part iv metabolism introduction to metabolism€¦ · part iv metabolism introduction to metabolism...
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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