PART IV MetabolismIntroduction to Metabolism

• Living organisms are not at equilibrium• entropy <-> enthalpy• Require energy input• Metabolism• exergonic reaction are coupled to endergonic processes• Phototrophs / Chemotrophs• Over our lifespan, we eat tons of nutrients and drink some 20,000 liters of water

Metabolic pathways

•Series of consecutive enzymatic reactions•Converge on common intermediates•Anabolic / catabolic•ATP and NADH are major free energy sources

ATP and NADPH are the sources of freeenergy for biosynthetic reactions

5 principal characteristics of metabolicpathways

• irreversible -> confers directionality to a pathway

• catabolic and anabolic pathways must differ• every pathway has a first committed step• all metabolic pathways are regulated, rate-

limiting step• occur in specific cellular locations,

intracellular, organs

Overview ofcatabolism

Metabolic Functions of Eukaryotic Organelles

Organic reaction mechanismsBiochemical reactions are generally catalyzed by anenzyme4 categories of reactions:

• group-transfer reactions• oxidation and reductions• eliminations, isomerizations, and rearrangements• reactions that make or break carbon-carbon bonds

Models of C—H bond breaking

(mostly redoxreactions)

(transfer toNAD+)

Biologically important nucleophilic andelectrophilic groups. (a) Nucleophiles

(excess of electrons)

Biologically important nucleophilic andelectrophilic groups. (b) Electrophiles

Types of metabolic group-transferreactions. (a) Acyl group transfer

i.e. peptide bond hydolysis by chymotrypsin

Types of metabolic group-transferreactions. (b) Phosphoryl group transfer

in-line addition inversion of configuration

Types of metabolic group-transferreactions. (c) Glycosyl group transfer

The phosphoryl-transfer reactioncatalyzed by hexokinase

chiral due to isotopicsubstitution

Oxidations and Reductions•Redox reactions involve the loss or gain of electrons•Frequent electron acceptor is NAD+

•Terminal acceptor in aerobes is O2, two step reductionby FADH2 (Pauli rule)• reduced = gains electrons; oxidized = loses electrons

The molecular formula and reactions of thecoenzyme flavin adenine dinucleotide (FAD)

Vit B2

Possible elimination reaction mechanisms usingdehydration as an example

i.e. dehydration resulting in the formation of a C=Cdouble bond, e.g. enolase, fumarase

Possible elimination reaction mechanisms usingdehydration as an example

Mechanism of aldose–ketose isomerization

Most prominent biochemical isomerization reaction, hydrogen shift, base-catalyzed, e.g. phosphoglucose isomerase,Racemization / epimerization

Examples of C—C bond formation and cleavagereactions. (a) Aldol condensation

Addition of a nucleophilic carbanion to an electrophiliccarbon atom (aldehyde, keton, ester, CO2)

Examples of C—C bond formation and cleavagereactions. (b) Claisen condensation ester

Examples of C—C bond formation and cleavagereactions. (c) Decarboxylation of beta-keto

acid

i.e. fatty acid degradation (beta-oxidation)

Stabilization of carbanions. (a) Carbanions adjacent to carbonylgroups are stabilized by the formation of enolates

(b) Carbanions adjacent to carbonyl groups hydrogen bonded togeneral acids are stabilized electrostatically or by charge

neutralization

Stabilization of carbanions. (c) Carbanions adjacent toprotonated imines (Schiff bases) are stabilized by the

formation of enamines

(d) Metal ions stabilize carbanions adjacent to carbonylgroups by the electrostatic stabilization of the enolate

Experimental approaches to the study ofmetabolism

How does one know what is written here ?

Key question with regard to metabolic conversion:

1. What is the sequence of reactions ?

2. What is the mechanism ?

3. How is it controlled ?

ToolsMetabolic inhibitors, growth studies, biochemical genetics• pathway intermediates accumulate in the presence ofinhibitors,e.g. glycolysis• genetic defects cause accumulation of intermediates,e.g. phenylketonuria•Metabolic blocks induced by mutagens / geneticselection of auxotrophs, e.g. arginine biosynthesis•Genetic manipulation of higher organisms, e.g. knockoutmice, expression of cratine kinase

IsotopesIsolated organs, cells,and subcellular organelles

Pathway for phenylalanine degradation

accumulates in urine

Pathway of arginine biosynthesis indicating thepositions of genetic blocks

Neurospora crassa auxotrophic mutantsin arginine biosynthesis

The expression of creatine kinase in transgenic mouseliver as demonstrated by localized in vivo 31P NMR

