Part III => METABOLISM and ENERGY

§3.1 Metabolic Pathways

§3.1a Overview of Metabolism

§3.1b High-Energy Compounds

§3.1c Redox Reactions

Section 3.1a:

Overview of Metabolism

Synopsis 3.1a

- Metabolism is the process through which living organisms break down nutrients (or synthesize biomolecules) to generate energy needed to drive various biochemical processes needed for the maintenance of life—ie the sum of all chemical reactions in a living organism!

- In metabolic processes, nutrients can be broken down to generate energy (catabolism), or biomolecules can be synthesized from simpler chemicals (anabolism)

- Metabolic processes usually occur via a series of enzyme-catalyzed reactions that are collectively referred to as a “metabolic pathway”

- Nutrition (dietary intake) can be divided into two major categories:

1) Macronutrients Proteins/carbohydrates/lipids2) Micronutrients Vitamins/minerals

Characteristics of Vitamins (Organic Molecules)- Vitamins are small organic molecules that organisms are unable to

synthesize and thus must be obtained from diet

- Vitamins are essential for driving a multitude of chemical reactions

- Vitamins can be divided into two groups: water-soluble and fat-soluble

(intestinal)

(Hydrophilic)

(Lipophilic)

(mM) (nM)

Major Minerals & Trace Elements (Inorganic Compounds)

- Minerals constitute naturally-occurring native elements and their compounds (inorganic)—egmetal ions, oxides, sulfides, and halides

- Like vitamins, minerals play a key role in driving a wide variety of cellular processes and enzyme-catalyzed reactions

- Minerals that are required in high concentration (in the mM range) are called “Major Minerals”

- Minerals that are required in low concentration (in the nM range) are called “Trace Elements”

Catabolic & Anabolic Pathways are Coupled!

- Metabolic pathways are a series of connected enzymatic reactions

- Reactants, intermediates and products of such reactions are referred to as “metabolites”

- In catabolic pathways (degradation), the breakdown of nutrients results in the release of free energy—which is conserved either via synthesis of ATP from ADP, or reduction of NAD(P)+ to NAD(P)H

- In anabolic pathways (biosynthesis), ATP and NAD(P)H serve as major energy sources or “currencies” for the synthesis of biomolecules

Overview of Catabolism

- Catabolism of macronutrients (eg proteins, polysaccharides and triglycerides) usually converges on acetyl-CoA—where CoA is coenzyme A

- Acetyl-CoA is then further catabolized via the citric acid cycle to generate NADH and FADH2

- NADH and FADH2 are subsequently oxidized via oxidative phosphorylation to generate additional sources of free energy

Metabolic Functions of Eukaryotic Organelles

Metabolic pathways occur in specific cellular compartments—eg glycolysis occurs in the cytosol, whereas citric acid cycle is carried out in the mitochondrion

Organ-Specific Metabolic Profiles

Erythrocyte

Heart

Organ-Specific Sources of EnergyMajor organs are specialized in their ability to generate energy from only specific metabolites:

Kidneys- Glucose

Brain- Glucose (primary)- Ketone bodies (starvation)

Liver- Amino acids!!

Adipose Tissue- Glucose- Fatty acids

Skeletal Muscle- Glucose- Fatty acids- Ketone bodies

Heart Muscle- Fatty acids- Ketone bodies

Erythrocytes (no mitochondria!)- Carry all but use none—O2!- Meet their own energy needs via anaerobic glycolysis—

oxidation of glucose to lactate via fermentation (§3.2)

Major Characteristics of Metabolic Pathways

(1) Metabolic pathways are irreversible

- While most enzymatically-driven steps in a metabolic pathway operate near equilibrium (G 0), metabolic pathways usually harbor one or more exergonic steps that operate far from equilibrium (G << 0)

- Such thermodynamic behavior imparts directionality on metabolic pathways

(2) Metabolic pathways harbor a committed step

- Metabolic pathways are usually characterized by an exergonic step (G << 0) early in the pathway that “commits” the pathway to continue downstream

(3) Catabolic and anabolic pathways differ

- Given that the conversion of metabolite 1 to metabolite 2 via intermediate A is exergonic (G << 0), the same route cannot be used for the synthesis of metabolite 1 from metabolite 2 due to unfavorable thermodynamics

- Rather, a separate pathway must be employed to convert metabolite 2 back to metabolite 1 via intermediates X and Y

- Existence of such independent pathways is an important property of metabolic pathways as it allows independent control of degradation versus biosynthesis

Metabolic Flux

- Living organisms are thermodynamically open systems that tend to maintain a steady-state rather than reach equilibrium—doing so would equate to death!

