Popular in Cellular Molecular (Dr. Mallery and Dr. DiResta)
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Topic 7- Protein biochemistry Techniques & Procedures for Isolating Proteins Isolation and purification of a protein is based upon its chemical and physical properties: size, shape, charge, and solubility of the protein one wants to isolate Protein isolating techniques Fractionation & Isolation Methodologies for Cellular Molecules... Grind and Find 1st is cell & tissue homogenization methodologies uses mortar/pestle, homogenizers, sonicators to break open cells *Grind cells in tissue grinders or in beaters or sonicadors in an osmotically buffered medium with enzyme’s substrate and in an isotonic media on ice. They result in ruptured cells producing a liquefied cellular homogenate Centrifugation Applies centrifugation force to separate homogenate components produces a pellet (solid part) & supernatant (liquid portion) : pellent/supernatant - 1924 T. Svedberg (1926 Nobel Prize) invents analytical centrifuge - 1938 G. Beherens isolates nuclei by centrifugation - 1946 George Palade, Hogeboom & Schneider perfect differential centrifugation in sucrose - 1954 C. deDuve isolates lysosomes (1st organelle not defined by microscopy) 2 major types: Differential centrifugation vs. rate-zonal * Speeds from 100xg to 1,000,000 x Separation examples: - via differential centrifugation - Repeated centrifugations at increasingly higher speeds ** separates organelles by their mass and density 300g = whole cells & nuclei, 12,000g = mitochondria/chloroplasts 100,000g = microsomes, ribosome, etc... & by size (Velocity sedimentation) ** or by buoyant density… equilibrium sedimentation – density gradient centrifugation Comparing shallow and steep gradient Chromatography Separation of molecules based on differences in their structure &/or physical properties when interacting with a stationary support media. PARTITION chromatography.... developed by R.L.M Synge *Small MW molecules are partitioned between phases of 2 different solvents (water/alcohol) on a support media PAPER chromatography... uses cellulose as support media (Chlorophylls) THIN LAYER chromatography... media is silica gel on glass plates/columns Protein Separations Proteins can separate based upon their physical properties: size, charge, affinity for ligands and shape Column Chromatography **Done in cylindrical glass column, on permeable support media or matrix, which retards flow of selected molecules, while others pass through Kinds of glass column chromatography: - Some examples: 1. Ion exchange chromatography (charged ligands) – separates molecules with different charges. And column matrix retrads passing proteins of opposite charge DEAE cellulose [diethylaminoethyl cellulose] CM-cellulose [carboxymethyl cellulose] 2. Size exclusion chromatography (gel filtration) – which seperates on the basis of molecular size Affinity chromatography – based on biological activity, an inert media polymer with a ligand Ex. Antibody, enzyme substrate binds a specific protein Molecular imprint polymer chromatography – is a technique to create template-shaped cavities in polymer matrices with memory of the template molecules to be used in molecular recognition Some possible uses: Removing contaminants during manufacture: - fungal poison aflatoxin - in food processing - during purification of optical isomers - removal of unwanted substances in blood - as enzyme substitutes (active sites) - as sensors to sniff out toxins/pathogens... atrazine (a herbicide) & sarin gas - detection of date rape drugs Isolation, Visualization, &/or Separation of a protein... Proteins migrate in an electrical field at rates that depend upon their net charge, size, and shape Electrophoresis- Separation of proteins by charge and size in a support media (gel- polyacrylamide/starch knwn as PAGE), which separates the protein molecules on the basis of their size, conformation and charge Isoelectric focusing: Proteins are separated in a gel of a continuous pH gradient. They move to a point in gel where they equal to their isoelectric point (no net charge) SDS-PAGE: A polyacrylamide gel electrophoresis. Proteins are treated with ionic detergent to separate according to their size. SDS binds to protein **In protein chemistry, detergents are used to lyse cells (releasing soluble proteins) and solubilizing membrane proteins and lipids. 2D-electrophoresis: A combination of IF and SDS together DNA electrophoresis: separates polynucleotide strands by charge Identification of proteins presence and its quantification Identification is usually performed by spectrophotometry, they measure intensity of light beam before & after light passes through a liquid solvent with sample dissolved in it, compares the two light intensities over a range of wavelengths. Percent transmittance – ratio of intensity of light passing through the sample to the intensity of light shining on sample multiplied by 100 Absorbance – is the log of the transmittance Topic 8- Enzymology ENZYMOLOGY Enzymes comes from the Greek word “ensymo” which means in-leaven - Enzymes are catalytic proteins regulating reaction rates - Ex. they control metabolism - Enzymes are molecules (mostly protein vs. ribozymes) that accelerate or catalyze chemical reactions (A--->B) in cells by breaking old covalent bonds & forming new covalent bonds - A biological catalyst… but, different from a chemical catalyst - Have complex structure (sequence of aa’s) act only upon a specific substrate do not change direction (energetics) of rx. Catalysis = acceleration of rate of a chemical reaction via a catalyst - enzymes convert substrates to products w/o changing themselves Ex. cAMP protein kinase A - group of enzymes that transfer P from ATP to SER on proteins - (PKA) refers to a family of enzymes whose activity is dependent on the level of cyclic AMP (cAMP) in the cell. [low levels inactive] - PKA is also known as cAMP-dependent protein kinase EC (188.8.131.52) is a quarternary holoenzyme of 2 Regulatory and 2 Catalytic subunits 1 enzyme crystallized UREASE, in 1926 by James Sumner + 2 NH 2C0-NH 2 + 2 H O2 -----> 4 NH 4 + 2 CO 2 Sumner was the first to crystallize a protein fraction with catalytic activity - 1,000s of enzymes have been purified and crystallized (except for ribozymes, which also have catalytic activity)- all are proteins - Proof that a biological activity is due to an enzyme has usually been to note the loss of biological activity as a result of proteolytic digestion Enzyme reaction path E + S <--> [ES] <--> E + P - Enzymes catalyze reactions by lowering the energy of activation... Ea - There is no difference in free energy between an enzyme catalyzed reaction and an uncatalyzed reaction, but a non-enzyme catalyzed reaction requires higher energy input than an enzyme catalyzed reaction. Catalase 2H 2 2 ---> 2 H 2 + O 2 condition Ea (cal/mol) Rate (lt/mol/sec) no catalyst 18,000 1.0 x 10 -7 Fe catalyst 10,000 56.0 catalase 2,000 4.0 x 10 6 - Turnover number - number of substrate molecules converted to product per second for a single enzyme molecule Some