Energetics of carbohydrate and lipid metabolism 1
Metabolism: The sum of all the chemical transformations taking place in a cell or organism, occurs through a series of enzymecatalyzed reactions that constitute metabolic pathways. Each of the consecutive Steps in a metabolic pathway brings about a specific, small chemical change, usually the removal, transfer, or addition of a particular atom or functional group. The precursor is converted into a product through a series of metabolic intermediates called metabolites. 2
The combined activities of all the metabolic pathways that interconvert precursors, metabolites, and products of low molecular weight, M r <1,000 (intermediary metabolism). Catabolism (Degradative phase) : Organic nutrient molecules (carbohydrates, fats, and proteins) are converted into smaller, simpler end products (such as lactic acid, CO 2, NH 3 ). Catabolic pathways release energy, some of which is conserved in the formation of ATP and reduced electron carriers (NADH, NADPH, and FADH 2 ); the rest is lost as heat. 3
Anabolism(biosynthesis): Small, simple precursors are built up into larger and more complex molecules, including lipids, polysaccharides, proteins, and nucleic acids. Anabolic reactions require an input of energy, generally in the form of the phosphoryl group transfer potential of ATP and the reducing power of NADH, NADPH, and FADH. 2
Some metabolic pathways are linear, and some are branched. Catabolic pathways are convergent and anabolic pathways divergent. Some pathways are cyclic: one starting component of the pathway is regenerated in a series of reactions that converts another starting component into a product. Most cells have the enzymes to carry out both the degradation and the synthesis of the important categories of biomolecules fatty acids.
Both anabolic and catabolic pathways to be essentially irreversible, the reactions unique to each direction must include at least one that is thermodynamically very favorable, a reaction for which the reverse reaction is very unfavorable. Metabolic pathways are regulated at several levels from within the cell and from outside. The most immediate regulation is by the availability of substrate; when the intracellular concentration of an enzyme s substrate is near or below K m, the rate of the reaction depends strongly upon substrate concentration.
The number of metabolic transformations taking place in a typical cell. Most cells have the capacity to carry out thousands of specific, enzyme-catalyzed reactions: Examples: Transformation of a simple nutrient such as glucose into amino acids, nucleotides, or lipids. Extraction of energy from fuels by oxidation. Polymerization of monomeric subunits into macromolecules.
The reactions in living cells fall into one of five general categories usually proceed by a limited set of mechanisms and often employ characteristic cofactors : (1) Oxidation-reductions; (2) Reactions that make or break carbon carbon bonds; (3) Internal rearrangements, isomerization's, and eliminations, (4) Group transfers, and (5) Free radical reactions. Reactions within each 10
Two phases of glycolysis. For each molecule of glucose that passes through the preparatory phase (a), two molecules of glyceraldehyde 3-phosphate are formed; both pass through the payoff phase (b). Pyruvate is the end product of the second phase of glycolysis. For each glucose molecule, two ATP are consumed in the preparatory phase and four ATP are produced in the payoff phase, giving a net yield of two ATP per molecule of glucose converted to pyruvate. 11
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ATP Formation Coupled to Glycolysis During glycolysis some of the energy of the glucose molecule is conserved in ATP, while much remains in the product, pyruvate. The overall equation for glycolysis is For each molecule of glucose degraded to pyruvate, two molecules of ATP are generated from ADP and P i. We can now resolve the equation of glycolysis into two processes 14
The conversion of glucose to pyruvate (exergonic) The formation of ATP from ADP and Pi, (endergonic) The sum of Equations 14 2 and 14 3 gives the overall standard freeenergy change of glycolysis, ΔG s 15
Fermentation: The general term for such processes, which extract energy (as ATP) but do not consume oxygen or change the concentrations of NAD + or NADH. In case of insufficient oxygen NAD + is regenerated from NADH by the reduction of pyruvate to L-lactate by lactate dehydrogenase at ph 7. The overall equilibrium of this reaction strongly favors lactate formation, as shown by the large negative standard free-energy change. 16
