Molecules to Cells PDF

Molecules to Cells Lecture 1: The Chemistry of life Prof. Vincent P. Kelly All matter in the universe is comprised of atoms Atoms are composed of subatomic particles Relevant subatomic particles include Neutrons (no electrical charge) Protons (positive charge) Electrons (negative charge) Neu...

Molecules to Cells Lecture 1: The Chemistry of life Prof. Vincent P. Kelly All matter in the universe is comprised of atoms Atoms are composed of subatomic particles Relevant subatomic particles include Neutrons (no electrical charge) Protons (positive charge) Electrons (negative charge) Neutrons and protons form the atomic nucleus Electrons form a “cloud” of negative charge around the nucleus Neutron mass and proton mass are almost identical and are measured in daltons Cloud of negative charge (2 electrons) ++ Nucleus Electrons −− ++ (a) (b) The periodic table of the elements shows the electron distribution for each element Valence electrons are those in the outermost shell, or valence shell The chemical behavior of an atom is mostly determined by the valence electrons Elements with a full valence shell are chemically inert Hydrogen 1H 2 Atomic number Helium 2He First shell Second shell Third shell He Atomic mass 4.003 Element symbol Electron distribution diagram Beryllium 3Li 4Be 5B 6C 7N 8O 9F 10Ne Lithium Boron Carbon Nitrogen Oxygen Fluorine Neon Sodium 11Na Argon Magnesium Aluminum 12Mg 13AI Silicon Phosphorus Sulfur Chlorine 14SI 15P 16S 17CI 18Ar  Electrons are organised into orbitals An orbital is the three- dimensional space where an electron is found 90% of the time Each electron shell consists of a specific number of orbitals Atoms with incomplete valence shells can share or transfer valence electrons with certain other atoms These interactions usually result in atoms staying close together, held by attractions called chemical bonds Ionic interactions Matter is made up of elements An element is a substance that cannot be broken down to other substances by chemical reactions A compound is a substance consisting of two or more elements in a fixed ratio A compound has characteristics different from those of its element i.e. emergent properties Organisms are composed of matter. Matter is anything that takes up space and has mass Atoms sometimes strip electrons from their bonding partners. An example is the transfer of an electron from sodium to chlorine After the transfer of an electron, both atoms have charges A charged atom (or molecule) is called an ion Na Cl NaCl Sodium Chlorine Sodium chloride +− Na Cl Na Cl Na Cl Na+ Sodium atom Chlorine atom Sodium ion Cl− Chloride ion (an anion) (a cation) Sodium chloride (NaCl) Covalent bonds Most of the strongest bonds in organisms are covalent bonds that form a cell’s molecules A covalent bond is the sharing of a pair of valence electrons by two atoms In a covalent bond, the shared electrons count as part of each atom’s valence shell A molecule consists of two or more atoms held together by covalent bonds A single covalent bond, or single bond, is the sharing of one pair of valence electrons A double covalent bond, or double bond, is the sharing of two pairs of valence electrons Hydrogen atoms (2 H) ++ ++ ++ Hydrogen molecule (H2) Nonpolar and polar covalent bonds δ+ H O H2O H δ+ δ− δ− Atoms in a molecule attract electrons to varying degrees Electronegativity is an atom’s attraction for the electrons in a covalent bond The more electronegative an atom is, the more strongly it pulls shared electrons toward itself In a nonpolar covalent bond, the atoms share the electron equally In a polar covalent bond, one atom is more electronegative, and the atoms do not share the electron equally Unequal sharing of electrons causes a partial positive or negative charge for each atom or molecule Dioxygen O2 is non-polar Weak Chemical Interactions-Hydrogen Bonds & Van der Walls interactions Many large biological molecules are held in their functional form by weak bonds A hydrogen bond forms when a hydrogen atom covalently bonded to one electronegative atom is also attracted to another electronegative atom In living cells, the electronegative partners are usually oxygen or nitrogen atoms If electrons are not evenly distributed, they may accumulate by chance in one part of a molecule Van der Waals interactions are attractions between molecules that are close together as a result of these charges Collectively, such interactions can be strong, as between molecules of a gecko’s toe hairs and a wall surface The reversibility of weak bonds can be an advantage Region of partial negative charge δ+ δ– δ– δ+ δ+ Hydrogen bond Polar covalent bond δ+ δ+ δ+ δ– δ– δ– The elements of life About 20–25% of the 92 natural elements are required for life (essential elements) Carbon, hydrogen, oxygen, and nitrogen make up 96% of living matter Most of the remaining 4% consists of calcium, phosphorus, potassium, and sulfur Trace elements are required by an organism in only minute quantities  Biological life can be studied at numerous levels 3 Communities 4 Populations 1 The Biosphere 2 Ecosystems 7 Tissues 6 Organs 5 Organisms 9 Organelles 10 Molecules 8 Cells Life can be studied at different levels, from molecules to the entire living planet This enormous range can be divided into different levels of biological organization Emergent properties result from the arrangement and interaction of parts within a system Energy flow and chemical cycling ENERGY FLOW Light energy comes from the sun. Plants take up chemicals from the soil and air. Plants convert sunlight to chemical energy. Organisms use chemical energy to do work. Chemicals pass to organisms that eat the plants. Heat is lost from the ecosystem. Decomposers return chemicals to the soil. Chemicals The Molecules of Life Nuclei containing DNA Sperm cell Egg cell Fertilized egg with DNA from both parents Embryo’s cells with copies of inherited DNA Offspring with traits inherited from both parents All living things are made up of four classes of large biological molecules: carbohydrates, lipids, proteins, and nucleic acids Macromolecules are large molecules and are complex Large biological molecules have unique properties that arise from the orderly arrangement of their atoms Enzymes are responsible for the assembly and disassembly of macromolecules and the processing of their intermediates. The synthesis and break down polymers is catalysed by enzymes A polymer is a long molecule consisting of many similar building blocks The repeating units that serve as building blocks are called monomers Carbohydrates, proteins, and nucleic acids are polymers Enzymes are specialized macromolecules that speed up chemical reactions such as those that make or break down polymers A dehydration reaction occurs when two monomers bond together through the loss of a water molecule Polymers are disassembled to monomers by hydrolysis, a reaction that is essentially the reverse of the dehydration reaction (a) Dehydration reaction: synthesizing a polymer 123 Short polymer Dehydration removes a water molecule, forming a new bond. 1234 Longer polymer (b) Hydrolysis: breaking down a polymer 1234 Hydrolysis adds a water molecule, breaking a bond. H2O 123 H Unlinked monomer H2O The Diversity of Polymers Components Nitrogenous base Phosphate group P Sugar Nucleotide (monomer of a polynucleotide) Examples Functions Stores hereditary information Various functions in gene expression, including carrying instructions from DNA to ribosomes DNA: Sugar = deoxyribose Nitrogenous bases = C, G, A, T Usually double-stranded RNA: Sugar = ribose Nitrogenous bases = C, G, A, U Usually single-stranded A cell has thousands of different macromolecules Macromolecules vary among cells of an organism, vary more within a species, and vary even more between species A huge variety of polymers can be built from a small set of monomers Components Examples Monosaccharides: glucose, fructose Disaccharides: lactose, sucrose Polysaccharides: Cellulose (plants) Starch (plants) Glycogen (animals) Chitin (animals and fungi) Functions Fuel; carbon sources that can be converted to other molecules or combined into polymers Strengthens plant cell walls Stores glucose for energy Stores glucose for energy Strengthens exoskeletons and fungal cell walls Monosaccharide monomer The protein side chains create a diversity of properties Components Amino acid monomer (20 types) Examples Enzymes Defensive proteins Storage proteins Transport proteins Hormones Receptor proteins Motor proteins Structural proteins Functions Catalyze chemical reactions Protect against disease Store amino acids Transport substances Coordinate organismal responses Receive signals from outside cell Function in cell movement Provide structural support Side chains (R groups) Back- bone Peptide bond H2O New peptide bond forming Amino end Peptide (N-terminus) bond Carboxyl end (C-terminus) Hydrogen bond Disulfide bridge Hydrophobic interactions and van der Waals interactions Ionic bond Polypeptide backbone Orbitals and bonding dictate a molecules shape and function A molecule’s size and shape are key to its function A molecule’s shape is determined by the positions of its atoms’ orbitals In a covalent bond, the s and p orbitals may hybridize, creating specific molecular shapes s orbital z Four hybrid orbitals Tetrahedron Hybrid-Orbital Model (with ball-and-stick model superimposed) Unbonded electron O pairs (a) Hybridization of orbitals Space-Filling Model Water (H2O) Ball-and-Stick Model O H 104.5o H HH Three p orbitals xy Methane (CH4) (b) Molecular-shape models HH CC HHHH HH Molecular shape determines activity Natural endorphin Carbon Hydrogen Nitrogen Sulfur Oxygen Morphine (a) Structures of endorphin and morphine Natural endorphin Morphine Endorphin receptors Brain cell (b) Binding to endorphin receptors Molecular shape determines how biological molecules recognize and respond to one another Opiates, such as morphine, and naturally produced endorphins have similar effects because their shapes are similar and they bind the same receptors in the brain Enzymes are proteins (sometimes RNA) that accelerate reactions Chemical reactions and enzymatic reactions are similar Chemical reactions make and break chemical bonds Chemical reactions are the making and breaking of chemical bonds The starting molecules of a chemical reaction are called reactants The final molecules of a chemical reaction are called products Photosynthesis is an important chemical reaction Sunlight powers the conversion of carbon dioxide and water to glucose and oxygen 2 H2 O2 2 H2O Reactants Chemical Products reaction Leaf Bubbles of O2 Reactants 6 CO2 Carbon dioxide 6 H2O Water Sunlight Products C6H12O6 6 O2 Glucose Oxygen Chemical reactions (and enzymatic reactions) are reversible 3H2 +N⇌2NH3  Products of the forward reaction become reactants for the reverse reaction The two opposite-headed arrows indicate that a reaction is reversible Chemical equilibrium is reached when the forward and reverse reactions occur at the same rate At equilibrium the relative concentrations of reactants and products do not change Cells are chemical factories The living cell is a miniature chemical factory where thousands of reactions occur Cellular respiration extracts energy stored in sugars and other fuels Cells apply this energy to perform work An organism’s metabolism transforms matter and energy, subject to the laws of thermodynamics Metabolism is the totality of an organism’s chemical reactions Metabolism is an emergent property of life that arises from orderly interactions between molecules External environment Animal body Digestion and absorption Heat Energy lost in feces Energy lost in nitrogenous waste Heat Carbon skeletons Bio- synthesis Heat Cellular respiration ATP Cellular work Heat Organic molecules in food Nutrient molecules in body cells Energy can exist in different forms Energy is the capacity to cause change Energy exists in various forms, some of which can perform work Energy can be converted from one form to another a. Kinetic