Biochemistry Sixth Edition Berg • Tymoczko • Stryer Chapter 15: Metabolism: Basic Concepts and Design Copyright © 2007 by W. H. Freeman and Company

Roadmap of Metabolic Pathways

Metabolism Metabolism – reactions occurring in a living system that produce and consume the energy needed for the organism to exist. Metabolic pathways. Metabolic reactions. High Energy bonds in compounds. Thermodynamics of reactions.

Metabolism Metabolism - the entire network of chemical reactions carried out by living cells Metabolites - small molecule intermediates in the degradation and synthesis of biopolymers Catabolic reactions - degrade biomolecules to create smaller molecules and energy Anabolic reactions - synthesize biomolecules for cell maintenance, growth and reproduction

Catabolism and Anabolism Catabolism Anabolism degradative synthetic oxidative reductive energy producing energy requiring (exergonic) (endergonic) makes pool molecules uses pool molecules produces NADH & uses NADPH almost NADPH exclusively

Energy Overview Energy distribution 1/3 2/3 nutrients ----> pool molecules ----> CO2, H2O, NH3  biomolecules

Pathways Metabolism includes all enzyme catalyzed reactions Metabolism can be subdivided into various areas: hexose shunt, electron transport, etc. The metabolism of the four major groups of biomolecules will be considered: Carbohydrates Lipids Amino Acids Nucleotides

Pathways Multiple-step pathways permit control of energy input and output Catabolic multi-step pathways provide energy in smaller stepwise amounts) Each enzyme in a multi-step pathway usually catalyzes only one single step in the pathway Control points occur in multistep pathways

Regulation Metabolism is highly regulated to permit organisms to respond to changing conditions Most pathways are irreversible Flux - flow of material through a metabolic pathway which depends upon: (1) Supply of substrates (2) Removal of products (3) Pathway enzyme activities

Levels of Regulation Direct regulation at the enzyme level (covalent or non-covalent). Regulation via external communication (hormonal). Regulation at the gene level (induction/repression).

Direct Regulation Feedback Inhibition: The product of a pathway controls its own synthesis by inhibiting an earlier step (the first step or the “committed” step in the pathway) . Feed-forward Activation: A metabolite early in the pathway activates an enzyme that appears later. Interconvertible enzyme activity can be rapidly and reversibly altered by covalent modification. E.g. protein kinases and protein phosphatases.

Glucose Metabolism Breakdown to small molecules and energy.

Metabolite Needed for formation of glycerol based phospholipids and to run the glycerol-P shuttle.

Adenosine Nucleotides Components of an energy system.

ATP An energy carrier considered to be common energy currency in a cell

Driving Forces behind the Energy of ATP Hydrolysis Resonance energy of reactants vs products. Charge repulsion of oxygens. Number of charges on oxygens. Solvation of reactants vs products. Entropy – number of reactant vs product molecules.

Phosphate Resonance pKas of phosphoric acid: 2.1, 6.9 and 12.3

Other High Energy Molecules

Go'of Hydrolysis

ATP Use Synthesis

Oxidation States Oxidation of triacylglycerols affords more energy than do carbohydrates.

Sources of Energy

Biological Redox Energy Electron Transport System (ETS) moves electrons from reduced coenzymes toward O2 This produces a proton gradient and a transmembrane potential Oxidative Phosphorylation is the process by which the potential is coupled to the reaction: ADP + Pi ATP

NAD+ Oxidizes GAP NADH carries electrons to the ETS.

Substrate Level Phosphorylation Substrate Level Phophoryation occurs When ATP is formed in a metabolic reaction.

Free Energy of Coupled Reactions 1,3-bisphosphoglycerate --- > 3-phosphoglycerate + Pi Go' = -49.4 kJ/mol ADP + Pi --- >ATP Go' = +30.5 kJ/mol 1,3-bisphosphoglycerate + ADP ---- > 3-phosphoglycerate + ATP Go' = -18.9 kJ/mol

Aerobic Oxidation Oxidative phosphorylation does not occur without electron transport.

Mitochondria Oxidation and electron transport Oxidative phosphorylation

NAD+ A two electron transfer agent NicotinamideNucleotide AMP = Adenine Nucleotide R = -PO3= for NADP+ AMP

Oxidation by NAD+ This side is the “A” face of the nicotinamide ring, the back side is the “B” face.

Oxidation by NAD+ A typically NAD+ oxidation is -OH to C=O

FAD A one electron transfer agent FMN = Flavin Mononucleotidein blue Note that this is ribitol. AMP in black

Oxidation by FAD FAD and FMN also accept two electrons but these enter the isoalloxazine ring one at a time.

