BIOL 1000 Lecture Notes

Week 2: Eukaryotic Cells Ribosomes: Millions of ribosomes scattered throughout the cytoplasm. They manufacture proteins. Eukaryotic ribosomes are also associated with the endoplasmic reticulum. Proteins manufactured by free ribosomes either remain in the cytosol or are imported into other organelles, such as the nucleus. Endoplasmic Reticulum: Portions of the nuclear envelope extend into the cytoplasm to form an extensive membrane-enclosed factory called the endoplasmic reticulum. Two regions: rough and smooth ER. The rough endoplasmic reticulum is named for its appearance in transmission electron micrographs. Ribosomes are attached to the membrane. The ribosomes associated with the rough ER synthesize proteins that function in the ER or will be shipped to another destination, such as a different organelle, the plasma membrane or secreted to the cell exterior. As the proteins are being manufactured, they move to the interior of the sac-like component of the rough ER. The interior of the ER is called the lumen. In the lumen of the rough ER, newly manufactured proteins undergo folding and other types of processing. Smooth ER does not contain ribosomes. The smooth ER makes membrane lipids such as cholesterol and phospholipids. The enzymes that synthesize these molecules are membrane proteins, although they are too small to see. Golgi Apparatus: The proteins that leave the rough ER must first pass through the Golgi apparatus before they reach their final destination. The Golgi consists of dozens of stacks of discrete flattened membranous sacs called cisternae. Which are stacked on top of each other like pancakes. Because of the way they are stacked. The Golgi has a distinct polarity or sidedness. The cis surface is closest to the nucleus and the trans surface is oriented toward the plasma membrane. The cis side receives the vesicles containing rough ER products, and the trans side ships them out to other organelles or to the cell surface. Lysosomes: Animal cells contain organelles called lysosomes that function as recycling centres. Lysosomes contain about 40 different enzymes, each specialised for hydrolyzing different types of macromolecules – proteins, nucleic acids, lipids or carbohydrates. The amino acids, nucleotides, sugars and other molecules that result from hydrolysis are exported from the lysosome via transport proteins in the organelle membrane. Once in the cytosol they can be used as sources of energy or building blocks for new molecules. The digestive enzymes inside lysosomes are collectively called acid hydrolases because under acidic conditions (pH of 5.0) they use water to break monomers from macromolecules. In the cytosol, where the pH is about 7.2, acid hydrolases would be less active. Endomembrane system: Composed of lysosomes, Golgi apparatus and the endoplasmic reticulum. It is a centre for producing, processing and transporting proteins and lipids in eukaryotic cells. For example, acid hydrolase is synthesized in the ER processed in the Golgi and shipped to the lysosome. Vacuoles: Only in plants and fungi. Compared with lysosomes, vacuoles are large. Vacuoles vary in size and function. Some contain digestive enzymes and serve as recycling centres; most are large storage containers. Some vacuoles contain hydrolases and play a similar role to the lysosome of animal cells, most of the vacuoles in plant and fungal cells act as storage depots. n many cases, ions such as potassium (K+) and chloride (Cl?), among other solutes, are stored at such high concentrations that they draw water in from the environment. Different specialized functions of storage. Peroxisomes: All eukaryotic cells have them. Contain enzymes involved in detoxifying reactive molecules, such as hydrogen peroxide. Peroxisomes are centres for reduction-oxidation (REDOX) reactions. For example, the peroxisomes in your liver cells contain enzymes that remove electrons from o oxidize the ethanol in alcoholic beverages. In animals and plants, the products of these reactions often include hydrogen peroxide which is highly reactive. If it escaped from the peroxisome, it would quickly react with and damage DNA, proteins and cellular membranes. This event is rare, however, because inside the peroxisome, the enzyme catalase quickly “detoxifies” hydrogen peroxide by catalyzing its oxidation to form water and oxygen. Mitochondria: Primarily responsible for supplying ATP in animals, plants and other eukaryotic cells. 2 membranes: the outer membrane defines organelles surface while the inner membrane forms a series of sac-like cristae. Mitochondrial matrix = solution enclosed within inner membrane. The chemical energy in carbs and fats is used to produce ATP. Most of the enzymes and molecular machines responsible for synthesizing ATP are embedded in the inner membrane. Each mitochondrion has man copies of a small chromosome called mitochondrial DNA (mtDNA) that is independent of the nuclear chromosomes. IT contains only a tiny fraction of the genes responsible for the function of the organelle – the other genes reside in the nuclear DNA. Among the genes present in mitochondrial DNA are those that encode RNAs for mitochondrial ribosomes. These ribosomes are smaller than those found in the cytosol, yet they still function to produce some of the mitochondrial proteins. Chloroplasts: Cells in plants and some protists possess organelles called plastids. Plastids begin as simple proplastids and then become specialized, depending on the cell type. In photosynthetic cells, the