terminology Cofactors: non-protein compounds, required for a protein's biological activity... such as small inorganic ions. Many metal ions: Cu, Mg, Mn, Fe, which act as activators &/or inhibitors of activity... Coenzymes : small non-protein ligands that catalyze reactions… - +/- electrons, transfer a group, form or break a covalent bond - Includes: *Vitamins *Lipo+c acid + + *NAD /NADP - redox coezymes (dehydrogenations) – H carrier and/or electron transfer *FAD – another redox coenzyme CoASH: acyl carrier via sulfhydryl Prosthetic group: large complex organic molecules, which may have catalytic activity (heme) Active site: portion of enzyme, which folds to precisely fit (trypsin and cAMP-PKA) the contours of a substrate via weak electrostatic interactions and facilitates bond reactivity Enzyme substrate complex: unique joining of enzyme and substrate at active site What does an enzyme substrate complex do? - Holds substrate out of aqueous solution - Holds substrate in specific orientation, close to transition state to allow reaction to occur - Reduces ability of free rotation and molecular collisions with non- reactive atoms - Allows amino acid side chains to alter local environment: can change ionic strength, pH, add or remove H-bonds to substrate while precisely holding substrate so that it can be acted upon - Ex. Rubisco Analogy: a nut and bolt held in your hand decreases the entropy of their binding over a random mix of nuts and bolts in a toolbox Mechanism of enzyme action (3 examples): The chemical reaction scheme by which an enzyme acts upon its substrate 1. Lysozyme example - An enzyme that cuts polysaccharide glycosidic bonds by hydrolysis (adds H O2 - Active site is a long groove, holding six sugar units. It has 2 acidic side chain (GLU & ASP) that hold the substrate - Breaks glycosidic bond via bond strain and distortion of GLU & ASP - Enzyme binding of substrate bends bonds from a stable state, lowering the Ea. The acidic side group of GLU provides a proton to attack a glycosidic bond - ASP favors hydrolysis of glycosidic bond 2. Protease - Hydrolysis of peptide bonds: serine proteases catalytic sites hold ser195, asp102 and his57 ( the OH of ser195 attacks the carbonyl carbon of peptide bond and transition state is held by hydrogen bonds 3. Catalytic action of cAMP dependent protein Kinase A of ATP delocalized by LYS and Mg , new bond forms between SER-OH REMEMBER: A proper shape of an enzyme is critical to its ability to catalyze a reaction Major classes of enzymes 1. Oxidoreductases (dehydrogenases) – catalyzes oxidation reduction, often using coenzyme as NAD /FAD - Ex. Alcohol dehydrogenase: ethanol + NAD + acetaldehyde + NADH 2. Transferases – catalyzes the transfer of functional group - Ex. Hexokinase: D-glu + ATP d-GLU-6-P + ADP 3. Hydrolases – Catalyzes hydrolytic reactions adds water across C- C bonds - Ex. Carboxypeptidase A 4. Lyases – Cleaves C-C, C-O, C-N & other bonds often generating a C=C bond or ring - Ex. Pyruvate decarboxylase: pyruvate acetaldehyde + CO 2 5. Isomerases (mutases) – catalyze isomerizations 6. Ligases – condensation of 2 substrates with splitting of ATP - Pyruvate Carboxylase Enzyme kinetics Defines the physical & chemical properties of enzyme by mathematical and/or graphical expression of the reaction rates of enzyme catalyzed reactions Characteristic Enzyme Kinetic Curves: How to determine if the reaction A B is enzymatic (observed enzyme kinetic reaction curves st 1. Rate (0.8 mL/O /m2n) vs. ( E ) – a classical 1 order linear plot 2. Rate vs. pH 3. Rate vs. Temperature 4. Rate vs. most characteristic curve (a plot of Velocity vs. Substrate rate of O 2roduction or rate of “dye” production) - Velocity vs. Substrate curve defines a rectangular hyperbola - At low substrate concentration, rate is directly proportional to the substrate concentration - At higher substrate concentration, rate declines giving a rectangular hyperbola st nd 1 and 2 order reaction kinetics are not sufficient to describe the rectangular hyperbola of enzyme reactions * In 1913 Leonor Michaelis and Maud Menten proposed a mathematical modeling of enzyme reactions using algebraic expressions and rate constants to define a rectangular hyperbola Some assumptions in the M&M derivation 1. Rate formation ES complex from E + P is negligible Ex. Can ignore the rate constant K 2. Rate limiting step is disassociation of ES to E + P = K (speed of dissociation) - Rate constant is the number of molecules converted by this reaction per unit time 3. An important state of the enzyme is term free enzyme which is able to react Derivation of Michaelis – Menten Enzyme kinetics *The derivation of equation occurs at a time when the rate of formation of ES complex is equal to rate of destruction (break down) - Ex. At equilibrium when [substrate] [enzyme] so that the tostl E is bound in ES complex and thus reaction works like a 1 order reaction enzyme catalyzed reaction; the rate limiting equation thus becomes destruction of ES * It would be easy if we could measure the concentration of [ES], say in a spectrophotometer, but its presence is fleeting so then the real function of M&M kinetics is to be able to express [ES] in terms of E & S alone, which are measurable quantities Km – The Michaelis Constant - Is applicable to enzyme reactions involving a single substrate is “inherent tendency” of reactants to interact chemically for that reaction - Is a constant that is independent of [E] and defined by [S] - Is a mathematical interpretation of an enzyme reaction - Is a measure of how efficiently an enzyme converts a substrate to product - Is the substrate concentration when enzyme velocity is equal to ½ Vmax *Km is a characteristic physical property for each and every different enzyme - It is independent of [E] and is independent of [S] - It measures “relative affinity” of an enzyme for its substrate *Suppose there’s more than 1 substrate for an enzyme - Kinases – enzymes that transfers phosphate groups from high- energy donor molecules, such as ATP, to specific substrates, each enzyme having its own Km Ex. One enzyme with 2 substrates each with following Km’s = 1 mg & 25 mg, thus one takes less substrate to reach same rate - Many enzymes have individual steps in a complex reaction sequences, each step has its own Km’s Ex. Km is a complex function of many individual rate constants - Not all enzymes are treatable by M&M kinetics. Most regulatory enzymes (multi-subunits) are not treatable by M&M kinetics Some ways to determine Km 1. By exploration from a graph of an M&M standard curve 2. By transformation of M&M curve graphically (greater accuracy) *Slope equals Km and y-intercept equals V-max Enzyme inhibition- reducing reaction rates velocity via binding of non- substrate molecule 2 classes of inhibitors: 1. Irreversible – inhibitor molecule can not be easily removed from enzyme, thereby reducing the total number of working enzyme molecules - Enzyme is physically altered by binding of inhibitor reducing its amount Ex.: Alkylating agents like idoacetamide, organophosphorus, some antibiotic drugs such as penicillin