Oxidation by FAD A typically FAD oxidation is -CH2-CH2- to -CH=CH-

Oxidized and Reduced Forms This is an isoalloxazine ring system

Coenzyme A An acyl transfer agent (forms a thioester) Note -PO3= on 3' of ribose An acyl transfer agent (forms a thioester)

Thioesters

Carriers and Coenzymes

Review of G Equations For the reaction: A + B C + D DG = DGo' + RT ln([C][D]/[A][B]) At standard state: All conc. are 1 M or 1 atm except [H+] and under these conditions: DG = DGo'

Review of G Equations For the reaction: A + B C + D At equilibrium: Keq = [C]eq[D]eq/[A]eq[B]eq and DG = 0, therefore: DGo' = -RT ln Keq For an oxidation-reduction reaction: DGo' = -nDEo'F (#e transferred)(cell potential)(Faraday’s const.)

Krebs Cycle Oxidations Also, there are two oxidative decarboxylations in the Kreb’s Cycle (citric acid cycle).

Free Energy of a Redox Reaction Oxidation Half-reaction: Half-Cell Potential Malate ---- > Oxaloacetate + 2 e + 2 H+ Eo' = +0.166 v Reduction Half-reaction: NAD+ + 2 e + 2 H+ ---- > NADH + H+ Eo' = -0.32 v Cell Reaction : Malate + NAD+ ---- > Oxaloacetate + NADH + H+ Cell Potential: Eo' = -0.154 v A cell reaction must contain an oxidation half-reaction and a reduction half-reaction to equate electron flow.

Free Energy of a Redox Reaction Go' = -nEo'F = -(2)(-0.154)(96480) = +29700 J/mol = +29.7 kJ/mol The equilibrium of this redox reaction lies far to the left. Cellular concentrations of the metabolites must be such that the overall G is negative in order for the reaction to proceed as written on the previous slide. For a redox reaction to proceed spontaneously, the cell potential must be positive.

Free Energy of a Redox Reaction Malate + NAD+ ---- > Oxaloacetate + NADH + H+ Which reactant is oxidized ? Which reactant is reduced ? Which reactant is the oxidizing agent ? Which reactant is the reducing agent ? Malate NAD+ NAD+ Malate

Reaction Types in Metabolism

Ligation with ATP

Isomerization

Group Transfer

Hydrolysis

Cleavage to form a Double Bond

Cleavage to form a Double Bond

Energy Charge of a Cell ATP + ½ ADP Energy Charge = ------------------------- ATP + ADP + AMP Limits are 0 and 1.0 If all is ATP, the energy charge = 1 If all is AMP, the energy charge = 0 ATP can be regenerated using adenylate kinase (this is a nucleoside monophosphate kinase): 2 ADP <===> ATP + AMP

Rate vs Energy Charge

Other ATP uses ATP can also be used to make other NTPs with nominal energy exchange using a nucleoside diphosphate kinase. ATP + NDP <===> ADP + NTP Other involvement of ATP: 1. Phosphate transfer to make high energy bond: Glutamine synthesis uses P from ATP Glu + ATP —> γ-PGlu + ADP, then NH3 displaces P to give Gln

Other ATP uses 2. PEP transfers P to make ATP: Enol-P (PEP) + ADP —> Pyr + ATP 3. Nucleotide transfer to make high energy bond: AMP from ATP combines with a fatty acid in making AcylSCoA catalyzed by acylSCoA synthetase (acyl thiokinase) during fatty acid activation. FA + ATP —> acyl-AMP + PPi, then CoASH displaces AMP to give acyl-SCoA

Effect of H+ on Keq pyruvate + NADH + H+ ----> lactate + NAD+ Keq' = Kapp = ------------------------- [pyruvate][NADH] so, Keq' = Keq (H+), where H+ is a reactant. similarly, Keq' = Keq /(H+), where H+ is a product.

FAD vs FAD-flavoprotein Electrons from succinate: FADH2 + CoQ < === > FAD + CoQH2 Go' for free FAD in solution: FAD + 2 H+ + 2 e- <===> FADH2 Eo' = -0.22v CoQ + 2 H+ + 2 e- <===> CoQH2 Eo' = +0.10v net FADH2 + CoQ <===> FAD + CoQH2 Eo' = +0.32v Go' = -nEo'F = -61.7 kJ/mol

FAD vs FAD-flavoprotein CoQ + FADH2 < === > CoQH2 + FAD Go' for FAD in a flavoprotein: FAD + 2 H+ + 2 e- <===> FADH2 Eo' = 0.00v CoQ + 2 H+ + 2 e- <===> CoQH2 Eo' = +0.10v net FADH2 + CoQ <===> FAD + CoQH2 Eo' = +0.10v Go' = -nEo'F = -19.3 kJ/mol This represents a difference in Go' of about 42 kJ/mol.

Table of Reduction Potentials

Biochemistry Sixth Edition Berg • Tymoczko • Stryer End of Chapter 15 Copyright © 2007 by W. H. Freeman and Company