Content of the present lecture 1) Chemical processes in live systems 2) Enzymes 3) Control of metabolism 4) Cell respiration 5) Photosynthesis Chemical processes in live systems Living things are different from nonliving matter ➢ create and maintain order ➢ perform never-ending stream of chemical reactions ➢ small organic molecules (AA, sugars, nucleotides, lipids) are modified to supply many other small molecules ➢ their are used to construct macromolecules that endow living systems with all of their most distinctive properties Chemical processes in live systems Cell is tiny chemical factory performs many millions of reactions every second Never-ending stream of chemical reactions Created by enzyme-catalysed reactions Metabolic pathway Interconnections of the pathways Long linear reaction of metabolic pathways are in turn linked to one another forming a maze of interconnected reactions that enable the cell to survive, grow, and reproduce The individual metabolic pathways Two opposing streams Catabolic pathways ➢ break down foodstuffs into smaller molecules ➢ produce energy and building blocks Anabolic pathways ➢ drive the synthesis of many other molecules that form the cell ➢ biosynthetic The second law of thermodynamics The degree of disorder only decrease Only for any isolated system ! A simple thermodynamic in cell ➢ a cell surrounded by a sea (rest of the universe) ➢ as the cell lives and grows, it creates internal order ➢ it constantly release heat energy (most disordered form) = increasing the intensity of molecular motion Interconversions of energy ➢ all energy forms are, in principle, convertible ➢ total amount of energy is conserved Photosynthetic organisms They use sunlight to synthesize organic molecules = photosynthesis Performed in plants, algae, and photosynthetic bacteria Photosynthesis Two stages ➢ capturing energy from sunlight and storing as chemical bond energy (waste product = oxygen) ➢ manufacturing of sugar from CO2 + water Oxidation Cell obtain energy by oxidation of organic molecules Enzymes Enzymes lower barriers … … the barriers that block chemical reactions Floating ball analogy 1/3 A barrier dam (activation energy) is lowered to represent enzyme catalysis reactants Floating ball analogy 2/3 The four walls of the box represent the activation energy barriers Floating ball analogy 3/3 A branching river with a set of barrier dams (yellow boxes) = series of enzyme-catalyzed reactions determines the exact reaction pathway How enzymes work ➢ each enzyme has an active site to which substrate binds ➢ an enzyme-substrate complex is formed ➢ reaction in the active site producing enzyme-product complex ➢ the product is then released, allowing the enzyme to bind further substrate molecules Free-energy change The free-energy change for a reaction determines whether it can occur ➢ free energy is marked G ➢ change of G must be negative = negative ΔG Reaction with a positive ΔG are energetically unfavourable, BUT ! Reaction coupling They are possible, if coupled with a second reaction with a negative ΔG The entire process must be negative Example Joining of two AAs create order in the universe Enjoy „molecular biology“ lecture for explanation Activated carrier molecules ➢ energy released by oxidation of food must be stored – as a chemical bond energy in activated „carriers“ ➢ the carriers difuse rapidly to the biosynthesis site ➢ energy in carriers is easily exchangeable Carrier molecules Activated carrier molecules are money to pay for reactions that otherwise could not take place Adenosine triphosphate = ATP Nicotinamide adenine dinucleotide (reduced) = NADH Nicotinamide adenine dinucleotide phosphate (reduced) = NADPH Principle of coupled chem. reaction A = direct oxidation of glucose to CO2 and water B = coupled reaction is analogue of carrier molecule C = carrier molecule is used in a variety of otherwise energetically unfavourable reactions ATP is most widely used hydrolysis ➢ hydrolysis of the terminal phosphate of ATP yields 11 and 13 kcal/mole of usable energy The large negative ΔG Example of a phosphate transfer reaction Transfer of the terminal phosphate in ATP to another molecule phosphorylation Biosynthesis driven by ATP hydrolysis Illustration of the formation of A-B in the condensation reaction Glutamine biosynthesis Catalysed by glutamine synthetase NADPH, a carrier of electrons ➢ produced in reactions of the general type (left) = two hydrogen atoms are removed from a substrate ➢ the oxidised form NADP+ receives one hydrogen plus one electron ➢ second proton is released into solution ➢ hydride ion has high energy, it can be easily transferred to other molecules (right) The structures of NADP+ and NADPH NAD+ and NADH are identical in structure to NADP+ and NADPH, respectively, except phosphate group ➢ Nicotinamide ring accepts two electrons together with a proton forming NADPH Coenzyme A is also an carriers ➢ carries an acetyl group thioester bond to acetate (macroergic) ➢ in the activated form is known as „acetyl CoA“ ➢ add two carbon units in the