- “Steady-state” implies that the rates of synthesis and degradation of metabolic intermediates within a cell are more or less equal such that their concentrations change little over time—eg the blood glucose level is maintained @ 1mg/ml!

- The rate of flow of metabolites through a metabolic pathway is referred to as “metabolic flux”—it is determined by the slowest or the rate-determining step in a metabolic pathway

- The metabolic flux of a pathway can be altered or modulated depending on the needs of the cell and/or organism

- This is usually achieved by altering the rate of the rate-determining step in a metabolic path via one of the following mechanisms:

(1) Allosteric Control(2) Feedback Inhibition(3) Feedforth Activation(4) Post-Translational Modification(5) Substrate Cycling(6) Gene Expression

Metabolic Flux: (1) Allosteric Control

- Many enzymes involved in controlling metabolic pathways are allosterically regulated by effectors—such as substrates and products resulting from within or other metabolic steps as well as coenzymes

- In allosteric control, effectors modulate enzymes by virtue of their ability to bind in an uncompetitive/mixed manner to an allosteric site away from the active site

- Such allosteric regulation may result in inhibition or activation of the enzyme

Metabolic Flux: (2) Feedback Inhibition

- Many enzymes involved in controlling metabolic pathways are reciprocally regulated by their products—the enzyme product or the final product of a pathway inhibits an enzyme involved in an earlier step so as to overhaul the metabolic flux

- Such inhibition may be achieved by the binding of product to the enzyme:1) active site in a competitive manner (competes with the substrate)2) allosteric site in an uncompetitive/mixed manner (may bind to both E and ES)

- Such regulatory control has come to be known as “negative feedback regulation”, or simply, “Feedback Inhibition”

Metabolic Flux: (3) Feedforth Activation

- Enzymes may be activated by their own substrates—the enzyme E1 substrate may augment E1 activity, usually by inhibiting enzyme E2 involved in inhibiting E1 through mechanisms such as post-translational modification (PTM)

- Such inhibition is usually achieved by the binding of enzyme E1 substrate to an allosteric site in enzyme E2 in an uncompetitive/mixed manner (may bind to both E and ES)

- Such regulatory control is called “Feedforth activation”

SE1

P

E2

Metabolic Flux: (4) Post-Translational Modification

- Many enzymes involved in controlling metabolic pathways are subject to post-translational modifications (PTMs)—also called covalent modification—such as phosphorylation

- Phosphorylation on residues such as serine, threonine, and/or tyrosine can enhance or inhibit the enzyme activity

- Such PTM is ultimately controlled by extracellular signals such as hormones

Metabolic Flux: (5) Substrate Cycling

- In many metabolic pathways, a forward reaction is opposed by a reverse step—eg consider the overall conversion of substrate A into product D via intermediates B and C

- In what is referred to as “substrate cycle”, enzyme f converts B to C but enzyme r does the opposite

- Thus, enzymes f and r essentially act in a concerted manner to cycle B to C and back again—hence substrate cycling!

- Substrate cycling provides an highly sensitive regulatory mechanism in that the overall metabolic flux can be allosterically modulated not only via enhancement of enzymatic activity of f but also by inhibition of enzymatic activity of r and vice versa!

- Substrate cycling thus renders metabolic pathways more sensitive to the concentrations of allosteric effectors than would otherwise be possible via regulation of a unidirectional step

Metabolic Flux: (6) Gene Expression

- In response to metabolic needs, the cellular concentrations of many enzymes involved in metabolic pathways can be modulated via gene expression, thereby enhancing metabolic flux

- List the categories of macronutrients and micronutrients required for mammalian metabolism and provide examples of each

- Explain the roles of ATP and NADPH in catabolic and anabolic reactions

- Give some reasons why enzymes are essential for the operation of metabolic pathways

- Explain the metabolic significance of reactions that function near equilibrium and reactions that function far from equilibrium

- Discuss the mechanisms by which the flux through a metabolic pathway can be controlled. Which mechanisms can rapidly alter flux?