energy is energy associated with motion b. Thermal energy is the kinetic energy associated with random movement of atoms or molecules. Heat is thermal energy in transfer between objects c. Potential energy is energy that matter possesses because of its location or structure d. Chemical energy is potential energy available for release in a chemical reaction Molecules to Cells Lecture 2: Enzymes & catalysis Prof. Vincent P. Kelly The Laws of Energy Transformation  Thermodynamics is the study of energy transformations In an open system, energy and matter can be transferred between the system and its surroundings Organisms are open systems A closed system will exchange energy with its surroundings but not matter An isolated system, such as that approximated by liquid in a thermos, is unable to exchange energy or matter with its surroundings Life is governed by the laws of thermodynamics Chemical energy in food (a) First law of thermodynamics Kinetic energy (b) Second law of thermodynamics Heat CO2 + H2O The First Law of Thermodynamics According to the first law of thermodynamics, the energy of the universe is constant. Energy can be transferred and transformed, but it cannot be created or destroyed The first law is also called the principle of conservation of energy The Second Law of Thermodynamics During every energy transfer or transformation, some energy is unusable and is often lost as heat. According to the second law of thermodynamics, Every energy transfer or transformation increases the entropy of the universe Entropy is a measure of molecular disorder, or randomness Free Energy, Stability, and Equilibrium Free energy is a measure of a system’s instability, its tendency to change to a more stable state During a spontaneous change, free energy decreases and the stability of a system increases Equilibrium is a state of maximum stability A process is spontaneous and can perform work only when it is moving toward equilibrium More free energy (higher G) Less stable Greater work capacity In a spontaneous change The free energy of the system decreases (∆G < 0) The system becomes more stable The released free energy can be harnessed to do work Less free energy (lower G) More stable  Less work capacity (a) Gravitational motion (b) Diffusion (c) Chemical reaction Reactions in a closed and open system Reactions in a closed system eventually reach equilibrium and can then do no work Cells are not in equilibrium; they are open systems experiencing a constant flow of materials A defining feature of life is that metabolism is never at equilibrium Catabolic pathways release energy by breaking down complex molecules into simpler compounds. Cellular respiration, the breakdown of glucose in the presence of oxygen, is an example of a pathway of catabolism Anabolic pathways consume energy to build complex molecules from simpler ones. For example, the synthesis of protein from amino acids is an anabolic pathway ∆G < 0 ∆G = 0 ∆G < 0 ∆G < 0 ∆G < 0 Free-Energy Change, ΔG in an reaction A living system’s free energy is energy that can do work when temperature and pressure are uniform, as in a living cell Biologists want to know which reactions occur spontaneously and which require input of energy To do so, they need to determine the energy and entropy changes that occur in chemical reactions The free-energy change of a reaction tells us whether or not the reaction occurs spontaneously, generates energy (exergonic) or is not spontaneous requires energy (endergonic) Free Energy and Metabolism The concept of free energy can be applied to the chemistry of life’s processes An exergonic reaction proceeds with a net release of free energy (ΔG) and is spontaneous An endergonic reaction absorbs free energy (ΔG) from its surroundings and is nonspontaneous The change in free energy (ΔG) during a process is related to the change in enthalpy — change in total energy (ΔH)—change in entropy (ΔS), and temperature in Kelvin units (T) ΔG = ΔH − TΔS ΔG is negative for all spontaneous processes. Spontaneous processes can be harnessed to perform work Processes with zero or positive ΔG are never spontaneous (a) Exergonic reaction: energy released, spontaneous Reactants Energy Products Progress of the reaction Amount of energy released (∆G < 0) (b) Endergonic reaction: energy required, nonspontaneous Products Energy Reactants Progress of the reaction Amount of energy required (∆G > 0) Free energy Free energy The Activation Energy (EA) is the energy needed to reach the transition state 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 AB CD Transition state EA Reactants AB CD Progress of the reaction A ∆G < 0 CD Products B Free energy Enzymes are catalysts that lower the Activation Energy (EA) A catalyst is a chemical agent that speeds up a reaction without being consumed by the reaction An enzyme is a catalytic protein. For example, sucrase is an enzyme that catalyzes the hydrolysis of sucrose Enzymes speed up metabolic reactions by lowering energy barriers (EA) The active site can lower an EA barrier by i. orienting substrates correctly ii. straining substrate bonds providing a favorable microenvironment iii. covalently bonding to the substrate Sucrase OH2O HO OH Sucrose Glucose Fructose (C12H22O11) (C6H12O6) (C6H12O6) Course of reaction without enzyme Reactants Course of reaction with enzyme EA without enzyme EA with enzyme is lower ∆G is unaffected by enzyme Products Progress of the reaction Free energy Animation of an enzymes catalysed reaction Even in an exergonic reaction, energy is needed initially to break the bonds (EA) that acts as a barrier to ‘runaway reactions’. In catalysis, enzymes or speed up specific reactions by lowering the EA barrier Enzymes do not affect the change in free energy (ΔG); instead, they hasten reactions that would occur eventually © 2018 Pearson Education Ltd. Spontaneous and non-spontaneous steps in a metabolic pathway ΔG < 0 ΔG 0 Enzyme 1 A Reaction 1 Starting molecule Enzyme 2 B Reaction 2 Enzyme 3 C Reaction 3 D Product Endergonic Reactions: ATP allows transfer of energy from catabolic to anabolic reactions ATP is a renewable resource that is regenerated by addition of a phosphate group to adenosine diphosphate (ADP) The energy to phosphorylate ADP comes from catabolic reactions in the cell The ATP cycle is a revolving door through which energy passes during its transfer from catabolic to anabolic pathways Energy from catabolism (exergonic, energy-releasing processes) ADP P i Energy for cellular work (endergonic, energy-consuming processes) ATP H2O ATP (adenosine triphosphate) is the cell’s energy shuttle ATP is composed of ribose (a sugar), adenine (a nitrogenous base), and three phosphate groups The bonds between the phosphate groups of ATP’s tail can be broken by hydrolysis. Energy is released from ATP when the terminal phosphate bond is broken  This release of energy comes from the chemical change to a state of lower free energy, not from the phosphate bonds themselves Most energy coupling in cells is mediated by ATP (a) The structure of ATP Adenine Triphosphate group (3 phosphate groups) (b) The hydrolysis of ATP Adenosine triphosphate (ATP) PPP H2O PP Pi Energy Adenosine Inorganic diphosphate (ADP) phosphate Ribose ATP allows work to be performed in a cell ATP powers cellular work by coupling exergonic reactions to endergonic reactions A cell does three main kinds of work: i. Chemical work—pushing endergonic reactions ii. Transport work—pumping substances against the direction of spontaneous movement iii. Mechanical work—such as contraction of muscle cells To do work, cells manage energy resources by energy coupling, the use of an exergonic process to drive an endergonic one (a) The structure of ATP Adenine Triphosphate group (3 phosphate groups) (b) The hydrolysis of ATP Adenosine triphosphate (ATP) PPP H2O PP Pi Energy Adenosine Inorganic diphosphate (ADP) phosphate Ribose Example of how ATP can promote non-spontaneous endergonic reaction In the cell, the energy from the exergonic reaction of ATP hydrolysis can be used to drive an endergonic reaction Overall, the coupled reactions are exergonic ATP drives endergonic reactions by phosphorylation, transferring a phosphate group to some other molecule, such as a reactant The recipient molecule is now called a phosphorylated intermediate NH3 NH2 Glu ∆GGlu = +3.4 kcal/mol Glu Glutamic acid Ammonia Glutamine (a) Glutamic acid conversion to glutamine Glu ATP Glutamic acid NH3 1 P ADP 2 NH2 ADP Pi Glu Glu Phosphorylated Glutamine intermediate (b) Conversion reaction coupled with ATP hydrolysis ∆GGlu = +3.4 kcal/mol Glu NH3 ATP NH2 ADP Pi Glu ∆GATP = –7.3 kcal/mol (c) Free-energy change for coupled reaction ∆GGlu = +3.4 kcal/mol +∆GATP = –7.3 kcal/mol Net ∆G = –3.9 kcal/mol ATP hydrolysis can be used to power non-spontaneous reactions The three types of cellular work (mechanical, transport, and chemical) are powered by the hydrolysis of ATP Transport and mechanical work in the cell are also powered by ATP hydrolysis ATP hydrolysis leads to a change in protein shape and binding ability Transport protein ATP P (a) Transport work Vesicle Solute ADP Pi Pi Solute transported Cytoskeletal track ADP Pi Protein and vesicle moved ATP ATP Motor protein (b) Mechanical work 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 While bound, the activity of the enzyme converts substrate to product The reaction catalyzed by each enzyme is very specific The active site is the region on the enzyme where the substrate binds 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-substrate Enzyme complex Catalysis in the Enzyme’s Active Site In an enzymatic reaction, the substrate binds to the active site of the enzyme Enzymes are extremely fast acting and emerge from reactions in their original form Very small amounts of enzyme can have huge metabolic effects because they are used repeatedly in catalytic cycles The rate of an enzyme-catalyzed reaction can be sped up by increasing substrate concentration When all enzyme molecules have their active sites engaged, the enzyme is saturated If the enzyme is saturated, the reaction rate can only be sped up by adding more enzyme 1 Substrates enter active site. Substrates 6 Active site is available for new substrates. Enzyme 5 Products are released. 