proplastids mature into chloroplasts. Chloroplasts convert solar energy into chemical energy during photosynthesis. Proplastids can become amyloplasts: store starch in the plants root cells or chromoplasts: where some flower pigments are deposited. Like the mitochondrion, the chloroplast is surrounded by a double membrane. Unlike mitochondria, however, there are no cristae extending from the inner membrane into the inter­ior. Instead, a third membrane forms an independent network of hundreds of flattened, sac-like structures called thylakoids throughout the interior. Most thylakoids are arranged in interconnected stacks called grana. Many of the pigments, enzymes, and macromolecular machines responsible for converting light energy into chemical energy are embedded in the thylakoid membranes The region between thylakoids and the inner membrane, called the stroma, contains enzymes that use this chemical energy to produce sugars. Like mitochondria, each chloroplast contains copies of its own circular chromosome and small ribosomes that manufacture some, but not all, of the organelle’s proteins. For photosynthesis. Cytoskeleton: Extensive system of protein fibres gives the cell its shape and structural stability. Involved in moving the materials within the cell as well as the cell itself. It organizes all the organelles and other cellular structures into a cohesive whole. Eukaryotic Cell Wall: In plants and fungi only. Located outside of plasma membrane and furnishes a durable outer layer that gives structural support to the cell. Nucleus: Physically separates the nuclear genome from the rest of the cell. Protection for DNA providing instructions for everything in our bodies and also needs to be passed down to daughter cells. Surrounded by a double membrane (2 lipid bilayers: nuclear envelope) Nuclear lamina: attached to inner membrane. Gives protection and shape. Composed of intermediate filaments. Provides structural support to the nucleus. Nuclear DNA exists as Chromatin. DNA becomes wound around things called histones. DNA wraps around all these histones and DNA with these histone proteins and non-histone proteins = chromatin. DNA is never on its own. 2 chromosomes – 1 from mom – 1 from dad. Chromatin: DNA plus associated proteins (histone and non-histone proteins) 2 types: Euchromatin and Heterochromatin Euchromatin: DNA is very loosely packed. Important when you need access to the genes. Genes active and transcription occurring. DNA needs to be free if you need to be reading it. Found when making what that gene is coding for. Heterochromatin: Genes are inactive. Cell does not need to make whatever the gene is coded with. DNA is densely packed. Eukaryotic DNA packing – Histones: Green is DNA, purple is histones. 4 histones and 4 behind them. They form an octamer. 8 histones. Some of DNA winding around it. Not all DNA, we have a lot of DNA. In the middle is histone 1. Job is to pull all octamers together. Binding of histone H1 causes nucleosomes to package into a coiled structure called solenoid or 30nm chromatin fibre. Nucleolus: A non-membrane bound structure composed of proteins and nucleic acids. Synthesis of ribosomal RNA and ribosomal subunit assembly. Where the RNA molecules found in ribosomes are manufactured and the large and small ribosomal subunits are assembled. rRNA = encoded for on DNA. Nuclear envelope: Has nuclear pores. Important because things need to get in and out of the nucleus. Must be controlled tightly. Nuclear transport: the cell must control what moves into and out of the nucleus. Nuclear Pore Complex: Made up of multiple proteins. Come together to form transport system across the double membrane system. Allows only certain materials in and out of nucleus. How does it know which are allowed in to the nucleus? Nucleoplasmin is a protein that enters nuclear pore and goes into nucleus. Radioactive labelled tails ended up in nucleus, whereas core that was radioactively labelled stayed in the cytosol. There is something on the tail fragments that is not on the core fragments, a signal that tells nuclear pore that it is allowed in the nucleus. What is this signal called? It is a single sequence. Called the nuclear localization signal or NLS. Made up of amino acids. NLS is very important to not be mutated signal sequence. Found that molecule that the molecule no longer entered the nucleus and they remained in the cytosol. The sequence is very important. Endomembrane system: Nuclear membrane, ER, Golgi, lysosomes, vesicles, vacuoles and plasma membrane. Collection of interrelated internal membranous sacs that divide the cell into functional and structural compartments called organelles. Internal- inside of the cell. Membranes are so similar to one another. If you take a piece of one it can fuse into another. Example: vesicle is being made from the ER membrane. It fuses with Golgi apparatus. – membrane that was a part of the ER is now a part of the Golgi. Golgi to plasma membrane within vesicle. Thing inside vesicle went outside of cell. Only possible because of similar membranes. Basically, moves via vesicles. Organelles are membrane bound and have some sort of special function to them. Rough ER has unique functions from Golgi and nuclear membrane etc. How did you come up from evolution with similar endo membranes? Ancient eukaryotic cell only had plasma membrane then at some points ended up being infolding of plasma membrane. Developed further and ended up breaking off of plasma membrane leading to internal membrane system that eventually evolved