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Enzymes Lecture 4 Catalytic proteins Recap... - Proteins are the key functional molecules in life - Proteins have many structures, resulting in a wide range of functions - A proteins specific conformation determines how it functions Enzymes • Are a type of protein that acts as a catalyst-speeding up chemical reactions • A catalyst is defined as a chemical agent that changes the rate of a reaction without being consumed by the reaction • Enzymes are biological catalysts Chemical reactions • Chemical reactions involve the breaking and forming of bonds, this requires energy • Activation energy is the initial investment of energy needed to start a reaction • In most chemical reactions this is provided in the form of heat • humans need to maintain a temperature of 37˚C • Enzymes provide the activation energy for reactions to proceed at normal temperatures in biological systems Enzymes • What enzymes do therefore is: Reactants Transi6on state Products (ready to react) + catalyst Catalysts act here • Biochemists call ‘reactants’ substrates Substrate Transi6on state Products (ready to react) Enzymes act here + enzyme + lipase • Enzymes are substrate-‐specific – each substrate fits into the enzyme’s ac6ve site Eg. Sucrase 1 Active site is available for a molecule of substrate, the reactant on which the enzyme acts. 2 Substrate binds to enzyme. Substrate (sucrose) (sucrase) Transi6on state (sucrase) Glucose OH Fructose Enzyme (sucrase) H 2O H O (sucrase) 4 Products are released. 3 Substrate is converted to products. Chemical reaction involved: Hydrolysis of sucrose involves the breaking of the bond between glucose and fructose the breaking of a bond in the water molecule the forming of 2 new bonds How do they work? • Complex molecules have the potential to breakdown spontaneously (2nd law of thermodynamics= the universe tends towards disorder) • They continue to exist because the initial activation energy required to breakdown can’t be reached to initiate a breakdown reaction • An enzyme lowers the activation energy required for a reaction enabling the substrates to absorb enough energy even at a moderate temperature to reach transition state Enzyme shape • Enzymes are substrate specific – The substrate fits like a key in a lock – When the substrate and enzyme are joined the catalytic action of the enzyme converts the substrate to product • The specificity of an enzyme for a substrate results from the shape of the enzyme –due to a compatible fit between the active site and the substrate • The substrate binds to the “active site” of the enzyme -This is the catalytic centre of the enzyme - The active site is typically a pocket or groove on the surface of the enzyme Active site – groove on surface of enzyme Groove Eg. Lysozyme- an enzyme that Breaks down bacterial cell walls by recognizing and binding to specific molecules on the bacteria (a) A ribbon model Groove (b) A space-filling model Enzymes “active site” • Is usually formed by only a few amino acids • Is not rigid • The side chains of the aa’s in the enzyme interact with the chemical groups of the substrate- enzyme changes shape and fits even better around the substrate- INDUCED FIT • Induced fit brings the chemical groups of the active site into new positions enhancing the ability of the enzyme to catalyse the reaction • The rest of the protein structure provides the structural framework that determines the configuration of the active site Induced fit Induced fit 6 important Features of enzymes 1. Side chains of amino acids make up the ac6ve site 2. Substrates bind via weak interac6ons: hydrogen bonds, hydrophobic interac6ons 3. Enzymes act at op6mal pH and temperatures: Enzyme ac6vity Enzyme ac6vity 37oC 7.4 temp pH Excep6ons: Thermophiles 70⁰C Excep6ons: pH 2 in stomach – pepsin pH 8 in intes6ne -‐ trypsin Features of enzymes 4. Many require non-protein helpers to aid catalysis - ‘co-factors’ Eg: calcium, iron. • If the co-factor is organic it is a co-enzyme - Most vitamins are coenzymes Eg: vitamin C, NAD+ • Vitamin C is used by an enzyme to cross-link collagen. Vitamin C deficiency causes scurvy – a disease of dysfunctional cross-linking of collagen • NAD+ and Zinc are co-factors used by alcohol dehydrogenase Alcohol metabolism Enzyme 1 Co-factor= zinc Co-enzyme = NAD+ Enzyme 2 Alcohol dehydrogenase Acetaldehyde dehydrogenase ADH Alcohol NAD+ NADH Co-enzyme Acetaldehyde + 2x H NAD+ NADH Co-enzyme Acetate/ ace6c acid + H poisonous