An endergonic reaction (also called a heat absorb nonspontaneous reaction or an unfavorable reaction) is a chemical reaction in which the standard change in free energy is positive, and energy is absorbed. A reaction that is thermodynamically favored has a negative ΔG, so the products are at a lower energy than the reactants so that reaction will spontaneously occur. For ΔG to be negative, ΔH has to be small in the equation ΔG=ΔH TΔS. (exergonic reaction) 17
1- Phosphorylation of Glucose in glycolysis Use the energy of ATP Multiple isoforms of hexokinase exist in organisms (e.g., hexokinase I, II, III, and IV (glucokinase)). Nucleophilic oxygen at C6 of glucose attacks the last (γ) phosphate of ATP. ATP-bound Mg ++ facilitates this process by shielding the negative charges on ATP. Highly thermodynamically favorable/irreversible 18
2- Phosphohexose Isomerization Slightly thermodynamically unfavorable/reversible product concentration kept low by pairing with favorable next step to drive reaction forward. 3-2nd Priming Phosphorylation by Phosphofructokinase-1 This process uses the energy of ATP. Highly thermodynamically favorable/irreversible 4- Aldol Cleavage of F-1,6-bP by Aldolase Thermodynamically unfavorable/reversible 5- Triose Phosphate Interconversion by Triose Phosphate Isomerase Thermodynamically unfavorable/reversible 19
6- Oxidation of GAP by Glyceraldehyde-3-Phosphate Dehydrogenase Thermodynamically unfavorable/reversible 7-1st Production of ATP by Phosphoglycerate Kinase Highly thermodynamically favorable/reversible 8- Migration of the Phosphate Thermodynamically unfavorable/reversible 9- Dehydration of 2-PG to PEP Slightly thermodynamically unfavorable/reversible 10-2nd Production of ATP by Pyruvate kinase Highly thermodynamically favorable/irreversible 20
Only a Small Amount of Energy Available in Glucose Is Captured in Glycolysis Glycolysis G = 146 kj/mol 2 GLUCOSE Full oxidation (+ 6 O 2 ) G = 2,840 kj/mol 6 CO 2 + 6 H 2 O
Cellular Respiration Process in which cells consume O 2 and produce CO 2 Provides more energy (ATP) from glucose than glycolysis Also captures energy stored in lipids and amino acids Evolutionary origin: developed about 2.5 billion years ago Used by animals, plants, and many microorganisms Occurs in three major stages: - acetyl CoA production - acetyl CoA oxidation - electron transfer and oxidative phosphorylation
Respiration: Stage 1 Acetyl-CoA Production Generates some ATP, NADH, FADH 2 Carbohydrates release 1/3 of total potential CO 2 during Stage 1.
Respiration: Stage 2 Acetyl-CoA Oxidation Generates more NADH, FADH 2, and one GTP Remaining carbon atoms from carbohydrates, amino acids, and fatty acids are released during Stage 2.
Respiration: Stage 3 Oxidative Phosphorylation Generates the vast majority of ATP during catabolism
In Eukaryotes, Stages 2 and 3 Are Localized to the Mitochondria Glycolysis occurs in the cytoplasm. Citric acid cycle occurs in the mitochondrial matrix. Oxidative phosphorylation occurs in the inner membrane. Except succinate dehydrogenase, which is located in the inner membrane
The Citric Acid Cycle (CAC)
Sequence of Events in the Citric Acid Cycle Step 1: C-C bond formation between acetate (2C) and oxaloacetate (4C) to make citrate (6C) Step 2: Isomerization via dehydration/rehydration Steps 3 4: Oxidative decarboxylations to give 2 NADH Step 5: Substrate-level phosphorylation to give GTP Step 6: Dehydrogenation to give FADH 2 Step 7: Hydration Step 8: Dehydrogenation to give NADH
One Turn of the Citric Acid Cycle
Direct and Indirect ATP Yield TABLE 16-1 Reaction Stoichiometry of Coenzyme Reduction and ATP Formation in the Aerobic Oxidation of Glucose via Glycolysis, the Pyruvate Dehydrogenase Complex Reaction, the Citric Acid Cycle, and Oxidative Phosphorylation Number of ATP or reduced coenzyme directly formed Glucose glucose 6-phosphate 1 ATP 1 Fructose 6-phosphate fructose 1,6-bisphosphate 1 ATP 1 2 Glyceraldehyde 3-phosphate 2 1,3-bisphosphoglycerate 2 NADH 3 or 5 b 2 1,3-Bisphosphoglycerate 2 3-phosphoglycerate 2 ATP 2 2 Phosphoenolpyruvate 2 pyruvate 2 ATP 2 2 Pyruvate 2 acetyl-coa 2 NADH 5 2 Isocitrate 2 α-ketoglutarate 2 NADH 5 2 α-ketoglutarate 2 succinyl-coa 2 NADH 5 2 Succinyl-CoA 2 succinate 2 ATP (or 2 GTP) 2 2 Succinate 2 fumarate 2 FADH 2 3 2 Malate 2 oxaloacetate 2 NADH 5 Total 30-32 Number of ATP ultimately formed a a This is calculated as 2.5 ATP per NADH and 1.5 ATP per FADH 2. A negative value indicates consumption. b This number is either 3 or 5, depending on the mechanism used to shuttle NADH equivalents from the cytosol to the mitochondrial matrix; see Figures 19-30 and 19-31.
