The living cell is a miniature chemical factory where thousands of reactions occur. The cell extracts energy and applies energy to perform work Some organisms even convert energy to light, as in bioluminescence  Enzyme 1 Enzyme 2 Enzyme 3 A B C D Reaction 1 Reaction 2 Reaction 3 Starting Product molecule  Enzymes are proteins that act as bio-catalysts that speed up the rates of biochemical reactions without themselves undergoing changes or without being consumed by the reaction. can reuse  Enzymes speed up metabolic reactions by lowering energy barriers. enzymes give the shortcut  An example of an enzyme-catalyzed reaction is hydrolysis of sucrose by the enzyme sucrase.  Sucrase Sucrose Glucose Fructose (C12H22O11) (C6H12O6) (C6H12O6)  The Activation Energy Barrier Every chemical reaction between molecules involves bond breaking and bond forming. The initial energy needed to start a chemical reaction is called the free energy of activation, or activation energy (EA). Activation energy is often supplied in the form of thermal energy that the reactant molecules absorb from their surroundings. the minimum energy is reequired, to lower down the expectation energy  A B C D Transition state A B Free energy EA C D Reactants A B G  O C D Products Progress of the reaction How Enzymes Lower the EA Barrier ? Enzymes catalyze reactions by lowering the EA barrier. create shortcut, lower down the activation energy Enzymes do not affect the change in free energy (∆G); instead, they hasten reactions that would occur eventually.  Course of reaction EA without without enzyme enzyme EA with enzyme is lower Free energy Reactants free energy Course of G is unaffected reaction by enzyme with enzyme Products Progress of the reaction  Substrate Specificity of Enzymes The reactant that an enzyme acts on is called the enzyme’s substrate. The enzyme binds to its substrate, forming an enzyme- substrate complex. The active site is the region on the enzyme where the substrate binds. some functional role also aid in the binding Induced fit of a substrate brings chemical groups of the active site into positions that enhance their ability to catalyze the reaction.  Substrate Active site Enzyme Enzyme-substrate complex (a) (b) Catalysis in the Enzyme’s Active Site In an enzymatic reaction, the substrate binds to the active site of the enzyme The active site can lower an EA barrier by ◦ Orienting substrates correctly sth will drive the substrate into the enzyme ◦ Straining substrate bonds require less energy to convert into product ◦ Providing a favourable micro-environment some cofactor, and some functional group helps create the flavoured environment ◦ Covalently bonding to the substrate  1 Substrates enter active site. 2 Substrates are held in active site by weak interactions. Substrates Enzyme-substrate complex Active site Enzyme  1 Substrates enter active site. 2 Substrates are held in active site by weak interactions. Substrates Enzyme-substrate complex 3 Active site can lower EA and speed up a reaction. Active site Enzyme 4 Substrates are converted to products. Figure 8.15-3 1 Substrates enter active site. 2 Substrates are held in active site by weak interactions. Substrates Enzyme-substrate complex 3 Active site can lower EA and speed up a reaction. 6 Active site is available for two new substrate molecules. Enzyme 5 Products are 4 Substrates are released. converted to products. Products Effects of Local Conditions on Enzyme Activity An enzyme’s activity can be affected by ◦ General environmental factors, such as temperature and pH how the enyme perform the best, enzyme stability and enyzme optimizaton are different ◦ Chemicals that specifically influence the enzyme such as cofactors, inhibitors   Effects of Temperature and pH Each enzyme has an optimal temperature in which it can function. bonding makes the enzyme strong  Each enzyme has an optimal pH in which it can function.start with ideal temperature and pH, 37 and 7 Optimal conditions favour the most active shape for the enzyme molecule.  Optimal temperature for Optimal temperature for typical human enzyme (37°C) enzyme of thermophilic (heat-tolerant) Rate of reaction bacteria (77°C) 0 60 20 80 40 100 120 Temperature (°C) (a) Optimal temperature for two enzymes Optimal pH for pepsin Optimal pH for trypsin (stomach (intestinal enzyme) enzyme) when study pH, must Rate of reaction use different buffer, certain buffer work at certain range 0 1 5 2 3 4 6 7 8 9 10 pH (b) Optimal pH for two enzymes Figure 8.16a Optimal temperature for Optimal temperature for typical human enzyme (37°C) enzyme of thermophilic (heat-tolerant) Rate of reaction bacteria (77°C) 0 60 20 40 80 100 120 Temperature (°C) (a) Optimal temperature for two enzymes  Optimal pH for pepsin Optimal pH for trypsin (stomach (intestinal enzyme) enzyme) Rate of reaction 0 1 5 2 3 4 6 7 8 9 10 pH (b) Optimal pH for two enzymes   Cofactors Cofactors are non-protein enzyme helpers. Cofactors may be inorganic (such as a metal in more to metal ionic form) or organic An organic cofactor is called a coenzyme more to organic Coenzymes include vitamins   Enzyme Inhibitors Competitive inhibitors bind to the active site of an enzyme, competing with the substrate. Non-competitive inhibitors bind to another part of an enzyme, causing the enzyme to change shape and making the active site less effective. Examples of inhibitors include toxins, poisons, pesticides, and antibiotics. for treatment, (a) Normal binding (b) Competitive inhibition (c) Noncompetitive inhibition Substrate Active site Competitive inhibitor Enzyme Noncompetitive inhibitor The Evolution of Enzymes Enzymes are proteins encoded by genes. Changes (mutations) in genes lead to changes