into endomembrane system. Functions of Endomembrane System: Synthesis, modification, transport and secretion of proteins Involves rough ER, Golgi, and eventual fusion of vesicle from Golgi to plasma membrane and secretion out of cell. Insulin is a peptide hormone, produced by pancreas beta cells. It regulates glucose levels in the body, by signalling cells to absorb glucose from the blood. Diabetes mellitus is a result of defective insulin production/function. Human insulin is made of two chains of amino acids held together by disulfide bonds. Gene for insulin is on chromosome 11. How do we go from gene to insulin to secretion? Gene for insulin is located inside nucleus. Starts with transcription. Gene transcribed, now have mRNA transcript for insulin, going to be exported through nuclear pore into the cytosol. Once it is in the cytosol, it is going to connect with free ribosomes. They are going to start to translate the mRNA. Eventually, they will attach to the ER. (only do that if whatever is being translated is going to be secreted from cell or stay in ER, as is the case with insulin). Insulin is translated as preproinsulin as single chain. At the level of the ER (as it goes into lumen), the signal sequence of amino acids is going to be cut off and formation of disulfide bonds, giving a proinsulin. Proinsulin will then be moved to the Golgi vesicle. Eventually will have modified How does the cell know which proteins to make in cytoplasm and which on RER? Key is the signal sequence. Sequence of amino acids that tells the cell that this protein that’s being made needs to be finished at the level of the rough ER. How do we get cytoplasmic ribosome from within cytosol directed to the ER? GO OVER AND WATCH RIBOSOMES TARGETING PROTEIN TO THE ER!!!!! The Golgi complex “tags” proteins for sorting to their final destinations. Protein modification (glycoprotein, lipoprotein) Still in its proinsulin structure at this point. Proinsulin packed in vesicle; vesicle contains protease. Protease cuts disulfide bonds of chain B and A. They separate and insulin is formed. Exocytosis Insulins to be secreted from the cell are transported to the membrane in secretory vesicles which release their contents to the exterior by exocytosis. Endocytosis Vesicles also form by the reverse process, endocytosis, which brings molecules into the cell from the exterior. Endocytosis and exocytosis balance each other out. If only endocytosis, reduction of membrane, exocytosis increase. Translation in ER > Protein is exported from ER via vesicle created from ER membrane > Vesicle containing protein fuses with Golgi > Vesicle containing protein leaves Golgi in vesicle made from Golgi > Vesicle fuses with plasma membrane Synthesis of lipids and detoxification of toxins Smooth ER membranes have no ribosomes. Synthesizes lipids that become part of cell membranes. In the liver, smooth ER converts drugs, poisons, and toxic by-products into substances that can be tolerated or more easily removed from the body. Transportation and breakdown (digesting) of large biomolecule-containing particles. Lysosomes: Contain hydrolytic enzymes to break down macromolecules, low acidic pH, originate from the Golgi. Each type of enzyme works optimally at specific pH. Receptor-mediated endocytosis: when molecule binds to receptors, it causes formation of vesicle. (ENDOCYTOSIS) In membrane of endocytic vesicle, we now have the receptors and molecules specific to receptors. Fuses with early endosome (early stages of lysosome). This is made from Golgi membrane. Vesicle fuses to early endosome. In the early endosome, an acidic environment is created because in endosome we have proton pumps. The protons are being brought into the endosome and creates acidic environments. This causes the molecule that was brought in to be released. Becomes a late endosome. Contains hydrolytic enzymes from Golgi via a vesicle that was created from Golgi membrane. Within the Golgi the hydrolytic enzymes are tagged. Tagging tells vesicle to go to late endosome to deliver hydrolytic enzymes. It becomes a lysosome. The molecule brought in is now being broken down into its original form and can be used by the cell. Another pathway in which items are brought in is called phagocytosis. This is where we have a large engulfing of material. No longer a receptor there. A vesicle is created and whatever is in it gets brought in. Goes to phagosome and to lysosome. Fuses with lysosome. Material inside gets degraded, broken down, by hydrolytic enzymes in lysosome which can be used by cells. Example amoeba. Amoeba eats bacterium, goes in and around the bacteria. Creates large vesicle by phagocytosis. Becomes a phagosome. Delivered to the lysosome. Phagosome fuses to lysosome (because membranes are so similar) hydrolytic enzymes in the lysosome with phagosome are going to break down bacteria and then those bacteria can be used for what ever the organism needs it for. Stars over again. Whatever was not needed is let go. Another example – immune system. We have macrophages (type of white blood cell) engulfs invaders. Delivered to lysosome to be destroyed. Macrophage traps bacteria, engulfs then thru phagocytosis. Brought to lysosome, then hydrolytic enzymes break down bacteria into original constituents and can be used again or excreted. Next pathway is within the cell. Autophagy. Organelle that’s not working properly so we want to degrade it back to original components so it can be used again. Vesicle formed around damaged organelle. Delivered to lysosome and fused