2 Substrates are held in active site by weak interactions. Enzyme-substrate complex 3 The active site lowers EA. Products 4 Substrates are converted to products. Effects of Temperature and pH The ‘local environment’ has effects on enzyme activity An enzyme’s activity can be affected by general environ- mental factors, such as temperature pH Interacting chemicals Each enzyme has an optimal temperature in which it can function Each enzyme has an optimal pH in which it can function Optimal conditions favor the most active shape for the enzyme molecule and are often reflective of the conditions in which the organism live (a) Optimal temperature for two enzymes Optimal temperature for Optimal temperature for typical human enzyme (37oC) enzyme of thermophilic (heat-loving) bacteria (75oC) 0 20 40 60 80 100 120 Temperature (oC) (b) Optimal pH for two enzymes Pepsin (stomach Trypsin (intestinal enzyme) enzyme) 0 1 2 3 4 5 6 7 8 9 10 pH Rate of reaction Rate of reaction Cofactors bind to an apoenzyme to facilitate a reaction NAD+ and NADP+ are coenzymes that have a critical role in redox reactions Cofactors are nonprotein enzyme helpers Cofactors may be inorganic (such as a metal in ionic form) or organic An organic cofactor is called a coenzyme Coenzymes include vitamins. Small quantities of these vitamins must be consumed in order for our enzymes to function correctly. Many cofactors will sit in the enzyme active site and assist the binding of the substrate. An inactive enzyme without the cofactor is called an apoenzyme, while the complete enzyme with cofactor is called a holoenzyme. Enzyme inhibitors can be used as drug molecules Competitive inhibitors bind to the active site of an enzyme, competing with the substrate Noncompetitive inhibitors bind to another part of an enzyme, causing the enzyme to change shape and making the active site less effective  Some examples of inhibitors are toxins, poisons, pesticides, and antibiotics (a) Normal binding Substrate Active site Enzyme (b) Competitive inhibition Competitive inhibitor (c) Noncompetitive inhibition Noncompetitive inhibitor Allosteric activation and inhibition of enzymes Regulation of enzyme activity helps control metabolism. Chemical chaos would result if a cell’s metabolic pathways were not tightly regulated A cell does this by switching on or off the genes that encode specific enzymes or by regulating the activity 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 Most allosterically regulated enzymes are made from polypeptide subunits, each with its own active site The enzyme complex 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 Allosteric enzyme with four subunits Regulatory site (one of four) Oscillation Non- functional active site Active site (one of four) Activator Active form Stabilized active form Inactive form Inhibitor inactive form Stabilized Feedback Inhibition controls cellular reactions to produce only what’s needed In feedback inhibition, the end product of a metabolic pathway shuts down the pathway Feedback inhibition prevents a cell from wasting chemical resources by synthesizing more product than is needed Isoleucine used up by cell Feedback inhibition Isoleucine binds to allosteric site. Active site no longer available; pathway is halted Active site available Initial substrate (threonine) Threonine in active site Enzyme 1 (threonine deaminase) Intermediate A Enzyme 2 Intermediate B Enzyme 3 Intermediate C Enzyme 4 Intermediate D Enzyme 5 End product (isoleucine) Co-operativity is another type of allosteric activation Inactive form Stabilized active form Substrate 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 Localization of Enzymes Within the Cell Structures (compartments) 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 Mitochondrion The matrix contains enzymes in solution that are involved in the second stage of cellular respiration. Enzymes for the third stage of cellular respiration are embedded in the inner membrane. 1 μm The Michaelis-Menton equation can be used to model enzyme reactions Vmax is the maximum velocity or rate at which the enzyme catalyzed a reaction. It happens when all enzyme active sites are saturated with substrate. Km is the concentration of the substrate where the velocity of the reaction is half maximal. Of enzyme concentration it is independent. A Michaelis–Menton plot shows substrate concentration [S] versus the rate of the reaction [V]. Km And Vmax can be determined from a Michaelis Menton plot. The unit of Km is moles per litre. The value of Km and Vmax can be acquired more correctly from a Lineweaver-Burk plot made by taking the reciprocal of the rate and substrate concentration. 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, particularly at the active site, can result in novel enzyme activity or altered substrate specificity Under environmental conditions where the new function is beneficial, natural selection would favor the mutated allele For example, repeated mutation and selection on the β-galactosidase enzyme in E. coli resulted in a change of sugar substrate under lab conditions Molecules to Cells Lecture 3: Membranes Prof. Vincent P. Kelly The plasma membrane is a complex mixture of lipid, protein and carbohydrate The plasma membrane is the boundary that separates the living cell from its surroundings The plasma membrane exhibits selective permeability, allowing some substances to cross it more easily than others Transport proteins are often responsible for controlling passage across cellular membranes Fibers of extra- cellular matrix (ECM) Glyco- protein Carbo- hydrates Glycolipid EXTRACELLULAR SIDE OF MEMBRANE Phospholipid Cholesterol Microfilaments of cytoskeleton Peripheral proteins Integral protein CYTOPLASMIC SIDE OF MEMBRANE Lipid molecules include fats, phospholipids and cholesterol Lipids are the one class of large biological molecules that does not include true polymers A unifying feature of lipids is that they mix poorly with water The most biologically important lipids are fats, phospholipids, and steroids Lipids consist mostly of hydrocarbon regions Fats are constructed from two types of smaller molecules: glycerol and fatty acids Glycerol is a three-carbon alcohol with a hydroxyl group attached to each carbon A fatty acid consists of a carboxyl group attached to a long carbon skeleton H2O Fatty acid (in this case, palmitic acid) Glycerol (a) One of three dehydration reactions in the synthesis of a fat Ester linkage (b) Fat molecule (triacylglycerol) Lipid molecules include fats, phospholipids and cholesterol Fats separate from water because water molecules hydrogen-bond to each other and exclude the fats In a fat, three fatty acids are joined to glycerol by an ester linkage, creating a triacylglycerol, or triglyceride  The fatty acids in a fat can be all the same or of two or three different kinds Fatty acids vary in length (number of carbons) and in the number and locations of double bonds Saturated fatty acids have the maximum number of hydrogen atoms possible and no double bonds Unsaturated fatty acids have one or more double bonds H2O Fatty acid (in this case, palmitic acid) Glycerol (a) One of three dehydration reactions in the synthesis of a fat Ester linkage (b) Fat molecule (triacylglycerol) The presence of double bonds affect the confirmation and properties of a lipid Fats made from saturated fatty acids are called saturated fats and are solid at room temperature Most animal fats are saturated Fats made from unsaturated fatty acids are called unsaturated fats or oils and are liquid at room temperature Plant fats and fish fats are usually unsaturated (a) Saturated fat Structural formula of a saturated fat molecule Space-filling model of stearic acid, a saturated fatty acid Phospholipids generate the internal and external membranes of cells In a phospholipid, two fatty acids and a phosphate group are attached to glycerol The two fatty acid tails are hydrophobic, but the phosphate group and its attachments form a hydrophilic head When phospholipids are added to water, they self-assemble into double-layered sheets called bilayers. At the surface of a cell, phospholipids are also arranged in a bilayer, with the hydrophobic tails pointing toward the interior The phospholipid bilayer forms a boundary between the cell and its external environment Choline Phosphate Glycerol Fatty acids Kink due to cis double bond (a) Structural formula Hydrophilic head Hydrophobic tails (c) Phospholipid symbol (b) Space-filling model (d) Phospholipid bilayer Hydrophobic tails Hydrophilic head Phospholipids form a bilayer in aqueous solution Hydrophilic head Hydrophobic tail Two phospholipids WATER WATER Phospholipids are the most abundant lipid in the plasma membrane Phospholipids are amphipathic molecules, containing hydrophobic (“water- fearing”) and hydrophilic (“water-loving”) regions The hydrophobic tails of the phospholipids are sheltered inside the membrane, while the hydrophilic heads are exposed to water on either side Cholesterol is an important component of cell membranes Artheriosclerosis Steroids are lipids characterized by a carbon skeleton consisting of four fused rings Cholesterol, a type of steroid, is a component in animal cell membranes and a precursor from which other steroids are synthesized A high level of cholesterol in the blood may contribute to cardiovascular disease The steroid cholesterol has different effects on the membrane fluidity of animal cells at different temperatures At warm temperatures (such as 37oC), cholesterol restrains movement of phospholipids At cool temperatures, it maintains fluidity by preventing tight packing Though cholesterol is present in plants, they use related steroid lipids to buffer membrane fluidity The Fluidity of Membranes (a) Unsaturated versus saturated hydrocarbon tails As temperatures cool, membranes switch from a fluid state to a solid state The temperature at which a membrane solidifies depends on the types of lipids Membranes rich in unsaturated fatty acids are more fluid than those rich in saturated fatty acids Membranes must be fluid to work properly; membranes are usually about as fluid as salad oil Cholesterol functions as a buffer for membrane fluidity Fluid Viscous Unsaturated tails prevent packing. Saturated tails pack together. (b) Cholesterol within the animal cell membrane Cholesterol reduces membrane fluidity at moderate temperatures, but at low temperatures hinders solidification. Cholesterol Variations in lipid composition of cell membranes of many species appear to be adaptations to specific environmental conditions Ability to change the lipid compositions in response to temperature changes has evolved in organisms that live where temperatures vary Integral membranes proteins expose hydrophobic regions to the phospholid bilayer N-terminus EXTRACELLULAR SIDE  α helix C-terminus CYTOPLASMIC SIDE δ– δ+ δ+ δ– Glycine (Gly or G) Methionine (Met or M) Alanine (Ala or A) Valine (Val or V) Leucine (Leu or L) Isoleucine (Ile or I) Proline (Pro or P) Peripheral proteins are bound to the surface of the membrane Integral proteins penetrate the hydrophobic core Integral proteins that span the membrane are called transmembrane proteins The hydrophobic regions of an integral protein consist of one or more stretches of nonpolar amino acids, often coiled into α helices Nonpolar side chains; hydrophobic Side chain (R group) Phenylalanine Tryptophan (Phe or F) (Trp or W) Dynamic structure of the cell membrane containing an integral protein In the fluid mosaic model, the membrane is a mosaic of protein molecules bobbing in a fluid bilayer of phospholipids Proteins are not randomly distributed in the membrane Membranes are held together mainly by weak hydrophobic interactions Most of the lipids and some proteins can move sideways within the membrane Rarely, a lipid may flip-flop across the membrane, from one phospholipid layer to the other Heterokaryon experiments were used to demonstrate the fluidity of the plasma membrane Mouse cell Membrane proteins Human cell Mixed proteins after 1 hour Hybrid cell Data from L. D. Frye and M. Edidin, The rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons, Journal of Cell Science 7:319 (1970). Membrane Proteins have a variety of essential functions (a) Transport Glyco- protein (d) Cell-cell recognition (e) Intercellular joining (f) Attachment to the cytoskeleton and extracellular matrix (ECM) i. Transport ii. Enzymatic activity iii. Signal transduction iv. Cell-cell recognition v. Intercellular joining vi. Attachment to the cytoskeleton and extracellular matrix (ECM) ATP Enzymes (b) Enzymatic activity Signaling molecule Receptor Signal transduction (c) Signal transduction Different proteins, often clustered in groups, embedded in the fluid matrix of the lipid bilayer Phospholipids form the main fabric of the membrane Proteins determine most of the membrane’s functions Cell-surface membranes can carry out several functions: Example of how cell-surface proteins are important in the medical field For example, HIV must bind to the immune cell-surface protein CD4 and a “co- receptor” CCR5 in order to infect a cell HIV cannot enter the cells of resistant individuals who lack CCR5 Drugs are now being developed to mask the CCR5 protein HIV Receptor Receptor (CD4) but no CCR5 (CD4) Co-receptor (CCR5) (a) HIV can infect a cell with CCR5 on its surface, as in most people. Plasma membrane (b) HIV cannot infect a cell lacking CCR5 on its surface, as in resistant individuals. Synthesis and Sidedness of Membranes and Membrane Carbohydrates Lipid bilayer Transmembrane Secretory glycoproteins Attached carbohydrate protein Golgi apparatus Vesicle Glycolipid Plasma membrane: Cytoplasmic face Extracellular face ER lumen Transmembrane glycoprotein Secreted protein Membrane glycolipid Blood- groups