Fats Provide Efficient Fuel Storage The advantage of fats over polysaccharides: Fatty acids carry more energy per carbon because they are more reduced. Fatty acids complex or carry less water because they are nonpolar. Glucose and glycogen are for short-term energy needs and quick delivery. Fats are for long-term (months) energy needs, good storage, and slow delivery.
Fatty Acid Oxidation Occurs in the Mitochondria in Three Stages Stage 1 consists of oxidative conversion of two-carbon units into acetyl-coa via oxidation with concomitant generation of NADH and FADH 2. involves oxidation of carbon to thioester of fatty acyl-coa Stage 2 involves oxidation of acetyl-coa into CO 2 via citric acid cycle with concomitant generation NADH and FADH 2. Stage 3 generates ATP from NADH and FADH 2 via the respiratory chain.
Stages of Fatty Acid Oxidation
The -Oxidation Pathway Each pass removes one acetyl moiety in the form of acetyl-coa.
Fatty Acid Catabolism for Energy For palmitic acid (C 16 ) Repeating the previous four-step process six more times (seven total) results in eight molecules of acetyl-coa. FADH 2 is formed in each cycle (seven total). NADH is formed in each cycle (seven total). Acetyl-CoA enters citric acid cycle and further oxidizes into CO 2. This makes more GTP, NADH, and FADH 2. Electrons from all FADH 2 and NADH enter ETF.
NADH and FADH 2 Serve as Sources of ATP TABLE 17-1 Yield of ATP during Oxidation of One Molecule of Palmitoyl-CoA to CO 2 and H 2 O Enzyme catalyzing the oxidation step β Oxidation Number of NADH or FADH 2 formed Acyl-CoA dehydrogenase 7 FADH 2 10.5 β-hydroxyacyl-coa dehydrogenase 7 NADH 17.5 Citric acid cycle Isocitrate dehydrogenase 8 NADH 20 α-ketoglutarate dehydrogenase 8 NADH 20 Succinyl-CoA synthetase Succinate dehydrogenase 8 FADH 2 12 Malate dehydrogenase 8 NADH 20 Total 108 Number of ATP ultimately formed a a These calculations assume that mitochondrial oxidative phosphorylation produces 1.5 ATP per FADH 2 oxidized and 2.5 ATP per NADH oxidized. b GTP produced directly in this step yields ATP in the reaction catalyzed by nucleoside diphosphate kinase (p. 516). 8 b
Oxidation-Reduction Reactions Reduced organic compounds serve as fuels from which electrons can be stripped off during oxidation.
Reversible Oxidation of a Secondary Alcohol to a Ketone Many biochemical oxidation-reduction reactions involve transfer of two electrons. In order to keep charges in balance, proton transfer often accompanies electron transfer. In many dehydrogenases, the reaction proceeds by a stepwise transfers of proton (H + ) and hydride (:H ).