and degraded from there. Plants don’t have lysosomes. So hydrolytic enzymes are found in the central vacuole. Part of the endomembrane system Created by the fusion of smaller vacuoles Within is solution containing many materials (e.g. ions, enzymes, metabolic waste, water) Vacuoles = vesicles Serves different functions that vary with plant species. Contain hydrolytic enzymes and degrade metabolic wastes Storage of material (pigments, poison) Store water, for turgidity and growth Mitochondria and chloroplast are energy transformers. They are NOT part of the endomembrane system which means their membranes are different, they cannot fuse vesicles from organelles associated with endomembrane system or plasma membrane. Mitochondria and Chloroplasts Morphology similar to bacteria and archaea Contain their own DNA – DNA shorter than nuclear DNA and is circular Contain own transcription and translation machinery Multiply by binary fission Contain an ETC on the inner membrane What does this suggest? Theory of endosymbiosis Origins of chloroplast and mitochondria Mitochondria developed from ingested aerobic bacteria Chloroplasts developed from cyanobacteria For whatever reason it was never broken down. The bacteria survived, both benefited, forming a symbiotic relationship; eventually host and bacterium became inseparable. Mitochondria are found in almost every type of cell. Chloroplasts are not. What does this tell us about the evolution of endosymbiosis? It likely occurred more than once. The first time with aerobic bacteria became mitochondria and occurred again with cyanobacteria by host cell that now contains ancient mitochondria. Now we have 2 different cell types, those who only have the ancient mitochondria and some cells that engulfed cyanobacteria as well that will have both cyanobacteria and ancient mitochondria into them. Lecture 11: The Cytoskeleton has 3 major functions: Function: Structural support Shape Motility (within cells, of cells, of organisms) Structure: Network of protein fibres and tubules Monomers polymers Motor proteins Filaments: Microtubules (tubulin monomer) Intracellular transport – movement within cell Organelle movement Cell motility (cilia and flagella) Intermediate filaments Cell specific Provide mechanical strength (keratin, lamina) Microfilaments (actin monomer) Cell shape Cell motility and muscle contraction Microtubules Hollow tube Composed of monomer tubulin Alpha tubulin and beta tubulin come together to form a dimer of tubulin. Structural polarity. Microtubules get added to plus end. Approx. 25nm thick Attached to CENTROSOME (microtubule organizing centre (MTOC) - cell centre) Close to nucleus Important for intracellular transport Important for mitosis Intermediate filaments Rope like structures Fibres wound into thicker cables Keratins, lamins or others 8-12 nm thick Protein structure varies with tissue specific Only associated with multicellular organisms Compose nuclear lamina Epithelial cells Hair (keratin) Anywhere mechanical strength required Microfilaments (actin filaments) 2 strand spiral shape, composed of protein called actin Actin comes together to make polymer. Structural polarity as well. Plus end and minus end. 5-7 nm thick Compose flexible networks primarily directly under the plasma membrane (cell cortex) Important for cell movements Lots found in microvilli (associated with structure) and muscle contraction Cytoskeleton is not a static structure Very dynamic meaning that it is in constant flux and change It is constantly being taken apart and re-built e.g. microtubules and microfilaments are constantly being disassembled and reassembled elsewhere in the cell POLYMERIZATION Assembly of monomers to extend the length of the filament Monomers added to the + end i.e. filament growth DEPOLYMERIZATION Disassembly of the filament Monomers removed from the + end Because at the plus end there is a GTP cap which promotes the growth of the microtubule. When the GTP cap is removed, disassembly occurs. The minus end is ALWAYS associated with the CENTROSOME. Only for microtubules!! Motor proteins Kinesin moves towards the + end of the microtubule (away from centrosome) Dynein moves towards to the - end of the microtubule (towards the centrosome) Motor molecules walk on microtubules Kinesin Walks along microtubules towards the fast growing or plus end Carries organelles, vesicles, microtubules Requires Exchange of ADP for ATP Hydrolysis of ATP Structure of kinesin: tail, stalk, head Tail is where an organelle or vesicle will be attached for carrying Molecular basis of Kinesin Walk When ATP is bound, we have a binding of the head to the microtubule, when ADP is bound, we have less of a binding/ loose binding of the head to the microtubule We need to move back foot forward and keep front foot in place. To do so, we must get rid of ATP that was holding back foot in place. ATP becomes hydrolyzed to ADP and Pi. weakens binding to microtubule and allows for conformational change that will swing back foot forward. But this is only possible if we remove front foots ADP with ATP. Conformational change swings foot forward and then ATP allows other foot to bind to microtubule. Constantly have motion occurring with exchange of ADP for ATP in front foot and ATP hydrolysis in back foot to provide a conformational change Lagging head: back head/foot, leading head: front head/foot Dyneins Another motor protein capable of moving on microtubules Travel in opposite direction of kinesin, towards the minus end of microtubule Transport