and glycolipids Membranes have distinct inside and outside faces The asymmetrical distribution of proteins, lipids, and associated carbohydrates in the plasma membrane is determined when the membrane is built by the ER and Golgi apparatus  Cells recognize each other by binding to molecules, often containing carbohydrates, on the extracellular surface of the plasma membrane Membrane carbohydrates may be covalently bonded to lipids (forming glycolipids) or, more commonly, to proteins (forming glycoproteins) Carbohydrates on the extracellular side of the plasma membrane vary among species, individuals, and even cell types in an individual The Permeability of the Lipid Bilayer A cell must exchange materials with its surroundings, a process controlled by the plasma membrane Plasma membranes are selectively permeable, regulating the cell’s molecular traffic Hydrophobic (nonpolar) molecules, such as hydrocarbons or oxygen and carbon dioxide, can dissolve in the lipid bilayer and pass through the membrane rapidly Hydrophilic molecules including ions and polar molecules do not cross the membrane easily Proteins built into the membrane play key roles in regulating transport Transport proteins allow passage of hydrophilic substances across the membrane Some transport proteins, called channel proteins, have a hydrophilic channel that certain molecules or ions can use as a tunnel Potassium ion Potassium ion channel protein Aquaporin is a membrane spanning protein that shuttles water Water movement through Aquaporin channel: video Channel proteins called aquaporins greatly facilitate the passage of water molecules Other transport proteins, called carrier proteins, bind to molecules and change shape to shuttle them across the membrane A transport protein is specific for the substance it moves Membrane selectivity: video Passive transport: diffusion across a membrane with no energy investment Diffusion is the tendency for molecules to spread out evenly into the available space Although each molecule moves randomly, diffusion of a population of molecules may be directional At dynamic equilibrium, as many molecules cross the membrane in one direction as in the other Substances diffuse down their concentration gradient, the region along which the density of a chemical substance increases or decreases No work must be done to move substances down a concentration gradient The diffusion of a substance across a biological membrane is passive transport because no energy is expended by the cell Molecules of dye Membrane (cross section) WATER Net diffusion (a) Diffusion of one solute Net diffusion Equilibrium Net diffusion Net diffusion Net diffusion Net diffusion Equilibrium Equilibrium (b) Diffusion of two solutes Effects of osmosis (across a artificial membrane) on water balance Water molecules can pass through pores, but sugar molecules cannot. This side has fewer solute molecules and more free water molecules. Water molecules cluster around sugar molecules. This side has more solute molecules and fewer free water molecules. Selectively permeable membrane Osmosis is the diffusion of water across a selectively permeable membrane Water diffuses across a membrane from the region of lower solute concentration to the region of higher solute concentration until the solute concentration is equal on both sides Osmosis Water balance of cells with no cell wall Tonicity is the ability of a surrounding solution to cause a cell to gain or lose water The tonicity of a solution depends on its concentration of solutes that cannot cross the membrane relative to that inside the cell Isotonic solution: Solute concentration is the same as that inside the cell; no net water movement across the plasma membrane Hypertonic solution: Solute concentration is greater than inside the cell; cell loses water Hypotonic solution: Solute concentration is less than inside the cell; cell gains water Cells without cell walls will shrivel in hypertonic solution and lyse (burst) in a hypotonic solution Hypertonic or hypotonic environments create osmotic problems for organisms that have cells without rigid walls Water balance of cells with cell walls Cell walls help maintain water balance A plant cell in a hypotonic solution swells until the wall opposes uptake; the cell is now turgid (firm) If a plant cell and its surroundings are isotonic, there is no net movement of water into the cell; the cell becomes flaccid (limp) In a hypertonic environment, plant cells lose water The membrane pulls away from the cell wall, causing the plant to wilt, a potentially lethal effect called plasmolysis Facilitated Diffusion: Passive Transport Aided by Proteins (a) A channel protein EXTRACELLULAR FLUID Channel protein CYTOPLASM In facilitated diffusion, transport proteins speed the passive movement of molecules across the plasma membrane Transport proteins include channel proteins and carrier proteins  Channel proteins provide corridors that allow a specific molecule or ion to cross the membrane Ion channels facilitate the transport of ions. Some ion channels, called gated channels, open or close in response to a stimulus. For example, in nerve cells, ion channels open in response to electrical stimulus Carrier proteins undergo a subtle change in shape that translocates the solute-binding site across the membrane. This change in shape can be triggered by the binding and release of the transported molecule Facilitated diffusion is still passive because the solute moves down its concentration gradient i.e. no energy required Carrier protein (b) A carrier protein Solute Solute The Need for Energy in Active Transport Passive transport Active transport Active transport requires energy, usually in the form of ATP hydrolysis, to move substances against their concentration gradients All proteins involved in active transport are carrier proteins Active transport allows cells to maintain concentration gradients that differ from their surroundings For example, an animal cell has a much higher potassium (K+) and a much lower sodium (Na+) concentration compared to its surroundings This is controlled by the sodium- potassium pump, a transport protein that is energized by transfer of a phosphate group from the hydrolysis of ATP Diffusion Facilitated diffusion ATP The sodium-potassium pump: a specific case of active transport i. Cytoplasmic Na+ binds to the sodium-potassium pump. The affinity for Na+ is high when the protein has this shape. ii. Na+ binding stimulates phosphorylation by ATP. iii. Phosphorylation leads to a change in protein shape, reducing its affinity for Na+, which is released outside. 6 5 4 EXTRACELLULAR [Na+] high FLUID [K+] low [Na+] low [K+] high Na+ 1 CYTOPLASM P ATP ADP Na+ Na+ Na+ Na+ Na+ 2 P Pi 3 Na+ Na+ P Na+ K+ K+ K+ K+ K+ K+ The sodium-potassium pump: a specific case of active transport iv. The new shape has a high affinity for K+, which binds on the extracellular side and triggers release of the phosphate group. v. Loss of the phosphate group restores the protein’s original shape, which has a lower affinity for K+. vi. K+ is released; affinity for Na+ is high again, and the cycle repeats. 6 5 4 EXTRACELLULAR [Na+] high FLUID [K+] low [Na+] low [K+] high Na+ 1 CYTOPLASM P ATP ADP Na+ Na+ Na+ Na+ Na+ 2 P Pi 3 Na+ Na+ P Na+ K+ K+ K+ K+ K+ K+ How Ion Pumps Maintain Membrane Potential ATP – + EXTRACELLULAR FLUID H+ H+ H+ Membrane potential is the voltage across a membrane Voltage is created by differences in the distribution of positive and negative ions across a membrane The cytoplasmic side of the membrane is negative in charge relative to the extracellular side Two combined forces, collectively called the electrochemical gradient, drive the diffusion of ions across a membrane A chemical force (the ion’s concentration gradient) An electrical force (the effect of the membrane potential on the ion’s movement) An electrogenic pump is a transport protein that generates voltage across a membrane. The sodium-potassium pump is the major electrogenic pump of animal cellsThe main electrogenic pump of plants, fungi, and bacteria is a proton pump, which actively transports hydrogen ions (H+) out of the cell H+ Proton pump CYTOPLASM – + H+ ++ H+ Coupled Transport by a Membrane Protein H+ ATP Proton pump H+ H+ H+ H+ – Sucrose + Cotransport occurs when active transport of a solute indirectly drives transport of other substances The diffusion of an actively transported solute down its concentration gradient is coupled with the transport of a second substance against its own concentration gradient H+ – – + H+ H+/sucrose cotransporter Sucrose Diffusion of H+ + H+ H+ –+  Exocytosis Small molecules and water enter or leave the cell through the lipid bilayer or via transport proteins Large molecules, such as polysaccharides and proteins, cross the membrane in bulk via vesicles Bulk transport requires energy In exocytosis, transport vesicles migrate to the membrane, fuse with it, and release their contents outside the cell Many secretory cells use exocytosis to export their products, especially cells of the digestive tract © 2018 Pearson Education Ltd. Endocytosis Phagocytosis EXTRACELLULAR FLUID Solutes Pseudopodium Pinocytosis Receptor-Mediated Endocytosis Receptor In endocytosis, the cell takes in macromolecules by forming vesicles from the plasma membrane Endocytosis is a reversal of exocytosis, involving different proteins There are three types of endocytosis i. Phagocytosis-cellular eating ii. Pinocytosis-cellular drinking iii. Receptor-mediated endocytosis Food or other particle Coated pit Coated vesicle Solutes Plasma membrane Coat protein Food vacuole CYTOPLASM Coated vesicle with specific solutes (purple) bound to receptors (red) Examples of the three types of endocytosis 5 μm Green algal cell Pseudopodium of amoeba Plasma membrane Coat protein An amoeba engulfing a green algal cell via phagocytosis (TEM) Pinocytotic vesicles forming (TEMs) In phagocytosis, a cell engulfs a particle in a vacuole The vacuole fuses with a lysosome to digest the particle  In pinocytosis, molecules dissolved in droplets are taken up when extracellular fluid is “gulped” into tiny vesicles In receptor-mediated endocytosis, binding of specific solutes to receptors triggers vesicle formation Receptor proteins, receptors, and other molecules from the extracellular fluid are transported in the vesicles Emptied receptors are recycled to the plasma membrane Top: A coated pit Bottom: A coated vesicle forming during receptor- mediated endocytosis (TEMs) 0.25 μm 0.25 μm Cholesterol intake as an example of receptor mediated endocytosis LDL LDL receptor Normal Mild Severe cell disease disease Human cells use receptor-mediated endocytosis to take in cholesterol, which is carried in particles called low-density lipoproteins (LDLs) Individuals with the disease familial hypercholesterolemia have missing or defective LDL receptor proteins Video summarizing membrane transport © 2018 Pearson Education Ltd. Molecules to Cells Lecture 4: Metabolism & major metabolic pathways Prof. Vincent P. Kelly Digestion Tongue Salivary glands Oral cavity Pharynx Esophagus Gallbladder Stomach Pancreas Small intestine Large intestine Rectum Anus Liver Digestion is the process of breaking food down into molecules small enough to absorb Mechanical digestion, chewing or grinding, increases the surface area of food Chemical digestion splits food into small molecules that can pass through membranes; these are used to build larger molecules In chemical digestion, the process of enzymatic hydrolysis splits bonds in molecules with the addition of water Chemical digestion in the human digestive system ORAL CAVITY, PHARYNX, ESOPHAGUS STOMACH SMALL INTESTINE (enzymes from pancreas) CARBOHYDRATE DIGESTION Polysaccharides Disaccharides (starch, glycogen) (sucrose, lactose) Salivary amylase Smaller Maltose polysaccharides PROTEIN DIGESTION Proteins Pepsin Small polypeptides Pancreatic trypsin and chymotrypsin Smaller polypeptides Pancreatic carboxypeptidase Small peptides Dipeptidases, carboxy- peptidase, and