Exercise 3.1a

Section 3.1b:

High-Energy Compounds

Synopsis 3.1b

- Organisms capture the free energy released on degradation of nutrients in the form of “high-energy” compounds such as ATP

- Subsequent breakdown of ATP is used to power otherwise endergonic reactions

- The “high-energy” of ATP is related to the large negative free energy change associated with the hydrolysis of its phosphoanhydride bonds

- ATP hydrolysis can be coupled to an endergonic reaction such that the net reaction is favorable

Need for Energy Currency

- Combustion of fuel (or oxidation of food) such as glucose is highly exergonic as it releases considerable amount of free energy (-2850 kJ/mol)

- How can such energy be harnessed to drive endergonic reactions?

- How about storing this released energy into “high-energy” compounds—or simply, energy reservoirs/carriers/wallets—as a form of energy “currency” to be used later to drive endergonic processes?

- Indeed, the cellular machinery couples the energy released from the oxidation of food to the synthesis of energy currencies such as ATP and NAD(P)H

- ATP and NAD(P)H can then be used to “pay for” cellular processes that are otherwise thermodynamically unfavorable!

Go = -2850 kJ/mol

+ Energy (ATP)

Hydrolysis of “High-Energy” Compounds- Being referred to as “high-energy”

compounds is highly misleading and an unfortunate misnomer

- Although the hydrolysis of “high-energy” compounds releases large amounts of free energy, such release of energy does not result from the breaking of their “high-energy” bonds

- On the contrary, bond-breaking is an endergonic not exergonic process—ie it consumes rather than releases energy!

- In essence, the “high-energy” compounds harbor “unstable” bonds that can be easily hydrolyzed into thermodynamically more stable (lower energy) products!!

- So what makes the hydrolysis of “high-energy” compounds such as ATP an highly exergonic process?!

Why Is ATP Hydrolysis So Exergonic?!Highly exergonic hydrolysis (-31 kJ/mol) of the terminal phosphoanhydride bond of ATP into ADP and inorganic phosphate (Pi) is due to three major factors:

(1) Lower electrostatic repulsion—ATP hydrolysis results in the minimization of electrostatic repulsions between the negatively charged O atoms of phosphoanhydride bonds

(2) Higher solvation energy—The products of hydrolysis (eg ADP and Pi) are better solvated than ATP due to more favorable hydrogen bonding interactions with water, thereby resulting in greater (more favorable) solvation energy

(3) Greater resonance stabilization—Since the electrons on terminal O atoms are more delocalized than the bridging phosphoanhydride O atoms, hydrolysis of the terminal phosphoanhydride bond of ATP replaces one bridging oxygen atom with two new terminal oxygen atoms resulting in greater electronic stabilization of ADP and Pi relative to ATP

Being a “high-energy” compound, ATP serves as the cell’s primary energy currency!

Inorganic

Phosphate (Pi)

ATP Hydrolysis Coupled to Synthesis of Glucose-6-P

- Being a “high-energy compound”, ATP can spontaneously transfer its terminal phosphoryl group (-PO3

2-) to other compounds with lower phosphate hydrolysis potential—eg glucose

- Phosphorylation of glucose to glucose-6-phosphate (glucose-6-P) is an endergonic process

- Coupling this endergonic process to exergonic hydrolysis of ATP easily overcomes the thermodynamic barrier with an overall G << 0

- The half-reactions shown above are purely hypothetical and do not actually occur!

- Rather, hexokinase catalyzes the transfer of the terminal phosphoryl group (-PO32-) from

ATP directly to glucose to generate glucose-6-P (the first step of glycolysis—see §3.2)

(PEP)

PEP Hydrolysis Coupled to Synthesis of ATP

- Phosphorylation of ADP into ATP is an endergonic process

- Coupling this endergonic process to exergonic hydrolysis of phosphoenolpyruvate (PEP) renders it thermodynamically feasible with an overall G << 0

- The half-reactions shown above do not actually occur!

- Rather, pyruvate kinase catalyzes the transfer of the phosphoryl group (-PO32-) from PEP

directly to ADP to generate ATP (the final step of glycolysis—see §3.2)—a process referred to as “substrate-level phosphorylation” due to the fact that the phosphoryl group is transferred to ADP from another compound or substrate (eg PEP) as opposed to the energy stored in the transmembrane chemical potential (proton) gradient utilized in “oxidative phosphorylation”—see §3.7

Flow of Phosphoryl Groups to and from ATP

Phosphoryl groups flow from the “high-energy” donors to “low-energy”

acceptors

The squiggle (~) denotes a “high-energy”

phosphate bond!

Glycolytic

intermediates

Energy reservoir in skeletal

muscle and brain

- What kinds of molecules do cells use as energy currency?

- Why is ATP a “high-energy” compound?