vesicles, organelles, involved with cilia and flagella movement Microtubule-mediated colour change in fish scale cells Pigments granule’s spread out along MTs Pigments granules moved to centrosome When fish become scared, they lose all colour, when they are calm their colour returns. Pigmentation within entire scale cell. Pigmentation becomes concentrated in the middle. Organelles are carried by kinesins and dynein’s. When melanosomes are brought towards centrosomes its the dynein’s carrying them there, when it brought out away from centrosome its kinesins carrying them there. Flagella and Cilia – eukaryotes Flagella Associated with microtubules and dynein’s Whip-like or oar-like movements Used for locomotion Cilia Associated with microtubules and dynein’s Shorter than flagella Occur in higher numbers Similar movements to flagella Move fluids over the cell surface Microtubules and dynein mediate the movement of cilia and flagella Microtubule 9+ 2 complex In between microtubule doublets are dynein’s. as dynein’s are walking towards minus end it causes these microtubule doublets to bend. First part of whipping motion. As soon as dynein’s let go, the curving of doublets turns to straight orientation (whip back) Bacterial vs eukaryotic flagella Flagella of bacterial and eukaryotic cells are analogous structures. (structures that have the exact same function but are not evolutionarily related) They have the same function (locomotion) but are not evolutionarily related. They evolved independently Bacterial – circular motion, rotor mechanism Eukaryotic – whipping motion, microtubule and dynein mechanism Actin filaments or microfilaments Motor protein associated with microfilaments is myosin. Myosin has motor domains – associate with actin filaments Motor domains bind and hydrolyze ATP Microfilaments – myosin Microtubules - kinesin and dynein Myosin moves towards the plus end of actin It does not move along the microfilament. It stays in the same position Instead its interaction with the actin filament moves the actin filament itself. When ATP is bound to motor head, there is a loose binding with act in filaments. Because of this is allows myosin head to move forward towards plus direction Different from kinesin – when ATP is bound, we do not have strong binding. Hydrolysis of ATP to ADP + Pi allows for tight binding to myosin head with actin filaments and also for POWER STROKE: When the head moves back towards the right direction. The head region is tightly bound to actin filament so when it swings back to right (power stroke) it takes the actin filament with it. Pushes actin filament towards minus end. Muscle contraction Myosin and actin work together to create muscle contraction Focus on skeletal muscle – muscle attached to the skeleton Myosin motor proteins make up myosin filament Motor domains interact with actin filaments on both sides Heads of myosin’s moving towards plus end, binding and then in a power stroke, pushing the actin filament towards the minus end. Same thing happens for all filaments. Entire half of the sarcomere pushes towards minus end. Same thing for other end. Two sides become closer together = contraction. Sarcomere becomes smaller as all myosin motor domains interact with filament and push towards minus end. Minus ends closer together Actin and myosin cause many types of motility: cytokinesis Cytokinesis in animals: actin-myosin interactions draw the membrane in, dividing a cell in two Constriction allows cell to be divided two daughter cells Actin and myosin cause many types of motility: amoeboid movement Pseudopodium pushed outwards Must build actin filaments along the leading edge (direction of movement) of the moving cell. Direction of movement Depolarization – disassembled and reassembled to the right/up/down. [VIDEO] Neutrophil becomes polarized and starts chasing bacteria. The bacteria bounce around by thermal energy, moving in a random path. Eventually, neutrophil engulfs bacteria by phagocytosis. Lecture 13 All processes require energy and require enzymes ENERGY AND ENZYMES What is energy? “Energy is the capacity to cause change.” The capacity to do work or the ability to move or elicit change in matter. Energy in a System Enthalpy (H) internal energy Potential energy: stored energy due to location/chemical structures (chemical bonds, i.e. specific arrangement of atoms). A water drop sitting at the top of a waterfall has a defined amount of potential energy, Ep (top). Kinetic energy – the energy associated with motion. As the drop of water falls some of this potential energy is converted to kinetic energy (the energy of motion), Ek Other forms of kinetic energy When the water drop strikes the rocks below, its potential energy is now much lower. The change in potential energy has been transformed into an equal amount of kinetic energy in the form of motion that exerts a force, thermal energy, and sound. Result: Ep (top) = Ep (bottom) + Ek (total) Conclusion: energy is neither created nor destroyed; it simply changes form. FIRST LAW of THERMODYNAMICS Energy cannot be created or destroyed but only converted from one form to another. CONSERVATION OF ENERGY What other forms of kinetic energy exist? Electrical Light Chemical Thermal Chemical energy Energy associated with chemical reacting e.g. burning of fossil fuels Energy is stored (potential energy) in the bonds of the molecules Energy stored in bonds Stored in the chemical bonds of molecules as a form of potential energy called chemical energy. How much energy is associated with different bonds? Has to