aminopeptidase Amino acids SMALL INTESTINE (enzymes from intestinal epithelium) Disaccharidases Monosaccharides Nucleotidases Nucleosides Nucleosidases and phosphatases Nitrogenous bases, sugars, phosphates Pancreatic amylases Disaccharides NUCLEIC ACID DIGESTION DNA, RNA Pancreatic nucleases Nucleotides FAT DIGESTION Fat (triglycerides) Pancreatic lipase Glycerol, fatty acids, monoglycerides Regulation of Appetite and Consumption Over nourishment causes obesity, which results from excessive intake of food energy with the excess stored as fat Obesity contributes to type 2 diabetes, cancer of the colon and breasts, heart attacks, and strokes Researchers have discovered several of the mechanisms that help regulate body weight Hormones regulate long-term and short- term appetite by affecting a “satiety center” in the brain i. Ghrelin, a hormone secreted by the stomach wall, triggers feelings of hunger before meals ii. Insulin from the pancreas and PYY, a hormone secreted by the small intestine after meals, both suppress appetite iii. Leptin, produced by adipose (fat) tissue, also suppresses appetite and plays a role in regulating body fat levels Ghrelin Insulin Leptin PYY Satiety center Absorption of nutrients in the small intestine The small intestine has a huge surface area due to villi and microvilli that are exposed to the intestinal lumen The enormous microvillar surface creates a brush border that greatly increases the rate of nutrient absorption Transport across the epithelial cells can be passive or active, depending on the nutrient Vein carrying blood to liver Blood capillaries Epithelial cells Large circular folds Villi Nutrient absorption Microvilli Epithelial Amino Fatty acids acids and and mono- sugars glycerides Fats Blood Lymph Muscle layers Villi Intestinal wall Lacteal Lymph vessel cell Video showing absorption across the small intestine Energy storage by the body The body stores energy-rich molecules that are not immediately needed In humans, excess energy is first stored in the liver and muscle cells in a polymer called glycogen, which is a branched chain form of glucose When glycogen stores become full, excess energy is stored in fat in adipose cells in the form of triglycerides. Triglycerides are highly reduced molecules (making multiple oxidations possible) and anhydrous (they exclude water) making them compact When fewer calories are taken in than expended, the human body expends liver glycogen first, then muscle glycogen and fat and subsequently proteins Metabolism in the “non-fasting” state Carbohydrates are broken down into glucose by various enzymes. Some glucose is used immediately, but the majority enters the blood stream triggering insulin release and the uptake of glucose into cells. Excess glucose is stored as glycogen in liver and muscle.  Fats are digested in the small intestine and packaged into lipoproteins. Excess fat is stored as droplets in fat cells. When fats are used as an energy source, they are broken down in cellular mitochondria through b- oxidation. Proteins are broken down into individual amino acids and used in body cells to form new proteins or to join the amino acid pool. Amino acids that are in excess of the body's needs are converted by liver enzymes into keto acids and urea. Keto acids may be used as sources of energy, converted into glucose, or stored as fat. Urea is excreted from the body in sweat and urine. Metabolism in the “fasting” state Carbohydrate, fats and protein are metabolized in separate processes into a common product called acetyl-CoA. Acetyl-CoA is a major metabolic pathways and is an important part of the process which creates the energy molecule called ATP (adenosine triphosphate) in the mitochondria. Approximately 2-4 hours after a meal, blood glucose concentration drops to normal baseline levels and a fasting state begins. This drop in blood sugar causes insulin levels to also decline. Another hormone, glucagon, is released and it triggers the release of glycogen and fatty acids to fuel the body until the next meal arrives. Metabolism in the “starvation” state After about 3 days of fasting, the stored glycogen in the liver and muscles is exhausted, insulin levels drop and the body ramps up its access to stored fat. As fatty acids flow into the blood stream, the liver takes the excess fats and more ketone bodies through ketosis. The muscles continue to burn fatty acids, but decrease their use of ketones. The ketone bodies then build up in the blood stream to a level at which the brain begins to oxidize them for fuel. As the brain uses the ketones, it needs less glucose, so the liver decreases the rate of gluconeogenesis. This helps preserve muscle tissue as the body doesn't need to break down the amino acids to convert them to glucose. Due to the ability to metabolise ketones, the human body can survive for long periods without eating. Video: Homeostasis through the regulating of blood sugar levels Glucose homeostasis is maintained by glycogen stores The hormones glucagon and insulin are both produced in the islets of the pancreas. Alpha cells make glucagon, and beta cells make insulin The synthesis and breakdown of glycogen is central to maintaining metabolic balance insulin and glucagon regulate the breakdown of glycogen into glucose The liver is the site for glucose homeostasis. A carbohydrate- rich meal raises insulin levels, which triggers the synthesis of glycogen. Low blood sugar causes glucagon to stimulate the breakdown of glycogen and release glucose Secretion of insulin by beta cells of the pancreas Insulin Transport of glucose into body cells and storage of glucose as glycogen Figure 42.21 Blood glucose level rises (such as after eating). NORMAL BLOOD GLUCOSE (70–110 mg glucose/ 100 mL)  Blood glucose level falls (such as after fasting). Blood glucose level rises. Breakdown of glycogen and release of glucose into blood Glucagon Secretion of glucagon by alpha cells of the pancreas Blood glucose level falls. Glycogen is a major form of energy storage Glycogen is a multibranched polysaccharide of glucose that serves as a form of energy storage, representing the main storage form of glucose in the body. A core protein of glycogenin is surrounded by branches of glucose units to form a globular granule that may contain up to 30,000 glucose units. The glycogen polymer has branch points every ten residues. Two types of alpha linkage are found, and a-1,4 and a-1,6-glycosidic bond Glycogen energy reserves can be quickly mobilized to meet a sudden need for glucose. The glycogen in humans is principally stored in the liver and skeletal muscle. Due to the large weight of muscle it holds greater amount of glycogen, however this glycogen is not available to the rest of the body. The liver glycogen stores function to maintain glucose homeostasis Glycogen synthesis and breakdown have a different purpose in liver versus muscle The liver cells use glycogen to control glucose levels. Glucose is useful for shorts bursts of energy without using oxygen. This differs from the breakdown of fats and amino acids which requires oxygen Once glucose is trapped in cells as glucose-6-phosphate, it can be converted to glucose-1- phosphate, which is then added to glycogen chains by two repetitive reactions. The primary, regulated enzyme in glycogenesis is glycogen synthase Glycogen phosphorylase is the key enzyme in glycogen breakdown i.e. control point. Muscles cannot release glucose because they do not express glucose-6-phosphatase enzyme Glucose can be formed through the process of gluconeogenesis Gluconeogenesis, the formation of glucose, allows the production of glucose from non- sugar molecules. The major non-carbohydrate precursors are lactate, pyruvate, glycerol and amino acids In mammals, gluconeogenesis principally occurs in the liver, kidney, intestine, and muscle. The liver preferentially uses lactate, glycerol, and glucogenic amino acids (especially alanine) while the kidney preferentially uses lactate, glutamine and glycerol. The intestine uses mostly glutamine and glycerol. The liver can use both glycogenolysis and gluconeogenesis to produce glucose, whereas the kidney only uses gluconeogenesis. Gluconeogenesis is not the reverse of glycolysis. Glycolysis is an exergonic process whereas gluconeogeneis is endergonic. Seven of the steps in glycolysis steps are at equilibrium. However, steps 1, 3 and 10 are exergonic and cannot be reverse.  Gluconeogenesis: Conversion of pyruvate to phosphoenolpyruvate Gluconeogenesis begins in the mitochondria with the formation of oxaloacetate by the carboxylation of pyruvate. This reaction also requires one molecule of ATP and is catalyzed by pyruvate carboxylase. This enzyme is stimulated by high levels of acetyl-CoA (produced in β-oxidation in the liver) and inhibited by high levels of ADP and glucose. To export oxaloacetate to the cytosol from the mitochondria, it has to be reduced to malate using NADH (not shown). In the cytosol, malate is again oxidized back to oxaloacetate using NAD+, where the remaining steps of gluconeogenesis take place. Oxaloacetate is decarboxylated and then phosphorylated to form phosphoenolpyruvate using the enzyme PEPCK. A molecule of GTP is hydrolyzed to GDP during this reaction. Cytosol Mitochondria Gluconeogenesis: Conversion of F1,6BP to F6P The next several steps in the reaction are at equilibrium and are the same as in glycolysis, except reversed. Fructose 1,6-bisphosphatase converts fructose 1,6-bisphosphate (F1,6BP) to fructose 6- phosphate (F6P), using one water molecule and releasing one phosphate Note that in glycolysis, phosphofructokinase 1 converts F6P and ATP to F1,6BP and ADP. In both glycolysis and gluconeogensis, this reaction is the rate-limiting step and is highly regulated by allostery. F2,6BP, AMP and Citrate all act to control this step. Cytosol Gluconeogenesis: Conversion of glucose-6-phosphate to glucose Glucose-6-phosphate is formed from fructose 6-phosphate by phosphoglucoisomerase (the reverse of step 2 in glycolysis). Glucose-6-phosphate can be used in other metabolic pathways or dephosphorylated to free glucose by glucose-6-phosphatase. Whereas free glucose can easily diffuse in and out of the cell, the phosphorylated form (glucose-6-phosphate) is locked in the cell, a mechanism by which intracellular glucose levels are controlled by cells. The final reaction of gluconeogenesis, the formation of glucose, occurs in the lumen of the endoplasmic reticulum, where glucose-6- phosphate is hydrolyzed by glucose-6- phosphatase to produce glucose and release an inorganic phosphate i.e. again it is not a simple reversal of glycolysis. Glucose is shuttled into the cytoplasm by glucose transporters located in the endoplasmic reticulum's membrane. Endoplasmic reticulum Digestion and absorption of fats: Breaking down fats in the gut In the lumen of the gut triglycerides aggregate into fat globules as a consequence of the aqueous environment i.e. they are hydrophobic The liver produces bile salts (cholesterol like molecules that are amphipathic meaning they are both hydrophilic and hydrophobic), that are stored in the gall bladder The bile salts break down the fat globules so that the triglycerides can be accessed The pancreases secretes lipases to breakdown fats into monoacylglycerols and fatty acids that can now be absorbed by epithelial cells of the gut.  FAT GLOBULE Bile salts 1 Bilesaltsbreak up fat globules, increasing exposure of triglycerides to hydrolysis. LUMEN OF SMALL INTESTINE FAT DROPLET Lipase Triglycerides Fatty acids Monoglycerides 2 Theenzyme lipase breaks triglycerides down to fatty acids and monoglycerides. Digestion and absorption of fats: uptake of fats by epithelial cells Epithelial cells absorb fatty acids and monoglycerides and then recombine them back into triglycerides These fats are coated with phospholipids, cholesterol, and proteins to form water-soluble chylomicrons Chylomicrons are transported into a lacteal, a lymphatic vessel in each villus Lymphatic vessels deliver chylomicron-containing lymph to large veins that return blood to the heart Triglycerides Phospholipids, EPITHELIAL cholesterol, CELL and proteins Chylomicron 3 Monoglycerides and fatty acids diffuse into epithelial cells and are re-formed into triglycerides. 