biosynthesis of larger molecules Carboxyl group transfer Carboxylated biotin ➢ transfer a carboxyl group in the production of oxaloacetate, a molecule needed for the citric acid cycle Pyruvate carboxylase ➢ the acceptor molecule is pyruvate ➢ synthesis of carboxylated biotin requires ATP ! Other activated carriers Activated carrier Group carried in highenergy linkage ATP Phosphate NADH, NADPH, FADH2 Electrons and hydrogen Acetyl CoA Acetyl group Carboxylated biotin Carboxyl group S-Adenosylmethionine Methyl group Uridin diphosphate glucose Glucose The synthesis of polymers is driven by ATP hydrolysis Condensation and hydrolysis ➢ The macromolecules of the cell are polymers formed from monomers by a condensation ➢ The macromolecules are broken down by hydrolysis The condensation reactions are all energetically unfavourable Synthesis of macromolecules The condensation step depends on energy from NTP hydrolysis The nucleic acids, proteins, and polysaccharides are produced by the repeated addition of a monomer onto one end of growing chain Alternative pathway of ATP hydrolysis Pyrophosphate is first formed ➢ this route release about twice as much free energy ➢ it forms AMP instead of ADP and then hydrolysed Synthesis of RNA or DNA ➢ multistep process driven by ATP hydrolysis For details visit our lectures in Molecular biology Control of metabolism Types of control mechanisms Allosteric regulation Feedback loop Cooperation Enzyme localisation Allosteric regulation 1) allosteric activation 2) allosteric inhibition http://www.mindcreators.com/DevelopmentalSim/ProteinRegulators.htm Allosteric activation An enzyme subject to allosteric activation is inactive in its uncomplexed form, which has a low affinity to its substrate Binding of an allosteric activator (green) stabilizes the enzyme in its high-affinity form, resulting in enzyme activity Allosteric inhibition An enzyme subject to allosteric inhibition is active in its uncomplexed form, which has a high affinity to its substrate (S) Binding of an allosteric inhibitor (red) stabilizes the enzyme in its low-affinity form, resulting in little or no activity Feedback loop ATP inhibition in katabolic metabolic pathways The metabolic pathway is inhibited by its own product Anabolic metabolic pathways – aminoacids synthesis Feedback loop substrate (threonine) threonine in active site threonine deaminase intermediate product A intermediate product B intermediate product C intermediate product D enzyme 2 enzyme 3 enzyme 4 enzyme 5 final product (isoleucine) feedback loop Ile in allosteric site the active site not bind Tre Feedback loop Feedback Inhibition of Biological Pathways.wmv Cooperation Similar to allosteric activation Substrate molecules can stimulate enzyme Substrate activate conformational changes in all subunits of nonactive enzyme substrate nonactive enzyme active enzyme Enzyme localisation ➢ Internal cellular structure is responsible for metabolic pathways separation ➢ Multienzyme complexes guarantee the correct order of individual reactions ➢ Some enzymes and enzyme complexes are build in membranes ➢ Other are in solutions inside organelles The typical example – respiratory enzymes in mitochondria Gene expression regulation Visit our lectures in Molecular biology Cell respiration Cells need energy Prokaryotic cells produce energy (ATP) in their plasma membrane So, what the eukaryotic plasma membrane does? 55 Plasma membrane in eukaryotic cells is reserved for the transport processes 56 Eukaryotes instead use the specialized membranes inside energy-converting organelles to produce most of their ATP 57 Energy-converting organelles mitochondria ➢ present in the cells of virtually all eukaryotic organisms plastids ➢ most notably chloroplasts which occur only in plants and algae 58 Internal membranes for ATP Mitochondria and chloroplasts contain large amount of internal membrane This internal membrane provides the framework for an elaborate set of electrontransport processes that produce most of the cell´s ATP 59 Chemiosmotic coupling Common pathway used by mitochondria, chloroplast, and prokaryotes to harness energy for biological purposes The term „chemiosmotic coupling“ reflect a link between the chemical bond-forming reaction that generate ATP („chemi“) and membrane-transport processes („osmotic“) 60 The coupling process electrochemical proton gradient ATP synthase 61 Other protein machine The electrochemical proton gradient also drive other membrane-embedded protein machines ➢ in eukaryotes special proteins couple H+ flow to transport metabolites in and out of the organelles ➢ in bacteria besides ATP synthesis and transport also drives rapid rotation of the bacterial flagellum 62 Electron transport chain Is formed by the entire set of proteins in membrane, together with the small molecules involved in the orderly sequence of electron transfers ➢ Electrons are transferred between one site and another by diffusible molecules that can pick up electrons at one location and deliver them to another ➢ For mitochondria, the first of these