do with configuration and electron positioning. Positioning of electrons in a chemical bond is related to the electronegativity of the atoms associated with that bond. Atom with higher electronegativity is going to have electrons closer to it. Ex. oxygen has a high electronegativity so the electrons in bond of OH are drawn closet to oxygen. Creates a very strong bond because sharing of electrons is unequal. This is POLAR. Equal sharing of electrons = NON-POLAR. Creates a weak bond. Strongest bonds have least amount of potential energy, weakest have most potential energy. Metabolism the Chemical Reactions of Life Catabolic metabolic pathways Breakdown complex molecules into simpler compounds E.g. starch broken down into glucose molecules RELEASE ENERGY (EXERGONIC REACTION) Anabolic metabolic pathways Use simple molecules to build more complex ones E.g. protein synthesis USE ENERGY (ENDERGONIC REACTION) How much energy is available for work? Gibbs Free energy (G): portion of a system’s energy that is available to do work (convertible energy) Free energy is determined by Enthalpy (H, internal energy) and Entropy (S) in a system ?G = ?H – T?S Convertible energy = system internal energy – system associated energy (nonconvertible) ? (delta) = change ?G = change in free energy ?H = change in enthalpy T = absolute temperature (degree Kelvin) ?S = change in entropy SECOND LAW of THERMODYNAMICS The entropy of a system and the surroundings will increase – energy will always become more spread out Energy spontaneously disperses from being localized to becoming spread out if it is not hindered from doing so. Entropy (S) – measure of energy dispersal Heat disperse heat loss in a system Energy becomes more spread out entropy increase Heat is released from the bear – localized area to outwards How does energy disperse? Energy becomes more spread out into a larger volume entropy increase Molecule motion increases in a system – energy becomes more spread out (each molecule can take more energy states) entropy increase Number of molecules increase in a system. Energy becomes more spread out into each molecule entropy increase When does entropy increase? When energy becomes more spread out More in volume (phase change) More in number (catabolic reaction) More in molecular motion Entropy – measure of energy dispersal The part of system energy that is associated with the system and unavailable for work Energy conversion can NEVER be 100% because of the entropy increase in energy transformation. Always some not converted or available to do work and that’s because of the entropy. In a biological system (cell)… As enthalpy increases, entropy decreases. As enthalpy decreases, entropy increases. If energy is very spread out, we have less potential energy. If energy id more localized we have more potential energy. All individual monomers have higher entropy compared to when they are together. Change in Gibbs Free Energy ?G = G final state – G initial state OR ?G = G in products – G in reactants Negative ?G Exergonic reaction: energy released in reaction Reaction begins in higher energy state and ends in lower energy state Spontaneous reactions! Motion of molecules is enough to start chemical reaction Positive ?G Endergonic reaction: energy required, non spontaneous Reaction begins in lower energy state and ends in higher energy state Energy is required and used, not released Exergonic and endergonic reactions tend to be coupled. The energy released from the exergonic reaction is used to drive the endergonic reaction it is coupled to. Energetic coupling and phosphorylation Phosphorylation: Adding of terminal phosphate of ATP to another molecule. Last phosphate can readily be removed and added onto another molecule and increase energy of molecule its been added to How much energy is released when that terminal phosphate is removed? Terminal phosphate is removed from hydrolysis End up with ADP and inorganic phosphate 30.5 kJ/mol are produced – released as heat (thermal energy) leads to warming of water When you couple this with a reaction this energy can be used for phosphorylation of associated molecule called substrate and drive an endergonic reaction The energy from this exergonic reaction is going to be used to drive an endergonic reaction and that terminal phosphate is going to be used in the phosphorylation of the substrate Energetic coupling and phosphorylation Glutamine synthesis (anabolic reaction (something is being built)) Glutamic acid + ammonia glutamine = Endergonic reaction ?Gglu = +3.4 kcal/mol + ?GATP = -7.3 kcal/mol Net ?G = - 3.9 kcal/mol Energy source = ATP enough energy plus some left over heat ATP is hydrolyzed to ADP and inorganic phosphate is going to be used to phosphorylate glutamic acid Gives ‘phosphorylated intermediate’ When you create new bond on a molecule you are changing its structure = conformational difference of phosphorylated intermediate compared to glutamic acid Structural difference allows ammonia to readily bind and for creation of glutamate None of this is possible unless phosphorylation and change in potential energy associated with acid Phosphorylation cannot occur on its own, enzymes need to help Enzyme = Glutamine synthetase Binding site brings two together to permit the phosphorylation of the glutamic acid If you have a ?G greater than 0, you have an endergonic reaction and anabolic reaction. Energy is USED! Enthalpy (H) increases and Entropy (S) decreases. If you have a ?G less than 0, you have an exergonic reaction and catabolic reaction. Energy