4 Triglycerides are incorporated into water-soluble particles called chylomicrons. 5 Chylomicrons enter lacteals and are carried away by lymph. LACTEAL Digestion and absorption of fats: transport to adipose and muscle cells Predominately triglycerides are stored in fat cells (adipose cells) as fat globules in the cytoplasm. Muscle cells also store triglycerides but for ATP production In the gut, the triglycerides are placed into carrier particles called Chylomicrons Chylomicrons arise solely from the intestine and contain 90% triglyceride primarily of dietary origin and other phospholipids, cholesterol, and fat-soluble vitamins and are coated with a layer of apolipoprotein (apo A and B types). The chylomicrons bind to the membranes of fat and muscle cells and then are again broken down to the fatty acids and monoglycerides They are then reformed back into fat globules to function as high energy stores Release of fatty acids from Adipose cells Adipose cells mobilise the triglycerides into free fatty acids and glycerol in response to glucagon or adrenaline The FAs are released into the blood Serum albumin picks up the FAs and brings them to target cells Overview of fatty acid metabolism in target cells Step 1 When the body needs energy, adipose cells mobilise the triglycerides into free fatty acids and glycerol, which are released into the blood. Serum albumin picks up the FAs and brings them to target cells Acetyl-CoA is both the terminal product of FA breakdown (a process called b-oxidation) and as the initial building block for their production (referred to as FA synthesis) Note that Acetyl-CoA cannot function as a building block for carbohydrates In the breakdown of FAs they must be activated by the addition of Coenzyme A (CoA), which is then exchanged for a Carnithine molecule so that the FA can pass across the mitochondrial membrane (this will be described in more detail later on) Step 2 Step 1: Uptake and activation of fatty acids Firstly, an enzyme on the outer membrane of the mitochondria called Fatty Acyl CoA synthetase activates the fatty acid by forming a thioester bond between the sulphur atom on CoA and the carboxyl carbon of the FA. The enzyme catalyses this reaction in two steps i. An AMP molecule is transferred onto the fatty acid with the release of pyrophosphate. The hydrolysis of pyrophosphate to inorganic phosphate provides the driving force for the reaction ii. An CoA molecule is attached to the FA displacing the AMP molecule In the cytoplasms of the target cells the FA must be activated and taken up into the mitochondrial of the cell to undergo b- oxidation. This process breaks them down to Acyl- CoA molecules that can be fed into the citric acid cycle, generating ATP Step 2: Translocation of fatty acids into the mitochondrial matrix For the oxidation of FAs to occur they must be transported from the cytoplasm to the inner mitochondrial space. Following activation of a long-chain fatty acid to a FA-CoA, the CoA is exchanged for carnithine by the enzyme carnithine palmityltransferase 1 (CPT-1). The FA-carnithine molecule is then transported to the inside of the mitochondrion through the action of the Carnithine AcylCarnithine Translocase (CACT) where a reversal exchange takes place through the action of CPT-2. Once inside the mitochondrion the FA-CoA can serve as a substrate for the β-oxidation machinery, which is described next..... Overview of fatty acid metabolism: breakdown FA breakdown and synthesis are the reverse of one another e.g. the inverse steps oxidation versus reduction FA breakdown is an oxidative process that consists of 4 steps. Electrons are extracted and a carbon bond is broken to yield a FA molecule that is two carbons shorter. It is a cyclic process that is repeated. To facilitate FA breakdown a CoA molecule is added (i.e. activation) making more reactive. Step-by step breakdown i. A hydrogen is extracted and a double bond is created ii. A hydroxyl group is added to the double bond giving an alcohol iii. The alcohol group is oxidised to a carbonyl group, a ketone iv. CoA cleaves the activated FA, removing 2 carbons and generating Acetyl-CoA (oxidation) (oxidation) Overview of fatty acid metabolism: Synthesis (oxidation) FA synthesis is a reduction process and occurs under conditions where energy supply is large Again synthesis is four steps beginning with an activated Malonyl-CoA unit and Acetyl-CoA Malonyl-CoA is a key inhibitor of FA oxidation and contols the switch between fatty acid synthesis and oxidation. Malonyl CoA inhibits the enzyme carnitine palmitoyltransferase 1 (CPT1) thereby stopping FAs from entering the mitochondria. (oxidation) Fatty acid synthesis To build FAs Acetyl-CoA is needed, which comes from the matrix of the mitochondria. Fatty acid synthesis takes place in the cytosol of the cell. Therefore, its necessary to move the Acetyl-CoA into the cytoplasm. Citrate synthase converts oxaloacetate to citrate and transports it across the membrane What promotes fatty acid synthesis. When ATP is high in the mitochondria it inhibits the CAA enzyme isocitrate dehydrogenase thereby increasing the citrate levels. Fatty acid synthesis Citrate is not used in FA synthesis it is recycled back to oxaloacetate The enzyme malate dehydrogenase converts oxaloacetate to malate via malate dehydrogenase using a molecule of NADH than can subsequently by converted to pyruvate and moved back into the mitochondria Malate enzyme generates the NADPH that will be used in fatty acid synthesis Note that oxaloacetate cannot translocate across the mitochondria. Molecules to Cells Lecture 4: Mitochondrial Respiration Prof. Vincent P. Kelly Life Is Work Living cells require energy from outside sources to do work The work of the cell includes assembling polymers, membrane transport, moving, and reproducing Animals can obtain energy to do this work by feeding on other animals or photosynthetic organisms  Energy flows into an ecosystem as sunlight and leaves as heat The chemical elements essential to life are recycled Photosynthesis generates O2 and organic molecules, which are used in cellular respiration Cells use chemical energy stored in organic molecules to generate ATP, which powers work ECOSYSTEM CO2 + H2O Photosynthesis in chloroplasts Cellular respiration in mitochondria Organic molecules + O2 Light energy Heat energy ATP ATP powers most cellular work Cellular respiration Catabolic pathways yield energy by oxidizing organic fuels Catabolic pathways release stored energy by breaking down complex molecules Electron transfer plays a major role in these pathways These processes are central to cellular respiration The breakdown of organic molecules is exergonic Fermentation is a partial degradation of sugars that occurs without O2 Aerobic respiration consumes organic molecules and O2 and yields ATP Anaerobic respiration is similar to aerobic respiration but consumes compounds other than O2 CYTOSOL Glucose Glycolysis Pyruvate No O2 present: Fermentation Ethanol, lactate, or other products O2 present: Aerobic cellular respiration Acetyl CoA MITOCHONDRION CITRIC ACID CYCLE Cellular respiration: glycolysis as an example The transfer of electrons during chemical reactions releases energy stored in organic molecules This released energy is ultimately used to synthesize ATP Cellular respiration includes both aerobic and anaerobic respiration but is often used to refer to aerobic respiration Although carbohydrates, fats, and proteins are all consumed as fuel, it is helpful to trace cellular respiration with the sugar glucose C6H12O6 +6O2 →6CO2 +6 H2O + Energy (ATP + heat) Glucose Pyruvate CYTOSOL PYRUVATE OXIDATION Acetyl CoA CITRIC ACID CYCLE OXIDATIVE PHOSPHORYLATION (Electron transport and chemiosmosis) ATP Oxidative Electrons via NADH GLYCOLYSIS Electrons via NADH and FADH2 ATP Substrate-level MITOCHONDRION ATP Substrate-level Redox Reactions: Oxidation and Reduction Chemical reactions that transfer electrons between reactants are called oxidation- reduction reactions, or redox reactions In oxidation, a substance loses electrons, or is oxidized In reduction, a substance gains electrons, or is reduced (the amount of positive charge is reduced) The electron donor is called the reducing agent The electron receptor is called the oxidizing agent becomes oxidized (loses electron) becomes reduced (gains electron) becomes oxidized becomes reduced Oxidation of methane has similarity to oxidation of organic fuel in cellular respiration Some redox reactions do not transfer electrons but change the electron sharing in covalent bonds An example is the reaction between methane and O2 During cellular respiration, the fuel (such as glucose) is oxidized, and O2 is reduced Organic molecules with an abundance of hydrogen are excellent sources of high- energy electrons Energy is released as the electrons associated with hydrogen ions are transferred to oxygen, a lower energy state Methane (reducing agent) Oxygen (oxidizing agent) Water Reactants becomes oxidized Products Energy becomes reduced Carbon dioxide becomes oxidized becomes reduced Stepwise Energy Harvest via NAD+ and the Electron Transport Chain NAD+ NADH Nicotinamide (reduced form) Nicotinamide (oxidized form) 2[H] (from food) Dehydrogenase Reduction of NAD+ Oxidation of NADH Dehydrogenase In cellular respiration, glucose and other organic molecules are broken down in a series of steps Electrons from organic compounds are usually first transferred to NAD+, a coenzyme As an electron acceptor, NAD+ functions as an oxidizing agent during cellular respiration Each NADH (the reduced form of NAD+) represents stored energy that is tapped to synthesize ATP The Electron Transport Chain extracts energy from redox reactions NADH passes the electrons to the electron transport chain Unlike an uncontrolled reaction, the electron transport chain passes electrons in a series of steps instead of one explosive reaction O2 pulls electrons down the chain in an energy- yielding tumble The energy yielded is used to regenerate ATP H2 +1⁄2O2 2H + 1⁄2O2 H2O (a) Uncontrolled reaction (b) Cellular respiration Explosive release of energy 2H+ + 2e– Controlled release of energy ATP ATP ATP 2 e– 2 H+ 1⁄2 O2 H2O Electron transport chain Free energy, G Free energy, G The three stages of cellular respiration  1. GLYCOLYSIS (color-coded blue throughout the chapter) 2. PYRUVATE OXIDATION and the CITRIC ACID CYCLE (color-coded light orange and dark orange) 3. OXIDATIVE PHOSPHORYLATION: Electron transport and chemiosmosis (color-coded purple) Enzyme ADP P Substrate Enzyme Product ATP Harvesting of energy from glucose has three stages 1. Glycolysis (breaks down glucose into two molecules of pyruvate) 2. The citric acid cycle (completes the breakdown of glucose) Oxidative phosphorylation (accounts for most of the ATP synthesis) The process that generates almost 90% of the ATP is called oxidative phosphorylation because it is powered by redox reactions A smaller amount of ATP is formed in glycolysis and the citric acid cycle by substrate- level phosphorylation For each molecule of glucose degraded to CO2 and water by respiration, the cell makes up to 32 molecules of ATP Substrate level phosphorylation Glycolysis harvests chemical energy by oxidizing glucose to pyruvate PYRUVATE CITRIC GLYCOLYSIS OXIDATION ACID OXIDATIVE PHOSPHORYL- ATION ATP CYCLE Energy Investment Phase Glucose 2 ATP used Energy Payoff Phase 4ADP+4P 2NAD+ + 4e– +4H+ Net 2ADP+2P 4 ATP formed 2 NADH+2H+ 2 Pyruvate + 2 H2O 2 Pyruvate + 2 H2O 2 ATP 2NADH+2H+ Glucose 4 ATP formed – 2 ATP used 2NAD+ + 4e– +4H+ Glycolysis (“sugar splitting”) breaks down glucose into two molecules of pyruvate Glycolysis occurs in the cytoplasm and has two major phases Energy investment phase Energy payoff phase Glycolysis occurs whether or not O2 is present Glucose ATP ADP Hexokinase 