in amino acid composition of an enzyme Altered amino acids in enzymes may alter their substrate specificity. the binding site, the temperature, the pH affect the enzyme performance the most Under new environmental conditions a novel form of an enzyme might be favoured. Two changed amino acids were Active site found near the active site. Two changed amino acids Two changed amino acids were found in the active site. were found on the surface. Regulation of enzyme activity helps control metabolism Chemical chaos would result if a cell’s metabolic pathways were not tightly regulated. control mechanism must be set, A cell does this by switching on or off the genes that encode specific enzymes or by regulating the activity of enzymes  Allosteric Regulation of Enzymes Allosteric regulation may either inhibit or stimulate an enzyme’s activity. Allosteric regulation occurs when a regulatory molecule binds to a protein at one site and affects the protein’s function at another site.   Allosteric Activation and Inhibition Most allosterically regulated enzymes are made from polypeptide subunits only activated once it is needed Each enzyme has active and inactive forms The binding of an activator stabilizes the active form of the enzyme The binding of an inhibitor stabilizes the inactive form of the enzyme (a) Allosteric activators and inhibitors (b) Cooperativity: another type of allosteric activation Allosteric enzyme Active site Substrate with four subunits (one of four) Regulatory site (one Activator Stabilized active of four) Inactive form Active form Stabilized active form form call for full swing Oscillation Non- Inhibitor Inactive form Stabilized inactive functional active site form (a) Allosteric activators and inhibitors Allosteric enzyme Active site with four subunits (one of four) Regulatory site (one of four) Activator Active form Stabilized active form Oscillation Nonfunctional active site Inhibitor Inactive form Stabilized inactive form (b) Cooperativity: another type of allosteric activation Substrate Inactive form Stabilized active form  Cooperativity is a form of allosteric regulation that can amplify enzyme activity One substrate molecule primes an enzyme to act on additional substrate molecules more readily Cooperativity is allosteric because binding by a substrate to one active site affects catalysis in a different active site © 2011 Pearson Education, Inc.  Identification of Allosteric Regulators Allosteric regulators are attractive drug candidates for enzyme regulation because of their specificity.we can contro this, so used for medication Inhibition of proteolytic enzymes called caspases may help in management of inappropriate inflammatory responses. like allergic EXPERIMENT Caspase 1 Active Substrate site SH SH Known active form Active form can bind substrate SH Allosteric binding site Allosteric Known inactive form Hypothesis: allosteric inhibitor inhibitor locks enzyme in inactive form RESULTS Caspase 1 Inhibitor Active form Allosterically Inactive form inhibited form EXPERIMENT Caspase 1 Active Substrate site SH SH Known active form Active form can bind substrate SH Allosteric binding site Allosteric Known inactive form Hypothesis: allosteric inhibitor inhibitor locks enzyme in inactive form RESULTS Caspase 1 Inhibitor Active form Allosterically Inactive form inhibited form   Feedback Inhibition In feedback inhibition, the end product of a metabolic pathway shuts down the pathway. inhibit the enzyme to produce by having the product, to save the energy, for sustainable Feedback inhibition prevents a cell from wasting chemical resources by synthesizing more product than is needed.  Initial substrate Active site (threonine) available Threonine in active site Enzyme 1 (threonine Isoleucine deaminase) used up by cell Intermediate A Active site of Feedback enzyme 1 is inhibition Enzyme 2 no longer able to catalyze the Intermediate B conversion of threonine to Enzyme 3 intermediate A; pathway is Intermediate C switched off. Isoleucine binds to Enzyme 4 allosteric site. Intermediate D Enzyme 5 End product (isoleucine) Specific Localization of Enzymes Within the Cell Structures within the cell help bring order to metabolic pathways. Some enzymes act as structural components of membranes. In eukaryotic cells, some enzymes reside in specific organelles; for example, enzymes for cellular respiration are located in mitochondria.  Mitochondria The matrix contains enzymes in solution that are involved in one stage of cellular respiration. Enzymes for another stage of cellular respiration are embedded in the inner membrane. 1 m Biochemistry of Enzyme  Why Enzymes? Higher reaction rates Greater reaction specificity Milder reaction conditions Capacity for regulation - - COO COO NH2 Metabolites have - - O COO many potential COO OH pathways of decomposition - O COO - - Chorismate COO COO OH mutase - OOC Enzymes make the O desired one most favourable NH2 OH Enzymatic Substrate Selectivity OH H H - OOC + NH3 - + OOC NH3 H - OOC + NH3 No binding OH HO OH H H Binding but no reaction H NH CH3 *Example: Phenylalanine hydroxylase How to Lower G? Enzymes organizes reactive groups into proximity How to Lower G? Enzymes bind transition states best  transition state How is TS Stabilization Achieved? need to be stable and flavourable  acid-base catalysis: give and take protons  covalent catalysis: change reaction paths  metal ion catalysis: use redox cofactors, pKa shifters  electrostatic catalysis: preferential interactions with TS helps to move forward  covalent catalysis: change reaction paths O O O O H2O + O CH3 H3C O - + - + 2 H H3C slow H3C O O O O O H3C O CH3 + N + - fast CH3 H3C O.. N.. O H H O -.. O + N N CH3 + H3C O - OH + H