form covalent link to enzyme active site 2. Reversible – enzyme activity may be restored by overcoming the effect of the inhibitors and are thus treatable by M&M kinetics * 2 major types of reversible: - Competitive - Non-competitive Competitive inhibition - Inhibitor binds to E and forms [EI] complex at the active site - Inhibitor often looks like substrate... fools active site & binds. - Extent of inhibition is concentration dependent, [inhibitor is often at fixed conc], thus it can be overcome if [S] is very high. Ex. [S] >>> [I] *One classical example is malonic acid, inhibition of SDH *Easy to demonstrate is via graphical plots ► shows Vmax is SAME, but Km value is increased Non-competitive inhibition - Inhibitor binds to E, forms an [EI] complex, not at the active site inhibitor often bears no structural relationship to substrate - Removes a net amount of active enzyme. Ex. lowers total [E] Ex. it cannot be overcome, even if [S] is very high easy to demonstrate via graphical plots ► shows Km is SAME & Vmax is different Some specific Examples of Native Enzyme Inhibition: 1. Irreversible Enzyme Inhibition & Mechanism of Action of some Inhibitors... a. Sarin gas: a nerve gas agent forms a covalent link to serine at active site of enzymes b. Antibiotics - a natural molecule (often made by bacterial cells) that can kill other bacterial cells (& without hurting eukaryotic cells: they're insensitive) - Ex. Penicillin- any one of a group of antibiotics derived from the fungus Penicillium, similar to bacterial peptidoglycans, which irreversibly binds at active site of peptidoglycan transpetidase cross linkage & reducing the enzyme's activity, weakening bacterial cell walls that results in rupturing & cell death. Cross-linkage Competitive Enzyme Inhibition and some Mechanisms of Drug Action ACE Inhibitors - drugs that bind to the enzyme’s active site & reduce its activity. - ACE - Angiotensin Converting Enzyme: a proteolytic enzyme that cuts Angiotensin I, a polypeptide of 10 amino acids into Angiotensin II (of 8 amino acids). - Angiotensin II promotes hypertension ( high blood pressure - HBP ) via vasoconstriction * In 1960's John Vane discovered teprotide in brazilian pit viper venoms, a nonapeptide (9aa = Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) which can functions as a competitive inhibitor by binding to the active site of the ACE enzyme. * Today there are a number of synthetic peptide ACE inhibitors, all called "prils" Another competitive inhibitor example: Viagra - The enzyme guanylate cyclase in response to increase nitric oxide (NO) converts GTP into cGMP, which leads to smooth muscle relaxation (vasodilation) of the internal cushions of the helicine arteries resulting in increased blood flow. The enzyme cGMP phosphodiesterase converts cGMP to 5'GMP favoring decreased blood flow and normalcy. The action of Viagra prevents the degradation of this cGMP by cGMP phosphodiesterase keeping the increased blood flow rates high. - Viagra is often a potent competitive inhibitor, via competitive to PDE-type 5, which then results in continued vasodilation of smooth muscle resulting in increased blood flow Topic 9- metabolic design The design of metabolism Cells do obey laws of chemistry and physics, therefore they are capable of transforming energy Energy in cells is housed in a molecule’s chemical bonds Cell’s possess “Chemical Potential Energy Cellular energy also occurs in such forms as chemical concentration gradients across membranes can diffuse from higher to lower Electrical gradients (potential differences) across membranes a separation of charge as much as 200,000 volts Cellular transformation of energy make up metabolism and the pathways Energy in Cell structure energy out What we need to be able to do is measured energy in systems (energy’s ability to do work) Willard Gibbs applied the principles of Thermodynamics to chemical systems to determine the energy content & changes within a chemical reaction. He derived the free energy equations ΔG = ΔH - T ΔS *ΔG is measure amount energy in system able to do work (to stay away from equilibrium)... *Disorder increases (thus entropy increases) when useful energy, that which could be used to do work, is dissipated as heat... *ΔG is a numerical measure of how far a chemical reaction is from equilibrium. Biological systems are ISOTHERMAL, e.g., held at constant temp/pressure (4 to ≈ 65 ) and thus in biological systems ΔH ≈ 0 Cellular systems tend to break down (become disordered) & their potential/kinetic energy is converted into heat Cells combat this slide toward disorder by continually consuming new energy (from sun) to repair/replace structures broken down. ENERGY IN ----> CELL STRUCTURE ----> ENERGY OUT How do we measure energy changes in cells FREE ENERGY ΔG = ΔH - T ΔS free energy enthalpy entropy *ΔG is a numerical measure of how far a reaction is from equilibrium *ΔG is measure amount energy in system able to do work (to stay away from equilibrium) Disorder increases (thus entropy increases) when useful energy, that which could be used to do work, is dissipated as heat o o Most Cells are ISOTHERMAL - (4 to @ 45 ) - thus ΔH = 0 (Function with a very narrow temp range) Thus, ΔG can predict the direction of cellular reactions (TOWARD EQUILIBRIUM and Maximum ENTROPY) CHEMICAL REACTION A <---> B Which Way? ΔG = ΔG0’ + R T ln [ p]/[ r] Change in free energy content of a reaction...depends upon: 1. energy is stored in molecule's covalent bonds 2. remember, temperature is negligible... cells are isothermal, i.e., ΔG = actual free energy ΔGo' = standard free energy [change under std conditions] R = gas constant ( 2 x 10 Kc/mol) o T = absolute temp (-273 K) ln = natural log (conversion l10= 2.303) at equilibrium ΔG = 0 and [p]/[r] = Keq *EXERGONIC REACTION - is one which releases free energy Product [B] <<< energy REACTANT [A] [stored in covalent bonds] ex: burning wood (cellulose) glucose monomers = potential energy breaks bonds, release heat & light ---> CO & H O 2 2 cell respiration - (heterotrophy) - cellular burning of glucose slower, multi-step process to capture & release energy.... as ATP *ENDERGONIC REACTION - requires input of energy for A --> B Product [B] >>>energy Reactant [A] ex: photosynthesis - (autotrophy) glucose made from CO + 2 O -2light---> C H O6 12 6 energy poor vs. energy rich Which way will this reaction go? Gibbs free energy equation (ΔG = ΔH - T ΔS) derivation to... ΔG = ΔG0’ + R T ln [p]/[r] and ΔG0’ = -RT ln Keq Which way this reaction goes is dependent upon existing concentrations? ΔG ’ @ [equilibrium] the Keq of DHAP/G3P is 22.4 ΔG ’ = -  lg 10 22.4 = -  (1.35) = - 1,842 cal/mole (R → L) ΔG = ΔG ’ + RT ln [P] / [R] but, when DHAP = 0.1M & G3P = 0.001M ΔG = -1842 c/m + (-1364) (lg 0.010 = (-1842) + (-1364)(-2) = + 886 cal/mole Thus under standard condition the reaction is favored from G3P toward DHAP (-ΔG), but under a specific cellular condition, where the ratio of reactant and products is changed, the reaction may not be favored, and may go in other direction from DHAP to G3P This is what happens in glycolysis, the