- Describe the ways an exergonic process can drive an endergonic process

Exercise 3.1b

Section 3.1c:

Redox Reactions

Synopsis 3.1c

- Oxidation of food—to generate energy—essentially involves a series of redox reactions

- Redox reactions involve the transfer of electrons between compounds—iethey couple reduction and oxidation

- The free energy of electrons is captured via generation of reduced compounds and released upon their oxidation—release of such free energy is needed to drive many metabolic reactions

- The tendency of a compound to gain electrons (or become reduced) is rationalized in terms of the so-called reduction potential ()—the higher the , the greater the tendency to be reduced!

- Free energy and reduction potential are negatively related—the greater the reduction potential difference (+) between two compounds, the more negative the free energy change (-G)

Reducing agent (electron donor)—loses electrons (or hydrogen) and becomes oxidized(its oxidation state increases!)—eg Fe2+ loses an electron and becomes oxidized to Fe3+

Oxidizing agent (electron acceptor)—gains electrons (or hydrogen) and becomes reduced(its oxidation state decreases!)—eg Fe3+ gains an electron and becomes reduced to Fe2+

Oxidizing and Reducing Agents

Copper half-cellIron half-cell

KNO3K+ NO3-

- Consider the following redox reaction:Fe3+ + Cu+ <=> Fe2+ + Cu2+

which occurs during the oxidation ofcytochrome c oxidase in the mitochondrion

- In the context of an electrochemical cell—adevice capable of generating electrical energyfrom chemical reactions—the above redoxreaction can be divided into two half-reactions:

Cu+ <=> Cu2+ + e-

Fe3+ + e- <=> Fe2+

- Simply put, the copper half-cell undergoes oxidation by liberating electrons (electron-rich),while the iron half-cell experiences reduction by consuming electrons (electron-deficient)

- Such redox pair of half-reactions thus sets up an “electron gradient” across the two half-cellsresulting in an electrical potential difference or electromotive force () that drives the flowof electrons from electron-rich half-cell (copper) to electron-deficient half-cell (iron) throughan external circuit (eg a conducting wire)—how can we measure ?

- A salt bridge (eg a soaked filter paper in an electrolyte such as KNO3) is necessary to preventthe build-up of charge difference across the two half-cells by enabling the flow of ions (eg K+

toward the iron half-cell, and NO3- toward the copper half-cell) to maintain electronic

neutrality—failure to do so will impede the flow of electrons between the half-cells!

Redox Half-Reactions

- Consider the generalized redox reaction:A + B+ <=> A+ + B ------ = ox - red => = B - A

where + = Indicative of the oxidized state of corresponding speciesA = Reduction potential of half-reaction of species A (vide infra)B = Reduction potential of half-reaction of species B (vide infra) = Reduction potential difference between oxidizing (ox) and

reducing (red) agents—ie species B and A, respectively

- The half-reactions can be expressed as follows (electrons flow from A to B):A <=> A+ + e- ------- A

B+ + e- <=> B ------- B

- The reduction potential () is a measure of the tendency of a species to undergoreduction (or to gain electrons)—the higher the value (more positive) of , thegreater the reduction tendency

- Thus, B > A, since species B (acting as an oxidizing agent) displays higher tendencythan species A to undergo reduction

- How can we relate the reduction potential difference () to the standard reductionpotential difference () and free energy change (G)?

Reduction Potential Difference ()

- is related to by the so-called Nernst equation:

= + [RT/zF]lnQ [1]

where = Reduction potential difference (V)

= Standard reduction potential difference (V)

R = Universal molar gas constant (1.99 cal/mol/K, or 8.32 J/mol/K)

T = Absolute temperature (K)

z = Number of electrons transferred in the redox reaction

F = Faraday constant (96,485 C/mol, or 96,485 J/V/mol) => 1C=1J/V

Q = Reaction quotient (similar to equilibrium constant)

- Q is defined as:Q = [Aox][Bred] / [Ared][Box] [2]

- is related to the free energy change (G) released by the redox reaction asfollows:

G = -zF [3]

- Thus, a positive indicates a favorable redox reaction and vice versa

Nernst Equation

Standard Reduction Potentials () - Half-reactions with large positive arestrong electron acceptors (oxidizing agents)and energetically favorable

- Half-reactions with large negative arestrong electron donors (reducing agents)

- Electrons spontaneously pass from half-reactions with lower to increasing

- Thus, both NADH and FADH2 serve aselectron donors to reduce O2 to H2O duringoxidative phosphorylation—the free energyreleased is harnessed for ATP synthesis