Blood circulation Broken down into carbon and water during the Krebs cycle Alcohol metabolism Enzyme 1 Co-factor= zinc Co-enzyme = NAD+ Enzyme 2 Alcohol dehydrogenase Acetaldehyde dehydrogenase less efficient Very efficient ADH Alcohol NAD+ NADH Co-enzyme Methanol Acetaldehyde + 2x H formaldehyde NAD+ NADH Acetate/ ace6c acid + H Co-enzyme poisonous Blood circulation • Women have less ADH in their stomach than men Broken down into carbon and water during the Krebs cycle • “Flush syndrome”: apparent in many east asians and american indians symptoms: headaches, nausea, vomiting, heart palpitations 50% of individuals from japenese descent show flush syndrome Features of enzymes 5. Many drugs inhibit enzymes • Drugs can compete with the substrate and prevent the reaction happening HMG-‐CoA Mevalonic acid Cholesterol ENZYME: HMG-‐CoA reductase Eg. Statins inhibit HMG-CoA Reductase, a key enzyme in cholesterol synthesis HMG-‐CoA – statins therefore lower cholesterol and protect or against heart disease. Lovostatin competes with the substrate for the active site lovosta6n HMG-‐CoA reductase Features of enzymes 5. Many drugs inhibit enzymes • Drugs can compete with the substrate and prevent the reaction happening HMG-‐CoA Mevalonic acid Cholesterol ENZYME: HMG-‐CoA reductase Eg. Statins inhibit HMG-Co Reductase, a key enzyme in cholesterol synthesis HMG-‐CoA – statins therefore lower cholesterol and protect or against heart disease. Lovostatin competes with the substrate for the active site lovosta6n HMG-‐CoA reductase Structural similarity between the substrate and the inhibitor Features of enzymes 5. Many drugs inhibit enzymes Biosynthesis of nucleotides for replicating cells: Folic acid Dihydrofolate reductase nucleotides enzyme Folic acid Substrate methotrexate structurally similar drug Features of enzymes 5. Many drugs inhibit enzymes Biosynthesis of nucleotides for replicating cells: Folic acid Dihydrofolate reductase nucleotides Toxic to rapidly dividing cells enzyme methotrexate Folic acid Substrate methotrexate structurally similar drug Methotrexate is often used in cancer therapy Features of enzymes 6. Cellular enzymes can be activated by “phosphorylation” - the addition of a phosphate group • It is other enzymes that actually add the phosphate group to the other enzyme, -these enzymes are called kinases • Kinases are said to “phosphorylate” the protein • Eg: Adrenalin activates Phosphorylase kinase which phosphorylates Glycogen Phosphorylase. -This enzyme in turn breaks down glycogen to release glucose Signal cascade Receptor protein Adrenaline (hormone protein) signals Phosphorylase Kinase (protein kinase enzyme) P GP GP Glycogen Phosphorylase Glycogen glucose P =Phosphate group Adrenaline results in the release of glucose which allows for rapid produc6on of energy Understanding Biology “reductionism” Reducing down complex reactions to their component parts genetic engineering helping biologists understand the finer details of complex functions “systems biology” link all of the knowledge that we gain from these techniques together to gain a greater understanding of how the system as a whole works “Reductionism” Biological systems are highly complex ???Can studying each protein separately contribute to our understanding of such complex systems??? ??Would it be possible to chemically synthesise a cell?? It is not enough to just know the components BUT understanding the behaviour of a complex integrated system is incredibly difficult A complex organism can not be analysed without taking it apart Genome sequencing is an example of reduc6onism – Human Genome Project – Reduc6onism = reducing complex systems to simpler components that are easier to study – Human organism breaks down into a code of 3billion leiers “Systems Biology” Goal: Understand how biological systems are func6onally integrated Systems biology – model of the dynamic behaviour of whole cells High performance computers analyse highly complex systems of known interac6ons “Systems map” of the interactions between proteins in a cell of drosophila. ~3500 proteins Outer membrane and cell surface CELL Cytoplasm Nucleus Eg. of an applica6on: The ability to predict probable side effects of drugs DNA > RNA > PROTEIN > TRAIT GFP Protein Trait: Green Fluorescence Winner of the nobel prize for chemistry 2008 With the aid of GFP-fusion proteins, we can watch processes that were previously invisible GFP as a molecular tool HIV transmission detected using GFP-HIV fusion proteins T cells