pathway shifts R/P ratios and pulls rx to G3P Many biological systems can lead to an increase in order... decrease in entropy ( ΔS < 0)? How does Metabolism create more order via cellular chemical reactions? COUPLED REACTIONS - which often involve... the linking of the hydrolysis of ATP (a favored reaction) to a thermodynamically unfavored reaction, thereby creating some biological order (greater molecular structure). If ΔG for the reaction B + C --> D is +, but if it less than the ΔG of ATP hydrolysis and phosphate transfer then the reaction may be driven to completion by coupling to ATP hydrolysis ATP hydrolysis Phosphate Transfer Synthesis of glutamine-by-glutamine synthetase Synthesis of sucrose by sucrose-6-phosphate synthase Most cells use ATP hydrolysis energy and couple it to processes as: *Conformational changes in enzyme, like kinases, which phosphorylate proteins converting then from inactive to active (& vice versa); energy gained in the stressed conformation is released, when the protein relaxes. *The ability to couple reactions is one of the unique properties of living organisms... "The secret of Life is not some "vital force", but the unique energy operations of the Second Law of Thermodynamic at the molecular level." Energy is the currency of biology. By harvesting electrons from a stunning range of starting materials, Earth’s organisms produce adenosine triphosphate (ATP), which powers biological reactions. In the case of mammals and most eukaryotes, sugars and other organic molecules are common electron sources, the oxidation of which drives ATP production. Bacteria and archaea can use a range of other chemicals, from sulfide to iron to ammonium. Cells take up these electron-rich molecules and capture their electrons, which jump down an electron transport chain in the mitochondrial or cell membrane. As electrons move along the membrane toward a final electron acceptor, protons are pumped from the cell’s interior to the exterior, setting up a chemical gradient. Finally, protons stream back into the cell, releasing the chemical pressure and generating ATP. With each energy-requiring reaction, from flagella construction to cell division and growth, cells draw upon their ATP bank. Topic 10 – Cell respiration Cellular energetics How cells make ATP? Molecularly its mainly by phosphorylation of ADP Cellularly its through organelle based processes in eukaryotes Heterotrophic Metabolism (mitochondria) Cell Respiration ("Oxidation of Foods”) Anaerobic & Aerobic Reactions... *Substrate Level Phosphorylation *Oxidative Level Phosphorylation Autotrophic Metabolism (chloroplast) Photosynthesis Photophosphorylation 3 Primary Molecular Mechanisms of ADP Phosphorylation 1. Substrate Level Phosphorylation – transfer of “P” to ADP or GDP 2. Chemiosomosis – Oxidative ( electron capture leads to) Phosphorylation of ADP Oxidation subst-H (glu) + NAD + o NADH red+ product (CO +2 H2O) NADH --- e transport H proton motive force --- ATP synthase ATP 3. Photosynthetic phosphorylation – sunlight provides energy to add P to ADP Light + H O + NADP NADPH –e H ATP synthase 2 ATP 1 st: Cellular Respiration (Heterotrophy) Oxidation of food molecules Evolution of anaerobic/ aerobic metabolism was a major step in the history of life on planet earth Defines as: series cytoplasmic & mitochondrial *Linked enzymatic pathways *Catalyzing stepwise OXIDATION of food molecules to make ATP and NADH physiological view: uptake of O 2 & release of CO 2 biochemical view: O red2ction & CO producti2n 3 Stages: 1. Digestion of food polymers [CH O] 2 n --> [CH O2 monomers 2. Production of AcoA (via pyruvateO glycolysis &/or FA-oxidation 3. Oxidation of AcoA to CO & 2 O 2rebs Cycle & Electron transport chain 4 reaction systems of cellular respiration: (glucose oxidation of eukaryotes) A combination of anaerobic and aerobic metabolic pathways Glyco-lysis (in the cytoplasm) Glucose pyruvate + NADH + ATP Krebs cycle (in the mitochondria) Acetyl-coenzymeA CO + H 2 + NA2H + GTP +FADH 2 Electron Transport Chain (in mitochondrial membrane) Passage of e’s from NADH/FADH to O 2 H O 2 H g2adient + ATP synthase (in mitochondrial membrane) Mitochondrial membrane 24 subunit protein, which makes ATP as + H move into mitoplasm with their chemical gradient GLYCO-LYSIS: Greek (glykos) = "sweet" + "splitting" Glycolysis is a metabolic pathway found universally in biological systems. It is the metabolic pathway which converts glucose via a series of reactions to 2 molecules of pyruvate. As a result of these reactions, a small amount of ATP and NADH are produced. Most of the metabolic energy derived from glucose comes from the entry of pyruvate into the citric acid cycle and oxidative phosphorylation. These pathways occur under aerobic conditions. Under anaerobic conditions, pyruvate can be converted to lactate in muscle or ethanol in yeast. Among the important findings determined as part of the elucidation of the glycolytic pathway were: The finding by Hans Buchner and Eduard Buchner that fermentation, the conversion of sucrose to ethanol, could occur in the absence of a living cell. The finding of a hexose bi-phosphate intermediate (fructose 1-6 biphos) in glycolysis The activities required for the reactions to occur were composed of a heat-labile, non-dialyzable substance (enzymes) and a heat-stable, dialyzable substance (coenzymes). Many scientists, including Gustav Embden, Otto Meyerhof, Carl Neuberg, Jacob Parnas, Otto Warburg, Gerty Cori, and Carl Cori, contributed to the complete determination of the pathway. Glycolysis is formally known as the Embden-Meyerhof- Parnas Pathway. Glycolysis takes place in the cytoplasm of the cell. The goal of the initial reactions of glycolysis is to convert glucose into fructose 1,6- bisphosphate. This traps glucose in the cell as glucose 6-phosphate and forms a phosphorylated compound (fructose 1,6- bisphosphate) that can be cleaved intophosphorylated 3-carbon intermediates - DHAP & 3PGALD. These 3-carbon units can then be used to generate ATP by substrate level phosphorylation and by making pyruvate & NADH for the citric acid cycle and oxidative phosphorylation. The first step in glycolysis is the phosphorylation of glucose by ATP to form glucose 6-phosphate. This reaction, catalyzed by the enzyme hexokinase, traps glucose in the cell. The second step in glycolysis is the isomerization of glucose 6- phosphate to fructose 6-phosphate. This converts the sugar from a 6- membered pyranose to the 5-membered furanosestructure and involves the conversion of an aldose into a ketose. This reaction is catalyzed by the enzyme phosphoglucose isomerase. The third step in glycolysis is a second phosphorylation to form fructose 1,6-bisphosphatecatalyzed by the enzyme phosphofructokinase. Phosphofructokinase is an allosteric enzyme controlled by ATP and other metabolites. The importance of the control ofphosphofructokinase will be discussed in a later lesson. Up to this point no energy in the form of ATP has been generated by glycolysis. Two ATP's have been used. The second stage of glycolysis involves the cleavage of the 