- For example, free energy (G) released bythe reduction of O2 by NADH via thefollowing redox reaction is:

0.5O2 + NADH + H+ <=> H2O + NAD+

G = -zFwhere = (+0.815V) - (-0.315V) = 1.13V

F = 96,485 J/V/molz = 2

thus G = -(2).(96,485 J/V/mol).(1.13V)=> G = -218 kJ/mol

Redox Players

Major redox players, or cofactors, involved in mediating electron transfer from reduced metabolites to other compounds include:

(1) Nicotinamide adenine dinucleotide (NAD+)

(2) Flavin adenine dinucleotide (FAD)

(3) Flavin mononucleotide (FMN)

(4) Coenzyme Q (CoQ)

(5) Heme (Haem in Imperial English)

(6) Iron-sulfur clusters (Fe-S)

Nicotinamide adenine dinucleotide (NAD+)

- Only the nicotinamide moiety ofNAD+ serves as the site of reversiblereduction

- NAD+ adopts only two oxidationstates (oxidized NAD+ and reducedNADH)—implying that it can onlyaccept a pair of electrons asopposed to an unpaired electron

Adenine

Nicotinamide

Redox Players: NAD+

REDUCTIONSTRUCTURE

Phospho-

anhydride

bond

phosphorylated

in NADP

Ribose

Ribose

Flavin adenine dinucleotide (FAD)

Unlike NAD, FAD can adopt three oxidationstates (oxidized FAD, radical FADH. andreduced FADH2)—it can accept both pairedand unpaired electrons!

Redox Players: FADREDUCTIONSTRUCTURE

Flavin

AMP

Ribitol is the reduced form of ribose—cf aldose

vs alditol (§1.2)

Phosphoanhydride bond

Ribose

Flavin mononucleotide (FMN)

FMN differs from FAD in that it lacks AMP!

Redox Players: FMNREDUCTIONSTRUCTURE

Like FAD, FMN can also adopt three oxidationstates (oxidized FMN, radical FMNH. andreduced FMNH2)—it can accept both pairedand unpaired electrons!

Flavin

Phosphoester

bond

Redox Players: CoQREDUCTIONSTRUCTURE

Like FAD and FMN, CoQ also adopts threeoxidation states (oxidized CoQ, radical CoQH.

and reduced CoQH2)—it can accept bothpaired and unpaired electrons!

Coenzyme Q (CoQ)

also known as ubiquinone

(lipophilic)

n

where n = 6-10

Redox Players: Heme

- Heme is comprised of an heterocyclic ring called porphyrin with an iron ion at itscenter—the redox properties of heme are largely owed to the ability of iron to undergotransition between an oxidized (Fe3+) and reduced (Fe2+) state

- Heme exists in several biologically important forms such as heme a, heme b and heme c

- Heme occurs as a co-factor in a wide variety of proteins such as Mb and Hb (heme a—see §2.4) and cytochromes a/b/c (hemes a/b/c—see §3.7)

- By virtue of the ability of iron to transition between an oxidized (Fe3+) and reduced(Fe2+) state, heme plays a key role in orchestrating a plethora of redox reactions

Redox Players: Fe-S Clusters

- Iron-sulfur (Fe-S) clusters are comprised of iron ionsbridged between sulfide ions and furthercoordinated by either cysteine or cysteine/histidinesidechain groups within protein chains

- In iron-sulfur proteins, the two most commonlyoccurring Fe-S clusters are [2Fe-2S] and [4Fe-4S]

- In [2Fe-2S] cluster, two iron ions are bridgedbetween two sulfide ions—and each iron ion isfurther coordinated to either two CYS or CYS/HISresidues to adopt a tetrahedral geometry

- In [4Fe-4S] cluster, four iron ions are bridgedbetween four sulfide ions carving out a cube-likestructure—and each iron ions is further coordinatedto a CYS or HIS to adopt a tetrahedral geometry

- By virtue of the ability of iron to transition betweenan oxidized (Fe3+) and reduced (Fe2+) state, the Fe-Sclusters play a key role in orchestrating the transferof electrons from reduced to oxidized compounds

- What are the metabolic roles of the coenzymes NAD+ and FAD?

- Explain why NADH and FADH2 are a type of energy currency in the cell

- Explain the terms of the Nernst equation

- When two half-reactions are combined, how can you predict which compound will be oxidized and which will be reduced?

- How is related to G?

Exercise 3.1c