1 Fructose 6-phosphate Phosphogluco- isomerase ATP Phospho- fructokinase Glucose 6-phosphate Fructose 1,6-bisphosphate Glyceraldehyde 3-phosphate (G3P) Isomerase 5 Dihydroxyacetone phosphate (DHAP) GLYCOLYSIS: Energy Investment Phase 23 Aldolase 4 ADP Glycer- aldehyde 3-phosphate (G3P) 2Pi Phospho- glycerokinase 7 Phospho- glyceromutase 8 9 2 NADH + 2 H+ phosphate dehydrogenase 6 2 ATP 2 ADP 2 H2O 2 Enolase 2 ATP 2 ADP 2 Pyruvate kinase 2 NAD+ Triose 2 2 2 1,3-Bisphospho- glycerate 3-Phospho- glycerate 2-Phospho- glycerate Phosphoenol- 10 Pyruvate pyruvate (PEP) GLYCOLYSIS: Energy Payoff Phase Oxidation of Pyruvate to Acetyl CoA PYRUVATE CITRIC GLYCOLYSIS OXIDATION ACID CYCLE OXIDATIVE PHOSPHORYL- ATION In the presence of O2, pyruvate enters a mitochondrion (in eukaryotic cells), where the oxidation of glucose is completed  Before the citric acid cycle can begin, pyruvate must be converted to acetyl coenzyme A (acetyl CoA), which links glycolysis to the citric acid cycle This step is carried out by a multienzyme complex that catalyzes three reactions 1. Oxidation of pyruvate and release of CO2 2. Reduction of NAD+ to NADH 3. Combination of the remaining two-carbon fragment and coenzyme A to form acetyl CoA CYTOSOL MITOCHONDRION Coenzyme A 13 2 CO2 Pyruvate Transport protein NAD+ NADH + H+ Acetyl CoA The Citric Acid Cycle PYRUVATE CITRIC GLYCOLYSIS OXIDATION ACID CYCLE ATP OXIDATIVE PHOSPHORYL- ATION The citric acid cycle, also called the Krebs cycle, completes the breakdown of pyruvate to CO2 The cycle oxidizes organic fuel derived from pyruvate, generating 1 ATP, 3 NADH, and 1 FADH2 per turn The citric acid cycle has eight steps, each catalyzed by a specific enzyme The acetyl group of acetyl CoA joins the cycle by combining with oxaloacetate, forming citrate The next seven steps decompose the citrate back to oxaloacetate, making the process a cycle The NADH and FADH2 produced by the cycle relay electrons extracted from food to the electron transport chain NADH NAD+ FADH2 FAD CITRIC ACID CYCLE ADP + Pi ATP 2 CO2 2 NAD+ 2 NADH + 2 H+ Acetyl CoA CoA + H+ CoA The electrons from NADH and FADH2 are fed into the electron transport chain  Following glycolysis and the citric acid cycle, NADH and FADH2 account for most of the energy extracted from food These two electron carriers donate electrons to the electron transport chain, which powers ATP synthesis via oxidative phosphorylation H2O NAD+ 8 Oxaloacetate Malate Acetyl CoA CoA-SH NADH HO +H+ 12 CITRIC 7 ACID CYCLE 3 NADH + H+ CO2 α-Ketoglutarate CO2 FADH2 Succinate 5 FAD Fumarate 6 CoA-SH 2 CoA-SH 4 NAD+ P NADH i GTP GDP Succinyl CoA ATP + H+ ADP Citrate Isocitrate NAD+ PYRUVATE CITRIC GLYCOLYSIS OXIDATION ACID CYCLE OXIDATIVE PHOSPHORYL- ATION ATP The Pathway of Electron Transport The electron transport chain is in the inner membrane (cristae) of the mitochondrion Most of the chain’s components are proteins, which exist in multiprotein complexes Electrons drop in free energy as they go down the chain and are finally passed to O2, forming H2O Electron carriers alternate between reduced and oxidized states as they accept and donate electrons Electrons are transferred from NADH or FADH2 to the electron transport chain  Electrons are passed through a number of proteins including cytochromes (each with an iron atom) to O2 The electron transport chain generates no ATP directly It breaks the large free-energy drop from food to O2 into smaller steps that release energy in manageable amounts NADH (least electronegative) 50 30 20 10 0 Cyt c1 Cyt c IV 2 e– NAD+ FADH2 Complexes I-IV each consist of multiple proteins with electron carriers. 40 FMN I Fe S 2 e– FAD Fe S II Q Cyt b Fe S Electron transport chain Cyt a Cyt a3 2 e– III 2H++1⁄2 O2 (most electronegative) H2O Free energy (G) relative to O2 (kcal/mol) Protein complex of electron carriers I H+ IV 2 H+ + 1⁄2 O2 H2O ATP synthase H+ NADH NAD+ ADP+ Pi (carrying electrons from food) ATP H+ 2 Chemiosmosis H+ H+ Cyt c Q III II FADH2 FAD 1 Electron transport chain Oxidative phosphorylation Certain electron carriers in the electron transport chain accept and release H+ along with the electrons In this way, the energy stored in a H+ gradient across a membrane couples the redox reactions of the electron transport chain to ATP synthesis The H+ gradient is referred to as a proton-motive force, emphasizing its capacity to do work Chemiosmosis: The Energy-Coupling Mechanism The energy released as electrons are passed down the electron transport chain is used to pump H+ from the mitochondrial matrix to the intermembrane space H+ then moves down its concentration gradient back across the membrane, passing through the protein complex ATP synthase H+ moves into binding sites on the rotor of ATP synthase, causing it to spin in a way that catalyzes phosphorylation of ADP to ATP This is an example of chemiosmosis, the use of energy in a H+ gradient to drive cellular work INTERMEMBRANE SPACE MITOCHONDRIAL MATRIX 3D structure of the ATP Synthase Accounting for the ATP produced by cellular respiration Electron shuttles span membrane CYTOSOL 2 NADH GLYCOLYSIS 2 NADH or 2 FADH2 2 NADH PYRUVATE OXIDATION 2 Acetyl CoA MITOCHONDRION Glucose 2 Pyruvate + 2 ATP + 2 ATP About 30 or 32 ATP Maximum per glucose: CITRIC ACID CYCLE OXIDATIVE 6 NADH 2 FADH2 PHOSPHORYLATION (Electron transport and chemiosmosis) + about 26 or 28 ATP During cellular respiration, most energy flows in this sequence: glucose → NADH → electron transport chain → proton-motive force → ATP About 34% of the energy in a glucose molecule is transferred to ATP during cellular respiration, making about 32 ATP The rest of the energy is lost as heat Fermentation and anaerobic respiration enable cells to produce ATP without oxygen Most cellular respiration depends on electronegative oxygen to pull electrons down the transport chain Without oxygen, the electron transport chain will cease to operate In that case, glycolysis couples with anaerobic respiration or fermentation to produce ATP Anaerobic respiration uses an electron transport chain with a final electron acceptor other than oxygen, for example, sulfate Fermentation uses substrate- level phosphorylation instead of an electron transport chain to generate ATP CYTOSOL Glucose Glycolysis Pyruvate No O2 present: Fermentation Ethanol, lactate, or other products O2 present: Aerobic cellular respiration Acetyl CoA MITOCHONDRION CITRIC ACID CYCLE Glucose GLYCOLYSIS 2 NAD+ 2 NADH + 2 H+ Glucose GLYCOLYSIS 2 NAD+ 2 NADH + 2 H+ NAD+ REGENERATION 2ADP+2Pi 2 ATP 2ADP+2 Pi 2 ATP 2 Ethanol (a) Alcohol fermentation 2 Lactate (b) Lactic acid fermentation NAD+ REGENERATION 2 Acetaldehyde 2 Pyruvate 2 CO2 2 Pyruvate  Alcohol fermentation and lactic acid fermentation Fermentation consists of glycolysis plus reactions to regenerate NAD+, which can be reused by glycolysis. Two common types are alcohol and lactic acid fermentation Comparing Fermentation with Anaerobic and Aerobic Respiration i. All use glycolysis to oxidize glucose and harvest the chemical energy of food ii. In all three, NAD+ is the oxidizing agent that accepts electrons during glycolysis iii. The processes have different mechanisms for oxidizing NADH to NAD+: In fermentation, an organic molecule (such as pyruvate or acetaldehyde) acts as a final electron acceptor, whereas in cellular respiration, electrons are transferred to the electron transport chain iv. Cellular respiration produces 32 ATP per glucose molecule; fermentation produces 2 ATP per glucose molecule Alcohol fermentation Glucose GLYCOLYSIS 2 NAD+ 2 NADH + 2 H+ 2ADP+2Pi 2 ATP 2 Ethanol (a) Alcohol fermentation NAD+ REGENERATION 2 Acetaldehyde 2 Pyruvate 2 CO2 In alcohol fermentation, pyruvate is converted to ethanol in two steps i. The first step releases CO2 from pyruvate ii. The second step produces NAD+ and ethanol Alcohol fermentation by yeast is used in brewing, winemaking, and baking lactic acid fermentation Glucose GLYCOLYSIS 2 NAD+ 2 NADH + 2 H+ 2 Pyruvate NAD+ REGENERATION 2 ADP + 2 P i 2 ATP 2 Lactate Lactic acid fermentation In lactic acid fermentation, pyruvate is reduced by NADH, forming NAD+ and lactate as end products, with no release of CO2 Lactic acid fermentation by some fungi and bacteria is used to make cheese and yogurt Human muscle cells use lactic acid fermentation to generate ATP during strenuous exercise when O2 is scarce An overview of fermentation: video Obligate anaerobes carry out fermentation or anaerobic respiration and cannot survive in the presence of O2  Yeast and many bacteria are facultative anaerobes, meaning that they can survive using either fermentation or cellular respiration In a facultative anaerobe, pyruvate is a fork in the metabolic road that leads to two alternative catabolic routes Molecules to Cells Lecture 6: Chloroplasts & Photosynthesis Prof. Vincent P. Kelly Life depends on photosynthesis The energy entering chloroplasts as sunlight gets stored as chemical energy in organic compounds Sugar made in the chloroplasts supplies chemical energy and carbon skeletons to synthesize the organic molecules of cells Plants store excess sugar as starch in chloroplasts and other structures such as roots, tubers, seeds, and fruits H2O O2 CO2 H2O Sucrose (export) ECOSYSTEM CO2 + H2O Photosynthesis in chloroplasts Cellular respiration in mitochondria Organic molecules + O2 Light energy Heat energy ATP ATP powers most cellular work The earth before life Earths atmosphere changed radically ~2.5 billion years ago The oldest known fossils are stromatolites (dating back 3.5 billion years). These rocks formed by the accumulation of sedimentary layers on bacterial mats Prokaryotes were Earth’s sole inhabitants for more than 1.5 billion years Most atmospheric oxygen (O2) is of biological origin. O2 produced by oxygenic photosynthesis reacted with dissolved iron and precipitated out to produce banded iron formations O2 accumulated gradually in the atmosphere from about 2.7 to 2.4 billion years ago, and then shot up rapidly to between 1% and 10% of its present level This “oxygen revolution” caused the extinction of many prokaryotic groups Some groups survived in anaerobic environments; others adapted using cellular respiration to harvest energy Colonization of land Animals Multicellular eukaryotes Present Humans.5 Origin of solar system and Earth 4.5 Prokaryotes Atmospheric oxygen 4 years ago 1 Billions of 1.5 3.5 Single-celled eukaryotes 32 2.5 1,000 100 10 1 0.1 0.01 0.001 0.0001 “Oxygen revolution” 43210 Time (billions of years ago) Atmospheric O2 (percent of present-day levels; log scale) The birth of eukaryotic life The oldest fossils of eukaryotic cells date back 1.8 billion years. Eukaryotes originated by endosymbiosis when a prokaryotic cell engulfed a small cell that would evolve into a mitochondrion Anaerobic host cells would have benefited from endosymbionts that could use oxygen as it built up in the atmosphere Over time, the host and endosymbionts would have become interdependent, forming a single organism All eukaryotes have mitochondria or remnants of mitochondria, but not all have plastids (chloroplasts and related organelles) that are typical of plants Serial endosymbiosis supposes that mitochondria evolved before plastids through a sequence of endosymbiotic events Mitochondria and plastids likely descended from bacterial cells; the original host is thought to be an archaean or close relative DNA Cytoplasm Plasma membrane Nuclear envelope Infolding of plasma membrane Nucleus Ancestral prokaryote Engulfed aerobic ER bacterium Mitochon- drion Ancestral eukaryote (a heterotroph) Engulfed photosynthetic bacterium Plastid Ancestral photosynthetic eukaryote Extant prokaryotes (still existing) have respiratory and thylakoid membranes Key evidence supporting an endosymbiotic origin of mitochondria and plastids: i. Inner membranes of both organelles are similar to plasma membranes of living bacteria ii. DNA structure and cell division are similar to bacteria iii. Both organelles transcribe and translate their own DNA iv. Ribosomes are more similar to