6-carbon fructose 1,6-bisphosphate to 3- carbon sugars followed by isomerizations. The generation of 3-carbon units from the 6-carbon sugar is catalyzed by the enzyme aldolase. In this reaction, dihydroxyacetone phosphate and glyceraldehyde 3- phosphate are generated. The glyceraldehyde 3-phosphate generated in this reaction can continue directly in the glycolytic pathway. Dihydroxyacetone phosphate must be converted to glyceraldehyde 3-phosphate in order to continue in the pathway. This isomerization is catalyzed by the enzyme triose phosphate isomerase. At equilibrium most of the 3-carbon sugar is in the form of dihydroxyacetone phosphate. However, the removal of glyceraldehyde 3-phosphate in further glycolytic reactions allows the formation of more glyceraldehyde 3-phosphate from dihydroxyacetone phosphate, shifting the equilibrium of the reaction. The next reaction of glycolysis generates a high potential phosphorylated compound, 1,3-bisphosphoglycerate. This compound is formed from glyceraldehyde 3-phosphate by the action of the enzyme glyceraldehyde 3-phosphate dehydrogenase. In this reaction, inorganic phosphate (P) ii incorporated into the C-1 position forming an acyl phosphate with NAD serving as the electron acceptor. The high energy potential of 1,3-bisphosphoglycerate is used to form ATP from ADP and P. Tiis reaction is carried out by phosphoglycerate kinase. These reactions result in the formation of NADH and ATP. All of the reactions can be seen in the diagrams in Panel 4-1 on page 112 of the first edition of ECB by Alberts, et al, 1998. The last part of glycolysis involves the formation of pyruvate and more molecules of ATP. This is accomplished by a rearrangement of 3- phosphoglycerate to form 2-phosphoglyceratefollowed by a dehydration to form phosphoenolpyruvate (PEP). The final nearly irreversible reaction is the formation of ATP and pyruvate catalyzed by the enzyme pyruvate kinase. Pyruvate is the precursor molecule for: 1. lactic acid ferementation (anaerobic respiration) 2. alcohol fermentation 3. aerobic respiration (tricarboxylic acid cycle - Krebs cycle) *10 step enzymatic pathway that converts hexose (C 6 --> 2 PYR (C ) +34 ATP (2 net) + 2 NADH - anaerobic = no direct requirement of oxygen, but can occur in oxygen's presence - cytoplasmic location (► seems too structured for randomness of aqueous solutions?) A - energy investment phase... (coupled Rx's) phosphorylation of low energy intermediates to high energy ones B - energy capture phase... * 2 key reactions types: A - redox reaction (glyceraldehyde3-P-dehydrogenase) B - two substrate level phosphorylations substrate-P + ADP ---> substrate + ATP GLYCO-LYSIS... Ancillary Pathways or how cells use products of glycolysis Fates of PYRUVATE... if anaerobic – "fermentations"... 1. alcoholic fermentation - via alcohol dehydrogenase 2. lactic acid respiration - via lactic acid dehydrogenase if aerobic - 3. Krebs Cycle - pyruvate enters mitochondrial Krebs Fates of NADH... = shuttles mitochondrial inner membrane is impervious to NAD+/NADH, but an evolutionary advantage is... shuttles moving e's from the cytoplasmic NADH's into mitochondrial NADH or FADH for eventual use in the ETC... 2 Alpha-Glycerol Phosphate Shuttle and Malate Shuttle We saw that one of the fates of the NADH made in glycolysis was to be recycled for use, by donating its proton and electrons to make either alcohol or lactic acid. If the electrons were not subsequently donated from the NADH, cells would eventually become depleted of working NAD, and energy metabolism would come to a halt at this enzymatic step. So there has been evolutionary pressure to select cells that can recycle this electron carrier to keep energy metabolism rolling along. In those tissues which do not carry out fermentations, this pair of electrons represents a certain amount of potential energy in the form of additional ATPs that could be made if the electron pair could be transferred to the electron transport chain inside the mitochondrial membranes. Unfortunately, the inner mitochondrial membrane isimpermeable to NAD & protons. Thus there are two pools of NAD in most cells. One is the inner mitochondrial pool and the other is the cytoplasmic pool of NAD. With the electron transport chain being sequestered in the inner membrane of the mitochondria, some cells have evolved a mechanism to move the electron pair, associated with the cytoplasmic pool of NAD, into the electron transport chain? SKELETAL MUSCLE & BRAIN tissues operate the GLYCEROL-3- PHOSPHATE SHUTTLE. The glycolytic pathway's cytosolic NAD accepts an electron pair and becomes NADH (+ H ). The electron pair is then transferred to dihydroxyacetone phosphate (DHAP). This step regenerates the NAD, thereby allowing the glycolytic pathway to continue its important operations using the oxidized form of this coenzyme (NAD). [see figure] Reducing the DHAP by addition of electrons converts it from DHAP to glycerol-3- phosphate. This newly formed compound easily migrates across the outer mitochondrial membrane. Again, the membrane is a barrier to proton and NAD flux, but not to glycerol-3-P. Once inside the mitochondria, the electron pair is donated from glycerol-3-P to FAD which becomes FADH , an2 which converts the glycerol-3-phosphate back into DHAP. The mitochondrial DHAP is free to wander back out into the cytosol. The FADH produced can cycle into the mitochondrial electron transfer chain and produce 2 ATP for each pair of electrons transferred from the cytosolic NADH. Two important points are demonstrated by the glycerol-3-phosphate shuttle: 1. the NAD is regenerated for use by glycolysis and 2. the potential energy of the cytoplasmic captured e's are realized to make ATPs. LIVER, KIDNEY & HEART MUSCLE tissue operate on the same principle, but use a different shuttle. This other pathway, which moves reducing equivalents from cytosol into mitochondria, is called theMALATE SHUTTLE, because malate is formed by oxidation of oxaloacetic acid - [OAA + NADH ---> malate + NAD]. The malate moves into the mitochondria, where it is converted back into OAA and NADH. [ malate + NAD ---> oxaloacetate + NADH ] The NADH can the be used by the electron transfer chain of the miotchondria to produce ATP. For each pair of electrons transferred from the cytosol to NADH, 3 molecules of ATP can be made. [seefigure] Note: a new value for ATP per NADH is 2.5 and ATP per FADH is 2 1.5. You may see the numbers ATP per NADH = 3 and ATP per FADH = 2 in some texts. For more information on the re-evaluation of 2 the number of ATP's/nucleotide coenzyme see Hinkle, et al. "Mechanistic stoichiometry of mitochondrial oxidative phosphorylation". Biochemistry 30:3576-82, 1991. The different values of 30 or 32 ATP/glucose depend on the method used to transport cytoplasmic NADH, formed by glycolysis, into the mitochondria, i.e. the shuttles. Electrons from glucose captured onto NADH are transferred into mitochondria using cytoplasmic via glycerol-3-P, where mitocgondrial G3PDH passes e's onto FADH 2 common to brain tissue, skeletal muscle, fungi & plants. KEY REACTIONS of