electron carriers is NAD+ 63 Electron-transport process Mitochondria convert energy from chemical fuels Chloroplasts convert energy from sunlight 64 Mitochondria-chloroplast differences Mitochondria ➢ convert energy from sunlight Chloroplast ➢ convert energy from chemical fuels ➢ drive electron transfer from carbohydrate to CO2 and water ➢ drive electron transfer from H2O to carbohydrate Inputs for mitochondrion are products of the chloroplasts 65 The mitochondrion Bounded by double membrane Cristae – infoldings of inner membrane that encloses matrix Matrix – inner semifluid containing respiratory enzymes 66 The mitochondrion function Glucose Pyruvate CO2 + H2O glycolysis + O2 ATP ATP ATP ATP ATP ATP 15x more ATP 67 Localization of mitochondria ➢ in some cells form long moving filaments ➢ in others they remain fixed in one position Near sites of high ATP utilization ➢ packed between myofibrils in a cardiac muscle cell ➢ wrapped tightly around the flagellum in a sperm 68 Citric acid cycle electron transport ATP synthesis 69 Citric acid cycle Molecular Biology of the Cell/Media/Animation/2.5 Citric Acid Cycle 70 Oxidative phosphorylation ➢ the process of ATP production from ADP + Pi ➢ it is performed on inner mitochondrial membrane 71 The general mechanism ➢ high energy electron pass along the electron-transport chain ➢ three respiration enzyme complexes pump H+ out ➢ the resulting electrochemical proton gradient across the inner membrane drives H+ back through ATP synthase ➢ the enzyme complex synthesize ATP in matrix 72 ATP synthase – a molecular turbine Molecular Biology of the Cell/Media/animation/14.4 ATP Synthase 73 Photosynthesis 75 6 CO2 + 6 H2O C6H12O6 + 6 O2 The chloroplast Chloroplasts are organelles in which photosynthesis occur ➢ They perform photosynthesis during the daylight hours ➢ The products of photosynthesis, NADPH and ATP are used to production of many organic molecules ➢ Based on biochemical and genetic evidence the chloroplasts are suggested as descendant of oxygen-producing photosynthetic bacteria that were endocytosed and lived in symbiosis with primitive eukaryotic cells 76 The chloroplast structure Bounded by double membrane Inner membrane infolded ➢ Forms disc-like thylakoids, which are stacked to form grana ➢ Suspended in semi-fluid stroma Green due to chlorophyll ➢ Green photosynthetic pigment ➢ Found ONLY in inner membranes of chloroplast 77 The chloroplast scheme stroma inner membrane outer membrane thylakoids with cavity inside 78 Electron micrographs 1/3 Wheat leaf cell containing chloroplasts, the nucleus, and mitochondria surrounded by a large vacuole 79 Electron micrographs 2/3 Single chloroplast 80 Electron micrographs 3/3 A high-magnification view of two grana (a stack of thylakoids) 81 Capturing energy in chloroplasts Two categories of reactions in plant 1) photosynthetic electron-transfer reactions (light) ➢ energy from sunlight energizes an electron in chlorophyll ➢ electron is transported along respiratory chain ➢ producing H+, ATP, NADPH and as waste product O2 2) carbon-fixation reaction (dark) ➢ ATP and NADPH serve as a source of energy and reducing power – to drive the conversion of CO2 to carbohydrate ➢ begins in chloroplast stroma and continue in cytosol 82 Photosynthesis in a chloroplast Formation of ATP, NADPH and O2 and conversion of CO2 to carbohydrates are separate processes although elaborate feedback mechanisms Several chloroplast enzymes required for carbon fixation are inactivated in the dark and reactivated by light-stimulated electron-transport processes 83 Carbon fixation Is catalysed by ribulose bisphophate carboxylase (Rubisco) 84 Calvin cycle ➢ 3 molecules of CO2 are fixed by Rubisco to 6 molecules of 3- P-glycerate ➢ cycle of reactions that regenerates the 3 molecules of ribulose-1,5-bis-P ➢ this leaves 1 molecule of glyceraldehyde 3-P 85 Calvin cycle - results Each CO2 molecule converted into carbohydrate consumes a total of 3 molecules of ATP and 2 molecules of NADPH 3CO2 + 9ATP + 6NADPH + water glyceraldehyde-3-phosphate + 8Pi + 9ADP + 6NADP+ 86 Central role of gly-3-P Glyceraldehyde-3-phosphate serves as a central intermediate in glycolysis ➢ It is exported to the cytosol where is converted into fructose-6-P or glucose-1-P ➢ these sugars are converted to another carbohydrates, especially to sucrose ➢ glyceraldehyde-3-phosphate that remains in the chloroplast is converted to starch in the stroma 87 C3 and C4 plants Compartmentalisation at low CO2 concentrations 88 Electron transfer Restoration of the photosynthetic centre Charge separation transfer from chlorophyll to electron transport chain in membrane 89 The structure of chlorophyll ➢ a magnesium atom is held in a porphyrin ring, which is related to the porphyrin ring that binds iron in heme ➢ process of energy conversion begins when a photon excites a chlorophyll molecule causing an electron in the chlorophyll to move from one orbital to another ➢ excited molecules return back by photochemical reaction 90 91 Photosynthetic Electron Transport and ATP Synthesis.avi