Reduction Potential Determines Flow of Electrons Reduction potential (E) affinity for electrons; higher E, higher affinity electrons transferred from lower to higher E E = (RT/nF)ln (K eq ) = G /nf E = E (e acceptor) E (e donor) G = nf E For negative G, need positive E E (acceptor) > E (donor)
TABLE 13-7a Half-reaction Standard Reduction Potentials of Some Biologically Important Half- Reactions E' (V) 1 / 2 O 2 + 2H + + 2e H 2 O 0.816 Fe 3+ + e Fe 2+ 0.771 NO 3 + 2H + + 2e NO 2 + H 2 O 0.421 Cytochrome f (Fe 3+ ) + e cytochrome f (Fe 2+ ) 0.365 Fe(CN) 6 3 (ferricyanide) + e Fe(CN) 6 4 0.36 Cytochrome a 3 (Fe 3+ ) + e cytochrome a 3 (Fe 2+ ) 0.35 O 2 + 2H + + 2e H 2 O 2 0.295 Cytochrome a (Fe 3+ ) + e cytochrome a (Fe 2+ ) 0.29 Cytochrome c (Fe 3+ ) + e cytochrome c (Fe 2+ ) 0.254 Cytochrome c 1 (Fe 3+ ) + e cytochrome c 1 (Fe 2+ ) 0.22 Cytochrome b (Fe 3+ ) + e cytochrome b (Fe 2+ ) 0.077 Ubiquinone + 2H + + 2e ubiquinol 0.045 Fumarate 2 + 2H + + 2e succinate2 0.031 2H + + 2e H 2 (at standard conditions, ph 0) 0.000 Crotonyl-CoA + 2H + + 2e butyryl-coa 0.015 Oxaloacetate 2 + 2H + + 2e malate 2 0.166 Source: Data mostly from R. A. Loach, in Handbook of Biochemistry and Molecular Biology, 3rd edn (G. D. Fasman, ed.), Physical and Chemical Data, Vol. 1, p. 122, CRC Press, 1976. a This is the value for free FAD; FAD bound to a specific flavoprotein (e.g., succinate dehydrogenase) has a different E' that depends on its protein environment.
TABLE 13-7b Half-reaction Standard Reduction Potentials of Some Biologically Important Half- Reactions E' (V) Pyruvate + 2H + 2e lactate 0.185 Acetaldehyde + 2H + + 2e ethanol FAD + 2H+ + 2e FADH 2 0.197 0.219 a Glutathione + 2H + + 2e 2 reduced glutathione 0.23 S + 2H + + 2e H 2 S 0.243 Lipoic acid + 2H + + 2e dihydrolipoic acid 0.29 NAD + + H + + 2e NADH NADP + + H + + 2e NADPH 0.320 0.324 Acetoacetate + 2H + + 2e β-hydroxybutyrate 0.346 α-ketoglutarate + CO 2 + 2H + + 2e isocitrate 0.38 2H + + 2e H 2 (at ph 7) 0.414 Ferredoxin (Fe 3+ ) + e ferredoxin (Fe 2+ ) 0.432 Source: Data mostly from R. A. Loach, in Handbook of Biochemistry and Molecular Biology, 3rd edn (G. D. Fasman, ed.), Physical and Chemical Data, Vol. 1, p. 122, CRC Press, 1976. a This is the value for free FAD; FAD bound to a specific flavoprotein (e.g., succinate dehydrogenase) has a different E' that depends on its protein environment.
NAD and NADP Are Common Redox Cofactors These are commonly called pyridine nucleotides. They can dissociate from the enzyme after the reaction. In a typical biological oxidation reaction, hydride from an alcohol is transferred to NAD +, giving NADH.
NAD and NADP Are Common Redox Cofactors
Formation of NADH Can Be Monitored by UV-Spectrophotometry Measure the change of absorbance at 340 nm Very useful signal when studying the kinetics of NAD-dependent dehydrogenases
Flavin Cofactors Allow Single Electron Transfers Permits the use of molecular oxygen as an ultimate electron acceptor flavin-dependent oxidases Flavin cofactors are tightly bound to proteins.