GLYCOLYSIS *Redox reaction involving NAD + *Substrate level phosphorylation Summary of GLYCOLYSIS 2 ATP to initiate pathway 2 substrate level phosphorylations makes 2 ATP (net), 2 NADH, and 2 PYRUVATE anaerobic (oxygen not required) & Fermentations (lactate & alcohol) & the Shuttles KREBS CYCLE [Citric Acid Cycle or Tricarboxylic Acid Cycle] A cyclical biochemical pathway resulting in aerobic oxidation of cellular fuels, such as carbs, fatty acids, & amino acids, while making CO , 2 O,2& ATP. Overview of aerobic respiration - includes pathways of Glycolysis, Krebs cycle, & Electron Transport System Overall reaction of Kreb's Cycle: acetyl-CoA + 3NAD + E-FAD + GDP + P + 2H O ----2 ---> CoASH + 3NADH + E-FADH + GTP2+ 2CO 2 Enzymes of Krebs cycle 5 dehydrogenases - ISDH, a KGDH, SDH, MDH, & PDH 2 hydratases - aconitatse & fumarase (+/- H O 2 1 thiokinase - succinyl-CoA 1 synthetase - citrate 2 multi-enzyme complexes - each with 60 proteins & 5 coenzymes each 1. pyruvate dehydrogenase 2. alpha ketoglutarate ► but prior to Krebs we need to get pyruvate into mitochondria Pyruvate Dehydrogenase Complex * Catalyzes the Oxidative Decarboxylation of an alpha-Keto acid 3 enzymes: 60 proteins subunits [is larger than a ribosome] A. pyruvate decarboxylase *12 dimers = 24 identical subunits B. lipoamide reductase transacetylase (reductase) *8 trimers = 24 identical subunits, each 3 lipoates C. dihydrolipoyl dehydrogenase *6 dimers 12 subunits with FAD PDH complex reactions A. Pyruvate decarboxylase B. Lipoamide reductase C. Dihydrolipoyl dehydrogenase 5 coenzymes: CoASH Lipoate Thiamine pyrophosphate E-FAD + NAD Coenzyme A is chemically a thiol [-SH], it can react with carboxylic acids to form thioesters, thus it functions as an acyl (acetyl) group carrier. The Kreb's cycle (citric acid cycle, tri-carboxylic acid cycle) is common pathway for oxidation of all fuel molecules including amino acids, sugars, and fatty acids. The main point of entry for carbon molecules is as acetyl CoA (2C) which condenses with oxaloacetate (4C) to form citrate (6C). The citric acid cycle generates the reduced coenzymes, NADH and FADH , as w2ll as GTP, and provides intermediates for biosynthetic reactions. The citric acid cycle occurs in the mitochondrial matrix of the cell. The reduced coenzymes enter oxidative phosphorylation (electron transfer chain) where the majority of the cell's ATP is synthesized, by the ATP synthase. [see overall figure] The first reaction to be considered is the oxidative decarboxylation of pyruvate to form acetyl CoA. This reaction, catalyzed by the enzyme pyruvate dehydrogenase complex, is not part of the citric acid cycle directly, but generates the acetyl CoA that enters the KC. This reaction provides the link between glycolysis and the KC. The reaction is as follows. Pyruvate + CoA + NAD + acetyl CoA + CO + 2ADH The Pyruvate Dehydrogenase Complex responsible for carrying out this reaction contains 3 kinds of enzymes and 5 different kinds of coenzymes. In a four-step reaction sequence pyruvate is converted into acetyl CoA. One of the important cofactors necessary for the activity of the pyruvate dehydrogenase complex is thiamine pyrophosphate (TPP). TPP is derived from the vitamin thiamine, also called vitamin B .1The function of TPP in the pyruvate dehydrogenase complex is to destabilize the bond between the carbonyl and carboxyl groups of pyruvate. TPP is also a cofactor for the transketolase enzymes. The structure of TPP is shown below. A deficiency in thiamine results in the disease called beriberi. The oral manifestations of thiamine deficiency include "old rose" colored tongue, some depapillation at the periphery of the tongue, and a deeper than expected red color of the oral mucosa. Lipoamide or lipoic acid contains 2 thiol groups, which are essential for its function as a cofactor. Lipoic acid is linked to the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex. The thiol groups can exist in the oxidized form or in the reduced form (2 free HS- groups). Because of its ability to undergo oxidative- reduction reactions, lipoate can serve as an electron carrier as well as an acyl carrier. The pyruvate dehydrogenase reaction depends on both of these functions. The mechanism of action of the PDH Complex is for the enzyme pyruvate decarboxylase to oxidatively decarboxylate the pyruvate by removing a CO . N2xt, the enzyme lipoamide reductase transacetylasetransfers the pyruvate carbon skeleton onto coenzyme- A. The third enzyme, dihydrolipoyl dehydrogenaseremoves a hydrogen onto NAD making NADH. The net result is the release of CO and t2e making ofNADH and acetyl-coenzyme-A for each pyruvate. Note the additional molecule of CO 2 and additional molecule of NADH formed by the pyruvate dehydrogenase reaction add to the totals of aerobic metabolism. However, these reaction products are not considered directly a part of the Krebs Cycle. After the production of acetyl CoA, the initial step of the KC is the condensation of oxaloacetate andacetyl CoA to form citrate. This reaction is catalyzed by the enzyme citrate synthase. The reaction involves a condensation of reactants to form the intermediate citryl CoA followed by a hydrolysis reaction yielding citrate and CoASH. The net reaction for +he citric acid cycle is: Acetyl CoA + 3 NAD + FAD + GDP + P + 2 H i 2 2 CO +23 NADH + FADH + GTP 2 H + CoA + Many of the reactions of the Krebs cycle result in the formation of important cellular molecules. Note: a new value for ATP per NADH is 2.5 and ATP per FADH is 2 1.5. You may see the numbers ATP per NADH = 3 and ATP per FADH =22 in some texts. The different values of 30 or 32 ATP/glucose depend on the method used to transport cytoplasmic NADH, formed by glycolysis, into the mitochondria, i.e. the shuttles. Summary 1. Pyruvate is converted to acetyl CoA by the pyruvate dehydrogenase complex 2. The pyruvate dehydrogenase complex contains several enzyme activities and cofactors and is the key step regulating the flow of 2 carbon fragments into the citric acid cycle. 3. Acetyl CoA condenses with oxaloacetate to form citrate as the first step of the Krebs cycle 4. One round (1 Acetyl-coA) of citric acid cycle results in the formation of 1 GTP, 3 NADH, 1 FADH , a2d 2 CO 2 Mechanism of Action of PDH Complex E1: pyruvate decarboxylase - thiamine pyrophosphate TPP removes COOH from pyruvate leaving 2 carbon fragment that binds the acyl fragment to the TPP. E2: lipomide reductase transacetylase - lipoate 2 carbon acyl group is transferred to one lipoamide arm, and then to the other, to position it for CoASH transfer. E3: dihydrolipoyl dehydrogenase- CoASH, FAD, NAD + acyl group is transferred to CoASH; the reduced lipoamides transfers 2H's to E-FAD --> E-FADH , and2then + E-FADH pa2ses H to NAD --> NADH The cycle itself - Key metabolic reactions of Krebs Cycle (including the PHD reaction) NAD is reduced (NADH – 3NADH/ACoA) Substrate level phosphorylation occurs GDP + P 2GTP/glu (=ATP) Decarboxylation (-COOH – 2x/AcoA; 1@ PDH step = 3) Acylation via CoASH (succinyl-CoA – 2x- akin to PDH) Thus each turn of the cycle (c6 2C3, thus