is RELEASED! Enthalpy decreases and entropy increases. Coupling is not always direct required energy carrier molecule. ATP is example of carrier molecule Activated carrier molecules Energy from exergonic reaction is picked up by carrier molecule carries the energy to the endergonic reaction that energy is then transferred and coupled to endergonic reaction carrier molecule no longer has energy goes back to exergonic reaction and picks up energy. Energetic coupling and electron transfer FAD – oxidized form When it picks up high energy electrons through process of cellular respiration its reduced to FADH2. electrons are always transferred with hydrogen NAD+ - oxidized form NADH - reduced form Where do we get out energy from? Break down organic molecules aka food Different types of food have diff amounts of chemical potential energy associated with them How do we convert food to usable energy aka ATP? Series of catabolic and anabolic chemical reactions Digestion glucose enters blood absorbed by body cells Glucose glycolysis pyruvate oxidation citric acid cycle electron transport chain NOT all energy released by a catabolic (exergonic) reaction is used for anabolic (endergonic) reactions SECOND LAW OF THERMODYNAMICS: during every energy transfer some energy is not available to do work. The entropy of the system increases (energy is more spread out) What form of energy is the unusable energy taking on? Thermal energy! Important for homeotherms who use the heat to maintain their internal temperature Metabolism helps maintain internal temperature Higher internal temp = higher metabolic rate Lower internal temp = lower metabolic rate Temperature increase = increase of metabolic rate … WHY? Lecture 14: Enzymes Class of proteins that catalyze chemical reactions Catalyze: to increase the rate of a chemical reaction Are not destroyed during the chemical reaction, therefore the same enzyme molecule can be used many times Substrate is specific to the enzyme How does an enzyme work? A region of enzyme, where substrate molecule binds – active site Usually a pocket or cleft on enzyme surface A region where catalysis occurs Substrate specificity – shape of the enzyme at substrate site allows enzyme specifically recognize and bind substrate Substrate bind to the active sites of the enzyme Enzyme – substrate complex forms and the enzyme alters shape slightly to form a tight bond with the substrate (induced fit) Product released and the enzyme is ready to catalyze another reaction How do enzymes catalyze reactions? LOWER THE ACTIVATION ENERGY (Ea) of a REACTION Enzymes DECREASE activation energy (Ea) Activation energy: amount of energy required to get a chemical reaction started Every chemical reaction required activation energy, even the ones that are spontaneous. They require energy in the form of thermal or kinetic = activation energy Rate of chemical reaction is increased when EA is decreased Need a lot of energy to push reaction forward to transition state. Enzyme specific to substrate brings down the amount of energy required to get chemical reaction going Enzymes are biological catalysts Lowering the activation energy of biological reactions Increasing the number of reactants that can reach the transition state Increasing the rate of reaction Does not change ?G (free energy) of the reaction, just changing amount of energy required to get reaction started How does enzyme decrease EA? Enzyme brings substrates close together Enzyme may serve as an ideal environment Enzyme may cause conformational changes Chymotrypsin – protease found in intestines Protease: class of enzymes that cut peptide bonds Enzymes are proteins Amino acids that make up the enzyme interacting with amino acids that make up the protein where the peptide bond has to be broken Mechanism of action Hydrophobic pocket is important for positioning the protein that is going to be coming in to have its peptide bond broken Amino acids: non polar, polar and charged polar Some of the non polar had aromatic ring to them. Aromatic ring is going to interact with hydrophobic pocket Enzyme lines up protein properly, if hydrophobic pocket is not there the protein will not line up properly and breaking of peptide bond will not occur. Now that it is lined up properly it is going to interact with serine… Cofactors Metals e.g. copper, zinc, iron Reversibly interact with enzymes Coenzymes Non protein organic molecules E.g. vitamins Reversibly interact with enzymes Examples: FAD and NAD+, both require types of vitamin D? Prosthetic groups Permanently associated with the enzyme E.g. retinal Enzyme function and environment pH Temperature pH and enzyme activity Chitinase Bacteria that live in a cool and neutral environment: optimal pH is 6 Bacteria that live in a hot and acidic environment: optimal pH is 3 Enzymes associated with certain environment evolved to work best in given environment. Cool and neutral = evolved to work at cool and neutral. Pepsin (stomach enzyme) very acidic environment pepsin works best at around pH of 2 Trypsin (intestinal enzyme) more neutral to basic, works best at pH of 8 Hydrolytic enzymes work best at pH of 5 Different enzymes work best at different pHs Temperature and enzyme activity Chitinase cool and neutral environment enzyme works optimally at relatively cooler temperature Hot and acidic environment enzyme works optimally at relatively hotter temperature As temp increases, so does activity of the enzyme – has to do with motion of molecules inside of bacteria. As temp increases, motion increases so