PART IV Metabolism
Introduction 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 free
energy for biosynthetic reactions
5 principal characteristics of metabolic
pathways
• irreversible -> confers directionality to a
pathway
• catabolic and anabolic pathways must differ
• every pathway has a first committed step
• all metabolic pathways are regulated, ratelimiting step
• occur in specific cellular locations,
intracellular, organs
Overview of
catabolism
Metabolic Functions of Eukaryotic Organelles
Organic reaction mechanisms
Biochemical reactions are generally catalyzed by an
enzyme
4 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 redox
reactions)
(transfer to
NAD+)
Biologically important nucleophilic and
electrophilic groups. (a) Nucleophiles
(excess of electrons)
Biologically important nucleophilic and
electrophilic groups. (b) Electrophiles
Types of metabolic group-transfer
reactions. (a) Acyl group transfer
i.e. peptide bond hydolysis by chymotrypsin
Types of metabolic group-transfer
reactions. (b) Phosphoryl group transfer
in-line addition
inversion of
configuration
Types of metabolic group-transfer
reactions. (c) Glycosyl group transfer
The phosphoryl-transfer reaction
catalyzed by hexokinase
chiral due to isotopic
substitution
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 reduction
by FADH2 (Pauli rule)
• reduced = gains electrons; oxidized = loses electrons
The molecular formula and reactions of the
coenzyme flavin adenine dinucleotide (FAD)
Vit B2
Possible elimination reaction mechanisms using
dehydration as an example
i.e. dehydration resulting in the formation of a C=C
double bond, e.g. enolase, fumarase
Possible elimination reaction mechanisms using
dehydration 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 cleavage
reactions. (a) Aldol condensation
Addition of a nucleophilic carbanion to an electrophilic
carbon atom (aldehyde, keton, ester, CO2)
Examples of C—C bond formation and cleavage
reactions. (b) Claisen condensation ester
Examples of C—C bond formation and cleavage
reactions. (c) Decarboxylation of beta-keto
acid
i.e. fatty acid degradation (beta-oxidation)
Stabilization of carbanions. (a) Carbanions adjacent to carbonyl
groups are stabilized by the formation of enolates
(b) Carbanions adjacent to carbonyl groups hydrogen bonded to
general acids are stabilized electrostatically or by charge
neutralization
Stabilization of carbanions. (c) Carbanions adjacent to
protonated imines (Schiff bases) are stabilized by the
formation of enamines
(d) Metal ions stabilize carbanions adjacent to carbonyl
groups by the electrostatic stabilization of the enolate
Experimental approaches to the study of
metabolism
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 ?
Tools
Metabolic inhibitors, growth studies, biochemical genetics
• pathway intermediates accumulate in the presence of
inhibitors,e.g. glycolysis
• genetic defects cause accumulation of intermediates,
e.g. phenylketonuria
•Metabolic blocks induced by mutagens / genetic
selection of auxotrophs, e.g. arginine biosynthesis
•Genetic manipulation of higher organisms, e.g. knockout
mice, expression of cratine kinase
Isotopes
Isolated organs, cells,and subcellular organelles
Pathway for phenylalanine degradation
accumulates in urine
Pathway of arginine biosynthesis indicating the
positions of genetic blocks
Neurospora crassa auxotrophic mutants
in arginine biosynthesis
The expression of creatine kinase in transgenic mouse
liver as demonstrated by localized in vivo 31P NMR
The conversion of [1-13C]glucose to glycogen
as 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 nitrogen
atoms in heme
Two possible pathways for the biosynthesis of
ether– and vinyl ether–containing phospholipids
The flow of a pulse of radioactivity from
precursor to product
Isolated organs, cells, and subcellular
organelles
Which organ, cell, subcellular organelle performs that
metabolic conversion ?
• Organ perfusion
• Tissue slices
• Cell sorter
• Tissue culture
Thermodynamics of phosphate compounds
Endergonic processes that maintain the living state are
driven by the exergonic reactions of nutrient oxidation
ATP the high-energy intermediate
Standard Free Energies of Phosphate Hydrolysis
of Some Compounds of Biological Interest
Some overall coupled reactions involving ATP. (a) The
phosphorylation of glucose to form glucose-6-phosphate
and ADP
Some overall coupled reactions involving ATP. (b) The
phosphorylation of ADP by phosphoenolpyruvate to form
ATP and pyruvate
Resonance and electrostatic stabilization in a
phosphoanhydride and its hydrolysis products
Why is the phosphoanhydrid
bond 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 1,3bisphosphoglycerate
2. Enol phosphate, i.e. phosphoenolpyruvate: ADP->ATP !
3. Phosphoguanidines
Competing resonances in
phosphoguanidines
The flow of phosphoryl groups from “high-energy”
phosphate donors, via the ATP–ADP system, to “lowenergy” phosphate acceptors
The phosphorylation of fructose-6-phosphate
by 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 diphosphate
kinase)
3. Many different physiological processes, e.g. protein
folding, translation
4. Orthophosphate / pyrophosphate cleavage, e.g. tRNA
charging
Pyrophosphate cleavage in the synthesis of an
aminoacyl–tRNA
Formation of ATP
1. Substrate-level phosphorylation, e.g.
phosphoenolpyruvate
2. Oxidative phosphorylation / photophosphorylation
3. 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 buffer
ATP + creatine <-> phosphocreatine + ADP, creatine
kinase
Serves 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 Some
Biochemically 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 similarly
maintained 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