occurs 2x) 4 protons passed to coe’s (3NADH & 1 FADH ) =26 NADH & 2 FADH )2 2 CO 2s are released = 4 CO ’2 1 GDP is phosphorylated to GTP (equvalent to ATP) = 2ATP equivalents Carbohydrates vs. Fats (as energy sources for cell respiration?) Fatty acid metabolism Oxidation Fatty Acids: triacylglycerol, fat/lipid droplet and fatty acid ►converts free fatty acids in blood into to Acetyl-CoA in the mitcohondria 3 Steps of Fatty Acid Oxidation cycle (Beta Oxidation) 1. Oxidation of COOH end of free fatty acid & linking FFA to CoASH H3C-H 2-H C2H C-2 C-2OOH 2. Transport of fatty acyl-coA into mitoplasm from cytoplasm H3C-H 2-H C2H C-2 C-2O-S-CoA 3. Oxidation of fatty acyl-coA into 2 carbon fragments of Acetyl-CoA H3C-H 2-H C2H C-2OOH + H 3-CO-S- CoA 4 enzymes of beta-oxidation cycle 1. Long fatty acyl-coA Synthetase (on outer mito. membranes) FA-COOH + ATP + CoASH <--> FAcoA + AMP + PP *converts cyotplasmic FFA to fatty-acyl-coA [c-c-c- c-ScoA] 2. Carnitine acyl-TRANSFERASE 1 (outer mito memb.) FAcoA + carnitine <-> Fatty acyl-carnitine + CoASH *transfers FAcoA to carnitine for transport across mito 3. Carnitine acyl-TRANSFERASE 2 (on mitoplasm side) Fatty acyl-carnitine + CoASH <--> FAcoA + carnitine *releases carnitine & leave FAcoA inside the mitoplasm 4. Fatty acyl-coA DEHYDROGENASE (in mitoplasm) *oxidizes FAcoA & reduces FAD and NAD+ in 4 steps Step 4 – of Beta- Oxidation cycle via fatty acyl coA dehydrogenase enzyme *sequence of steps for this mitochondrial dehydrogenase enzyme system... a) dehydrogenation with FAD --> FADH 2 b) hydration - addition of water c) dehydrogenation with NAD --> NADH . d) thiol clevage with CoASH *Release of a 2carbon fragment as Acetyl-CoA, feeds to Krebs cycle Net result: each turn of the cycle shortens a long chain fatty acid by 2 carbons generating 1 AcoA, 1 NADH and 1 FADH for 2ntry into Krebs cycle *So far little direct ATP (or equivalent GTP) has been made per Glucose (4 in glycolysis & 2 in KC) *Most of the ATP will be made in the Electron Transfer System of the mitochondria Topic 11- Chemiosmosis Mitochondrial Membrane Transport and the Electron Transport Chain Membranes = impermeant to most everything, esp to H + Outer membrane – porin (channel protein) diffuses molecules ≈ 5,000 daltons *Porins are single monomers with 16 stranded antiparallel beta barrel sheets forming a water-filled channel through which molecules < than 5,000d (MW) can move passively (ions, sugars, aa's) Inner membrane – 70% protein and 30% lipid contains: * Redox proteins of electron transport chain *ATP synthase * Many carrier proteins – phosphate translocases, ADP/ATP translocases, pyruvate/H symporter * a-glycerol P and malate shuttles enzymes * Lipid metabolism (Beta oxiadation enzymes) Mitochondrial origin was likely by endosymbiosis Mitochondrial DNA – Mitochondrial-DNA Human mitochondrial DNA has 16,569 np’s Only 13 out of 1,100 mitochondrial proteins are coded in the mitochondria. The rest are coded for by nucleus & made in cytoplasm. Gene transfers between plant plastids and nucleus Mitochondrial DNA also codes for some tRNA and rRNA Mitochondrial DNA gene functions: * 5 subunits of NADH dehydrogenase (complex I), * cytochrome oxidase subunits I, II, III (complex IV) * ATP synthase : subunits 6 & 8 (complex V), * RNA polymerase, & 22 tRNA's & 2 rRNA's Nuclear genes encoded components include: lipid, nucleotide, amino acid, & carbo metabolism, metabolism, heme & FeS synthesis, ubiquinone synthesis, proteases, chaperones, signal pathways, & DNA repair & replication. 1,000's mito copies per cell; maternally inherited; lots of short tandem repeat sequences; frequent point mutations; thus, sequence analysis can indicate phylogeny: Homoplasmic vs. Heteroplasmic mtDNA Mitochondrial genomes may not be uniform across cells of the body, but vary between different tissue types. It is assumed that from the beginning of life individuals are HOMOPLASMIC, meaning that within an individual, all the cells mitochondrial DNA (mtDNA) is the same. However, recent data suggests that each individual may be a mosaic of multiple cell [mt]DNA types, in different tissues. Using high throughput sequencing technology, molecular geneticist Nickolas Papadopoulos of the Ludwig Center for Cancer Genetic and Therapeutics and the Johns Hopkins Kimmel Cancer Center in Baltimore and his colleagues analyzed the mitochondrial genomes of a variety of tissues in 2 different people and the lining of the colon & 10 other tissues. In every individual, the researchers found at least 1 allele that differed between tissues, and one individual had as many as 14 HETEROPLASMES (varying mtDNA genomes). Once established, these findings may also affect more practical applications in forensics science, since the mtDNA in one tissue might vary from another tissue, caution must be used whencomparing a hair sample, for example, to blood. It's unclear why mtDNA is so variable. One reason may be thatmitochondria have a higher mutation rate than nuclear DNA or that the mitochondria have less effective DNA repair mechanisms. These findings are likely to spur future studies to further characterize the diversity in mitochondrial genomes and determine the mechanism underlying the variation. Mitochondrial aerobic cell respiration driven electron transport How electron transport works Oxidation-reduction (Redox) reactions involve the transfer of electrons from one substance to another. Redox reactions must occur together (in couples). One compound must lose electrons and the other gain electrons. The substance, which loses electrons is called the reducing agent while the substance which gainselectrons is called the oxidizing agent. The reduction potential is the measure of the ability of one compound to reduce another. For example, in the following reaction O2is the oxidizing agent (and is reduced) and NADH is the reducing agent (and is oxidized). Redox Potential Is a measure of tendency of molecular couple (acceptor/donor) to GAIN-LOSE e's - strong reducing agent (electron donor - NADH) has negative - E'o (redox potential) - strong oxidizing agent (electron acceptor - O2) has positive + E'o (redox potential) Redox Half-cell Free energy and redox potential Go = -n (f) ΔE’o NADH <---> NAD + H + 2e + - -0.32V (-320 millivolts) H O <---> ½ O + 2H + 2e + - +0.82V (+820 millivolts) 2 2 ΔGo' = - (1) (23.061 Kcal/volt) (1.14v) = - 26.29 Kcal *Theoretical P to O ratio for 1 NADH = 3 ATP = 7.3Kccal x 3 = 21.9 Kcal Electron Transport Chain and the order of its electron carrier molecules ETC is a series of electron CARRIER MOLECULES that that transfer e 's from a more negative redox potential to a more positive redox potential, while driving protons out of the mitoplasm into perimitochondrial space. --> Carriers are aligned linearly via increasing Redox Potential *From more electronegative [ - ] toward more electropositive to [ + ] and therefore by their energy differentials: NADH - nicotinamide adenine dinucleotide *FMN - Flavin mononucleotide amytal *FeS - iron sulfur protein FADH 2 flavin adenine dinucleotide *Q - coenzyme Q antimycin-A *cyto b - cytochrome b, cyto c1 - cytochrome c1 cyto c - cytochrome c cyto a - cytochrome a *cyto a3 - cytochrome a3 cyanide oxygen terminal electron acceptor --> Membranes themselves have no electrical charge, but instead they separate electrical charges making the membrane an insulator... an insulator that separates electric charges until used is a battery Major components of the electron transport chain Oxidative phosphorylation is the process by which ATP is formed as electrons are transferred from NADH or FADH to m2lecular oxygen (O ) 2 by a series of electron carriers. The energy released form the oxidation of glucose, fatty acids, and amino acids is stored as the reduced coenzymes NADH or FADH . The2e is a step by step transfer of electrons from NADH or FADH to s2ecific protein complexes, which are part of the electron transport chain. The ultimate acceptor of these electrons is O . 