The conversion of [1-13C]glucose to glycogenas observed by localized in vivo 13C NMR

Isotopes in Biochemistry

• Isotopes, atoms with different number of neutrons• used to label molecules without changing their chemical properties• used for in vivo NMR studies, 1H, 13C, 31P• radioactive isotopes (unstable), 3H, 14C, 32P, 35S

• alpha emitter (He)• beta (electrons), 3H, 14C, 32P; 0.0018. 0.155, 1.71 MeV• gamma (photons)• detection by

• proportional counting (Geiger, gas charge)• liquid scintillation counting (fluorescence)• autoradiography (film)

• half-lives• study precursor-product relation

Some Trace Isotopes of Biochemical Importance

Some Trace Isotopes of Biochemical Importance

The metabolic origin of the nitrogenatoms in heme

Two possible pathways for the biosynthesis ofether– and vinyl ether–containing phospholipids

The flow of a pulse of radioactivity fromprecursor to product

Isolated organs, cells, and subcellularorganelles

Which organ, cell, subcellular organelle performs thatmetabolic conversion ?

• Organ perfusion• Tissue slices• Cell sorter• Tissue culture

Thermodynamics of phosphate compounds

Endergonic processes that maintain the living state aredriven by the exergonic reactions of nutrient oxidationATP the high-energy intermediate

Standard Free Energies of Phosphate Hydrolysisof Some Compounds of Biological Interest

Some overall coupled reactions involving ATP. (a) Thephosphorylation of glucose to form glucose-6-phosphate

and ADP

Some overall coupled reactions involving ATP. (b) Thephosphorylation of ADP by phosphoenolpyruvate to form

ATP and pyruvate

Resonance and electrostatic stabilization in aphosphoanhydride and its hydrolysis products

Why is the phosphoanhydridbond a high energy bond ?•Resonance stabilization•Repulsion•Solvation energy

ATP is kinetically stable, i.e.not hydrolyzed

Hydrolysis of phosphoenolpyruvate

Other high-energy compounds

1. Acyl phosphates, i.e. acetyl phosphate or 1,3-bisphosphoglycerate

2. Enol phosphate, i.e. phosphoenolpyruvate: ADP->ATP !3. Phosphoguanidines

Competing resonances inphosphoguanidines

The flow of phosphoryl groups from “high-energy”phosphate donors, via the ATP–ADP system, to “low-

energy” phosphate acceptors

The phosphorylation of fructose-6-phosphateby ATP to form fructose-1,6-bisphosphate

and ADP

Consumption of ATP

1. Early stages of nutrient breakdown, e.g. glycolysis(hexokinase, phosphofructokinase)

2. Interconversion of nucleoside triphosphates, i.e.ATP + NDP -> ADP + NTP (nucleoside diphosphatekinase)

3. Many different physiological processes, e.g. proteinfolding, translation

4. Orthophosphate / pyrophosphate cleavage, e.g. tRNAcharging

Pyrophosphate cleavage in the synthesis of anaminoacyl–tRNA

Formation of ATP

1. Substrate-level phosphorylation, e.g.phosphoenolpyruvate

2. Oxidative phosphorylation / photophosphorylation3. Adenylate kinase AMP + ATP -> 2 ADP

Rate of ATP turnover

ATP is energy transmitter not reservoir !Consumption ca 3 mol; 1.5 kg/h, up to 10x on stress

Phosphocreatine provides a bufferATP + creatine <-> phosphocreatine + ADP, creatine

kinaseServes as an ATP generating system in in vitro

experiments

Oxidation - reduction reactions

• Electron transfer reaction (redox) are of immense biochemical importance• Reduction, gain of electrons• Oxiation, loss of electrons• Conjugate redox pair• Nernst equation• Measurements of redox potentials, relative to hydrogen half cell at pH=0• Concentration cells, e.g. across the plasma membrane, nerve cells

Example of an electrochemical cell

Standard Reduction Potentials of SomeBiochemically Important Half-reactions

Two examples of open systems in a steady state.(a) A constant flow of water in the river occurs

under the influence of the force of gravity

Thermodynamics of life

• Living systems are not at equilibrium (high entropy) unless they are dead• They are open systems at steady-state

The steady state of the biosphere is similarlymaintained by the sun

Thermodynamics of metabolic control

•Enzymes selectively catalyze required reactions•Many enzymatic reactions are near equilibrium•Pathway throughput is regulated by controlling enzymatic steps that are far from equilibrium