likelihood of molecules meeting up increases. Rate of reaction increases Our enzymes work best at 37 degrees Celsius whereas some bacterial enzymes work best at hotter enzymes After peak of temperature you have a sharp decline of activity. After peak and temp keeps increasing = sharp decline, WHY? Because of structure and function. Normal protein + extreme environment (heat, pH) denatured protein (breaks bonds and changes shape and function) Temp is becoming so high that protein structure changes Temperature increase = metabolic rate increase … up to a limit 242887428765500 Enzymes begin to denature at temps above approximately 40 degrees Celsius 24098252870200Enzymes DO NOT function properly Rate of chemical reactions decrease and metabolic rate decreases Regulating enzymes Concentration of substrate and enzyme Competitive inhibition Allosteric inhibition Allosteric activation Phosphorylation Concentration Rate of reaction, in part, based on substrate and enzyme concentration. If you have a very little amount o substrate but you have lots of enzymes the reaction rate will be based on amount of substrate u have To increase reaction must increase amount of substrate In this case, increase rate by increasing the concentration Because comes a point where all enzyme molecules are saturated Increase in rate of reaction with increase in substrate concentration as long as we have lots of enzyme. As more and more enzymes get busy the reaction rate decreases. We get to a point where the enzymes are all completely saturated with substrates. This leads to a plateau the reaction continues but at a plateaued rate. The only way to change rate of reaction is to add more enzymes to the system. Plateau is called the VMAX. If we continue to increase substrate concentration, the vmax will not change. Competitive inhibition Inhibiting molecule binds to active site, preventing the bonding of the substrate Inhibiting molecules COMPETE with substrate for active site. CONCENTRATION DEPENDENT Leads to decrease of reaction rate Amount of enzyme in active form vs amount in inactive form is completely concentration dependent. If you have lots of inhibiting molecule and tiny bit of substrate what is the likelihood that the inhibiting molecule will bind to site. But if you have tons of substrate higher change substrate is going to bind with active site. Allosteric inhibition and allosteric activation Non – competitive regulation Molecule binds to enzyme but not to the active site Molecule binds to an allosteric site Regulatory site: allosteric site Inhibiting and activating molecules Do not bind to active site Allosteric control of enzyme activity Allosteric inhibitor Two confirmations one where they have a high affinity for substrate and low affinity Non – competitive binding of molecules to enzyme to decrease affinity of substrate – enzyme interaction When an inhibiting molecule is not bound the enzyme is in a high affinity state which means its going to readily bind its substrate When it is bound to the regulatory site, we have a conformational change and this leads to enzyme being in a low affinity state meaning it wont readily bind the substrate Non competitive because binding to site other than active site Allosteric activator When allosteric activator is not bound, the enzyme is in a low affinity state which means it’s not going to bind its substrate When allosteric activator is bound to allosteric site, we have conformational change and enzyme has high affinity for substrate meaning its going to readily bind substrate Concentration dependent Which affinity state the allosteric enzyme is in depends on concentration of associated regulatory molecules If you have a allosteric enzyme associated with inhibitory molecule, if you have lots of concentration of inhibiting molecule in the system this means that you have increased interaction with allosteric enzymes and increase number of allosteric enzymes that have inhibitory molecule bound and thus in a low affinity state = inactive state If you have allosteric enzymes that are associated with activator molecule and a low concentration of activating molecules then you are going to have a high concentration of enzymes that are also in the low affinity state the only way to increase number of enzymes is by increasing concentration of activating molecule so there is an increased chance of it interacting with allosteric state and binding to it. Phosphorylation Phosphorylation changes the shape and activity of proteins Phosphorylation can either turn on or turn off particular enzymes. Phosphorylation changes the shape of activation loop (conformational change) causes enzyme to be active. Dephosphorylating it is going to make it inactive. Feedback control of enzyme activity Product of enzyme 3 interacts with enzyme 1 Allosteric activation Increase activity of enzyme 1 Increase the rate reaction catalyzed by enzyme 1 Positive feedback regulation (feedback loop) Increase of the reaction increase product Feedback inhibition A pathway involving 3 different enzymes Product is inhibiting molecule of enzyme 1 When product is bound to enzyme 1, enzyme is inactive, therefore the rest of the pathway does not occur. So, product is not being made, its going to slowly decrease its concentration. As you decrease concentration probability of inhibiting molecule binding to enzyme 1 is decreased. You end up with more of enzyme in an active state, the rest of the pathway can occur and now you have the product being produced. Threonine Isoleucine (inhibiting molecule)