2he electron-transport chain, also known as respiratory chain, is a series of linked electron carriers that transfer electrons from NADH and FADH to m2lecular oxygen (O )2 The respiratory chain is found in the inner mitochondrial membrane and is composed of 3 enzyme complexes and 2 mobile carriers, also known as respiratory assemblies. Respiratory assemblies are enzyme complexes of acceptor proteins, coenzymes, and metal ions, are located in the inner mitochondrial membrane. The respiratory assemblies are made up of 3 enzyme complexes, which are the sites of the proton pumps. Enzyme Complexes Mobile carriers NADH-Q reductase Q (ubiquinone) Cytochrome reductase Cytochrome c Cytochrome oxidase Summary 1. The transport of electrons from NADH and FADH provi2e "energy" for synthesis of ATP 2. Electrons from NADH & FADH are 2assed thru a series of electron transport complexes 3. Oxidation/reduction reactions are coupled 4. These electrons are passed to molecular oxygen as the ultimate acceptor 5. The integrity of the mitochondrial structure is essential for oxidative phosphorylation 6. Four enzyme complexes and 2 mobile carriers comprise the respiratory chain 7. NADH & FADH use2 for electron transport result from oxidation of foods, mainly glucose 8. As the electrons are transferred, protons are pumped into the inter-membrane space of the mitochondria resulting in the formation of a proton gradient. 9. The chemiosmotic hypothesis of Peter Mitchell states that a proton-motive force was responsible for driving the synthesis of ATP 10. Much experimental evidence supports the pumping of protons by the respiratory chain complexes as the primary energy conserving event of oxidative phosphorylation + Pyridine nucleotides NAD - enzyme bound hydrogen carriers. Accepts 2e’s and/or protons Flavoproteins FMN & FAD – protein bound hydrogen carriers Iron sulfur proteins FeS – non-heme iron electron carriers Ubiquinone (CoQ) – semiquinone & hydroquinone. Mobile membrane bound, non-protein hydrogen carriers Cytochromes – “colored proteins” with bound Fe atoms via iron porphyrin (heme) bound protein carriers How electron transport chain Mitochondrial Respiratory Assemblies I. NADH-Q reductase II. Succinate dehydrogenase III. Cytochrome-C-Reductase IV. Cytochrome Oxidase *ETC – passes electrons through ETC carrier proteins *PMF – proton motive force gradient Membrane potential difference *Δcharge = 140mV in(-) vs. out(+) Chemiosmosis (oxidative phosphorylation) Proton Motive Force Gradient – leads to synthesis of ATP + H gradient is generated by transfer of electron thru ETC Electrons go through series of redox proteins Electrons and H finally reduce O & 2ake H O 2 Mechanism – a fundamental energy mechanism that arose early in evolution and was retained (works like a fuel cell) Evidence: Fractionation and reconstitution. pH gradients and bacterio-rhodopsin Chemiosmosis in bacteria, mitochondria and chloroplasts ATP synthase Condenses ADP + Pi ---> ATP ATP synthase structure ATP synthase of liver mitochondria number about 15,000. *Made of 24 polypeptides with membrane & mitoplasmic pieces F1 5 polypeptides (nuclear DNA) 3α , 3β , 1γ , 1δ, & 1ε arranged like sections of grapefruit 3 catalytic sites for ATP synthesis - 1 on each β subunit F0 3 polypeptides in ratio of 1a, 2b, and 12c (C-ring) Binding Charge Mechanism of ATP Synthesis - A Rotary Motor 1. H+ movement changes binding affinity of F1's β-synthases's active site, thus when ADP & P bind to active site, they readily condense into ATP (removed from aqueous solution Keq = 1 and ΔG close to zero, thus ATP forms easily) 2. F1 active site is on β-subunits & it changes conformation* through 3 successive shapes (O-L-T) O - open - site has low affinity to bind ATP - thus releases it  L - loose - ADP & P loosely bound to site [1 & 2] T - tight - ADP & P tightly bound favoring condensation without water  3. Conformational changes result in rotation of subunits relative to central stalk (γ) α & β subunits of F1 form hexagonal ring that rotates around central axis γ stalk extends from F & interacts with 3 β's differently as it o o rotates thru 360 Proton Pathway 12 C-proteins reside in lipid bilayer (C-ring) C-ring is attached to γ stalk of F 1ubunit H+ diffuse through Fo half-channel rotating the 12C's of the Fo ring *Each C protein has a half-channel space with a neg charged aspartate C's bind H on pms side & via shape changes each C-rotates 30 CCW o + o next C in ring picks up H - thus 12 C's rotates ring cycles thru 360 + * Release of H into matrix happens at end of cycle 4 H moves ring 120 (γ stalk) shifts 120 --> β's change 4 H result in one ATP being made o rotation of C-ring drives γ stalk through 360 & ► 3 conformations of F1 (L-T-O) to make ATP Summary of cellular respiration Topic 12- Photosynthesis Photosynthesis defined as Light driven phosphorylation – production of ATP via photophosphorylation and a proton motive force ADP + P --> ATP & reduction of NADP --> NADPH Cellular process – in bacteria, blue-green algae, and eukaryotic cells with chloroplasts Capture of light energy by pigments – chlorophylls and accessory pigments Capture electrons as reducing power into NADPH – photophosphorylation via an electron transport chain Reduction of CO t2 CH O 2 2 Fundamental Reaction Mechanisms Light reactions (photo chemical reactions… non-enzymatic) Molecular excitation of chlorophyll by light results in charge separation via hydrolysis of 2 O releasing 2H and ½ O2 + Generation of proton motive force (H gradient) across thylakoid membranes Synthesis of ATP via an ATP synthase Reduction of NADP to NADPH within a photosynthetic ETC Dark reactions (thermo-chemical rxn’s) (temperature sensitive), thus enzymatic CO 2ixation via (reduction) reactions that are reverse of glycolysis Carboxylation rx: CO 2 RuBP --> 2 PGA [ 1C + 5C -- > 2 (3C) ] Reduction PGA with NADPH --> PGAL (glycolytic-like) Regeneration of RuBP via HMP (5C sugar) pathway --> RuBP 6CO + 12H O* --> C H O + 6H O + 6O* 2 2 6 12 6 2 2 Both reactions occur within the chloroplast Evolutionary Basis of photosynthesis * Origin of mitochondria and chloroplast may have been symbiotic Primary vs. seconda
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