bacterial than to eukaryotic ribosomes The evolution of eukaryotic cells allowed for a greater range of unicellular forms A second wave of diversification occurred when multicellularity evolved and gave rise to algae, plants, fungi, and animals 0.2 μm Respiratory membrane (a) Aerobic prokaryote 1 μm Thylakoid membranes (b) Photosynthetic prokaryote The Process that feeds the biosphere Plants and other photosynthetic organisms contain organelles called chloroplasts Photosynthesis is the process that converts solar energy into chemical energy within chloroplasts Directly or indirectly, photosynthesis nourishes almost the entire living world Autotrophs are “self-feeders” that sustain themselves (from CO2 and other inorganic molecules) without eating anything derived from other organisms Almost all plants are photoautotrophs, using the energy of sunlight to make organic molecules Photosynthesis occurs in plants, algae, certain other unicellular eukaryotes, and some prokaryotes Heterotrophs obtain organic material from other organisms. Almost all heterotrophs, including humans, depend on photoautotrophs for food and O2 (a) Plants (c) Unicellular eukaryotes (b) Multicellular alga (d) Cyanobacteria 40 μm (e) Purple sulfur bacteria 1 μm 10 μm Photosynthesis converts light energy to the chemical energy Chloroplasts are structurally similar to and likely evolved from photosynthetic bacteria The structural organization of these organelles allows for the chemical reactions of photosynthesis  Leaves are the major locations of photosynthesis in plants Chloroplasts are found mainly in cells of the mesophyll, the interior tissue of the leaf Each mesophyll cell contains 30–40 chloroplasts CO2 enters and O2 exits the leaf through microscopic pores called stomata A chloroplast has an envelope of two membranes surrounding a dense fluid called the stroma Leaf cross section Chloroplasts Vein Stomata CO2 O2 Chloroplast Mesophyll cell Mesophyll Thylakoid Outer membrane 20 μm Stroma Granum Thylakoid space Chloroplast Intermembrane space Inner membrane 1 μm Chloroplasts: The Sites of Photosynthesis in plants Stroma Granum Thylakoid space Thylakoid Outer membrane Intermembrane space Inner membrane 1 μm Thylakoids are connected sacs in the chloroplast that compose a third membrane system Thylakoids may be stacked in columns called grana Chlorophyll, the pigment that gives leaves their green color, resides in the thylakoid membranes Chloroplast Tracking Atoms Through Photosynthesis 6CO2 +12H2O+Lightenergy→C6H12O6 +6O2 +6H2O Photosynthesis is a complex series of reactions that can be summarized as the following equation shown on the left The overall chemical change during photosynthesis is the reverse of the one that occurs during cellular respiration Chloroplasts split H2O into hydrogen and oxygen, incorporating the electrons of hydrogen into sugar molecules and releasing oxygen as a by- product Reactants: 6 CO2 12 H2O Products: C6H12O6 6 H2O 6 O2 Photosynthesis as a redox process becomes reduced becomes oxidized Photosynthesis reverses the direction of electron flow compared to respiration Photosynthesis is a redox process in which H2O is oxidized and CO2 is reduced Photosynthesis is an endergonic process; the energy boost is provided by light The Two Stages of Photosynthesis: The light reactions Photosynthesis consists of the light reactions (the photo part) and Calvin cycle (the synthesis part) The light reactions (in the thylakoids) i. Split H2O ii. Release O2 iii. Reduce the electron acceptor NADP+ to NADPH iv. Generate ATP from ADP by photophosphorylation The light reactions convert solar energy to the chemical energy of ATP and NADPH Chloroplasts are solar-powered chemical factories. Their thylakoids transform light energy into the chemical energy of ATP and NADPH Light H2O Thylakoid Chloroplast ATP Stroma NADPH Pi REACTIONS LIGHT O2 NADP+ ADP + The Two Stages of Photosynthesis: The dark reactions The Calvin cycle (in the stroma) forms sugar from CO2, using ATP and NADPH The Calvin cycle begins with carbon fixation, incorporating CO2 into organic molecules Light H2O CO2 Thylakoid Chloroplast Stroma LIGHT REACTIONS NADP+ ADP + Pi ATP NADPH CALVIN CYCLE O2 [CH2O] (sugar) LIGHT REACTIONS Are carried out by molecules in the thylakoid membranes Convert light energy to the chemical energy of ATP and NADPH Split H2O and release O2 to the atmosphere CALVIN CYCLE REACTIONS Take place in the stroma Use ATP and NADPH to convert CO2 to the sugar G3P Return ADP, inorganic phosphate, and NADP+ to the light reactions The electrochemical properties of sunlight Light is electromagnetic energy, also called electromagnetic radiation Electromagnetic energy travels in rhythmic waves. Wavelength is the distance between crests of electromagnetic waves. Wavelength determines the type of electromagnetic energy The electromagnetic spectrum is the entire range of electromagnetic energy, or radiation Visible light consists of wavelengths (380 nm to 750 nm) that produce colors we can see. Visible light also includes the wavelengths that drive photosynthesis Light behaves as though it consists of discrete particles, called photons 10–5 nm 10–3 nm 1nm 1m 103 nm 106 nm (109 nm) 103 m 380 Visible light 450 500 550 600 650 700 750 nm Gamma rays X-rays UV Infrared Micro- waves Radio waves Shorter wavelength Higher energy Longer wavelength Lower energy Photosynthetic Pigments: The Light Receptors Light Chloroplast Reflected light Absorbed light Granum Transmitted light Pigments are substances that absorb visible light Different pigments absorb different wavelengths Chlorophyll a absorbs blue, violet and red light Chlorophyll b absorbs blue and orange light Wavelengths that are not absorbed are reflected or transmitted  Leaves appear green because chlorophyll reflects and transmits green light Measuring the absorption spectrum of chlorophyll pigments A spectrophotometer measures a pigment’s ability to absorb various wavelengths This machine sends light through pigments and measures the fraction of light transmitted at each wavelength An absorption spectrum is a graph plotting a pigment’s light absorption versus wavelength White Refracting Chlorophyll Photoelectric light prism solution tube Slit moves to pass light of selected wavelength. 1 23 Green light Galvanometer 4 0 100 The high transmittance (low absorption) reading indicates that chlorophyll absorbs very little green light. 0 100 The low transmittance (high absorption) reading indicates that chlorophyll absorbs most blue light. Blue light The absorption of visible light by chlorophyll pigments There are three types of pigments in chloroplasts: i. Chlorophyll a, the key light-capturing pigment ii. Chlorophyll b, an accessory pigment iii. Carotenoids, a separate group of accessory pigments The absorption spectrum of chlorophyll a suggests that violet-blue and red light work best for photosynthesis An action spectrum profiles the relative effectiveness of different wavelengths of radiation in driving a process. The action spectrum of photosynthesis was first demonstrated in 1883 by Theodor W. Engelmann He exposed different segments of a filamentous alga to different wavelengths Areas receiving wavelengths favorable to photosynthesis produced excess O2 allowing the growth of aerobic bacteria. Chloro- phyll a 400 Chlorophyll b Carotenoids 500 600 700 Wavelength of light (nm) (a) Absorption spectra 400 500 600 700 (b) Action spectrum Aerobic bacteria Filament of alga 400 500 600 700 (c) Engelmann’s experiment Rate of photosynthesis (measured by O2 release) Absorption of light by chloroplast pigments Structure of the chlorophyll pigments The action spectrum for photosynthesis is broader than the absorption spectrum of chlorophyll Accessory pigments, such as chlorophyll b, broaden the spectrum used for photosynthesis The difference in the absorption spectrum between chlorophyll a and b is due to a slight structural difference between the pigment molecules Accessory pigments called carotenoids may broaden the spectrum of colors that drive photosynthesis Some carotenoids function in photoprotection; they absorb excessive light that would damage chlorophyll or react with oxygen CH3 CH3 in chlorophyll a CHO in chlorophyll b Porphyrin ring: light-absorbing “head” of molecule; note magnesium atom at center Hydrocarbon tail: interacts with hydrophobic regions of proteins inside thylakoid membranes of chloroplasts; H atoms not shown Excitation of Chlorophyll by Light When a pigment absorbs light, it goes from a ground state to an excited state, which is unstable When excited electrons fall back to the ground state, excess energy is released as heat In isolation, some pigments also emit light, an afterglow called fluorescence Photon e– Excited state Heat Photon (fluorescence) Ground state Chlorophyll molecule (a) Excitation of isolated chlorophyll molecule (b) Fluorescence Energy of electron How a photosystem harvests light A photosystem consists of a reaction-center complex surrounded by light-harvesting complexes The reaction-center complex is an association of proteins holding a special pair of chlorophyll a molecules and a primary electron acceptor The light-harvesting complex consists of pigment molecules bound to proteins Light-harvesting complexes transfer the energy of photons to the chlorophyll a molecules in the reaction-center complex These chlorophyll a molecules are special because they can transfer an excited electron to a different molecule Photon Light- harvesting complexes Reaction- center complex Photosystem STROMA Primary electron acceptor e– Transfer of energy Special pair of chloro- phyll a molecules Pigment molecules THYLAKOID SPACE (INTERIOR OF THYLAKOID) Thylakoid membrane Structure of a photosystem Light H2O NADP+ ADP ATP NADPH CO2 CALVIN CYCLE [CH2O] (sugar) LIGHT REACTIONS O2 Chlorophyll (green) STROMA Protein subunits (purple) THYLAKOID SPACE A primary electron acceptor in the reaction center accepts excited electrons and is reduced as a result Solar-powered transfer of an electron from a chlorophyll a molecule to the primary electron acceptor is the first step of the light reactions Remember the purpose of the light harvesting process is to capture energy for the production of food Thylakoid membrane Structure of a photosystem There are two types of photosystems in the thylakoid membrane Photosystem II (PS II) functions first (the numbers reflect order of discovery). The reaction-center chlorophyll a of PS II is called P680 because it is best at absorbing a wavelength of 680 nm Photosystem I (PS I) is best at absorbing a wavelength of 700 nm. The reaction-center chlorophyll a of PS I is called P700 2 H+ H O + 2 complex NADP+ reductase NADPH Light 6 1/2 O2 3 1 e– Pc P700 Light e– 5 ATP Pigment molecules Primary electron acceptor e– 2 P680 Primary electron acceptor Pq e– Cytochrome Fd 8 NADP+ e– e– + H+ Photosystem II (PS II) Photosystem I (PS I) 4 Electron transport chain 7 Electron transport chain Linear electron flow through the photosystem During the light reactions, there are two possible routes for electron flow: cyclic and linear Linear electron flow, the primary pathway, involves both photosystems and produces ATP and NADPH using light energy. There are eight steps in linear electron flow: i. A photon hits a pigment in a light-harvesting complex of PS II, and its energy is passed among pigment molecules until it excites P680 ii. An excited electron from P680 is transferred to the primary electron acceptor (we now call it P680+) 2 H+ H O + 2 complex NADP+ reductase NADPH Light 6 1/2 O2 3 1 e– Pc P700 Light e– 5 ATP Pigment molecules Primary electron acceptor e– 2 P680 Primary electron acceptor Pq e– Cytochrome Fd 8 NADP+ e– e– + H+ Photosystem II (PS II) Photosystem I (PS I) 4 Electron transport chain 7 Electron transport chain Water ‘splitting’ by photosystem II 3. H2O is split by enzymes, and the electrons are transferred from the hydrogen atoms to P680+, thus reducing it to P680. P680+ is the strongest known biological oxidizing agent. The H+ are rel