Biochemistry

Important sources of fuel in the diet

How ATP drives chemical works
Metabolism is the sum of all chemical reactions that occur inside a cell
Anabolic processes power catabolic process.
Amphibolic process is both anabolic and catabolic
Importance of ATP as a short term energy sources
Average human body only stores a small amount of ATP
But much more ATP is required everyday
So ATP has to remade
Macronutrient fuel sources that supply substrates for regenerating ATP
Carbohydrates, protein, fat
Daily energy expenditure and basal metabolic rate
Anabolic and catabolic pathways
Energy from catabolism (exergonic: release energy) is used to power cellular work
(endergonic: consume energy)
Major fuel stores in the body

ATP-ADP cycle

 Phosphoanhydride bonds in ATP is

o Stable in water
o Hydrolysis of these bonds are exothermic
ATP was captured and fixed early in evolution
Negative charge want to repel each other making ATP unstable
Principles of bioenergetics
fG0
o Law of thermodynamics
1st law of thermodynamics
Energy cannot be created or destroyed, it only changes form
o U= Ufinal-Uinitial = q+w
o *Reactions happen at dilute solutions, assume
molecules behave like gas
Enthalpy (H) = U + PV
o Pressure and volume in most biological reactions are
constant
o H=U
o H=q + w
2nd law of thermodynamics
The universe is becoming increasingly disordered
Systems will progress towards increased entropy (S)
Time cannot be reversed
Non- equilibrium to equilibrium
*Life is a battle to avoid equilibrium
Organisms increase the entropy around them
Predicts whether a process with occur spontaneously
Ssystem + Ssurroundings = Suniverse
S is greater than or equal to q/T *constant temperature
H (change in energy of reaction)-TS (energy lost)= G (net change
in energy required)
G Gibbs free energy
Predicts whether a reaction is going to occur spontaneously
Predicts the maximum possible change (potential) in
concentrations between reactants and products or work

 More negative means more work can be done

Not predict the rate at which reactions will occur
Negative G0 value for ATP hydrolysis
The energy released from terminal phosphate by hydrolysis of anhydride bond is high
The free energy of formation (fG0) of ATP is greater than the sum of its products ATP
and Pi
G0 (kJmol-1) = -30.5 for hydrolysing third ATP
How free energy liberated by hydrolysis of ATP is used to carry out biological work
Metabolic reaction steps used to produce ATP
Pathway of glycolysis
Splitting of sugar
Glucose is trapped using phosphate (from 2 ATP)
o Rearranged
o Split into 2 3C molecules
o 2 more phosphates added
o 4 phosphates harvested
o Oxidation (strip electrons)
o 10 enzymes
o 2 ATP net gain
o 1 oxidation reaction for rearrangement and preparation for respiration
o 2 NADH
*Molecules must be rearranged to C-H for oxidation (C-C bonds not oxidised
directly)
o Electrons from these bonds ultimately flow to oxygen
LEOs a GERc
*Dehydrogenases do redox reactions
Detailed pathway
o Making glucose-6-phosphate
By hexokinase or glucokinase
1 ATP used
Negatively charged phosphate added
Prevent leaving cell
Changes shape so not recognised by GLUT
o Fructose-6-phosphate
Made by Phosphogluco-isomerase
o Fructose 1,6-bisphosphate
Made by Phosphofructokinase (PFK)
1 ATP used
*Glucose now has to go through catabolism
o Glyceraldehyde-3-phosphate
Split Fructose-1,6-bisphosphate using aldolase

Glyceraldehyde-3-phosphate and Dihydroxyacetone phosphate form

Triose phosphate isomerase interconvert between glyceraldyhyde-3phosphate and dihydroxyacetone phosphate
Glyceraldehyde-6-phosphate continues on glycolysis
1,3-Bisphosphoglycerate
Made by triose phosphate dehydrogenase
Add 2 inorganic phosphate
2NADH made from NAD+
Only oxidation reaction in glycolysis
*NADH + H+ made by glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)
3-Phosphoglycerate
Phosphoglycero-kinase makes it
2 ATP harvested
2-Phosphoglycerate
Made from phosphoglyceromutase
Phosphoenolpyruvate
Made from Enolase
2 water molecules removed
Pyruvate
Made from pyruvate kinase
Make 2 ATP

o
o

Roles of hexokinase and glucokinase in the first step of glycolysis

Hexokinase
o In most tissues
o Low Km
o Low Vm
o Inhibited by glucose 6-phosphate
Glucokinase
o Liver and Beta-cells
o High Km
o High Vm
o Not inhibited by glucose 6-phosphate
Km
o Reciprocal of affinity
o High Km means low affinity for glucose
Importance of substrate level phosphorylation
No oxygen is required
ATP is captured by substrate level phosphorylation in glycolysis
Importance of electron acceptor NAD+ for glycolytic pathway operation role of lactate
dehydrogenase in generating NAD+ in anaerobic conditions

 No oxygen
o NAD+ is limited in amount, so must be regenerated to start prevent glycolysis
stopping
Roles of the Cori cycle
Regulation of glycolysis
Hexokinase/ glucokinase
Phosphofructokinase
o Inhibited by high ATP
o AMP activates PFK
o Decreased pH (high H+)
o First committed step
Pyruvate kinase
Highly regulated
Irreversible in vivo
Overall pathway of CAC cycle

Before CAC
o Pyruvate goes through transport protein (pyruvate dehydrogenase (3 enzyme
complex))
Decarboxylated
Oxidised (make NADH)
CoA binds
*CoA made from vitamin B1
Makes Acetyl CoA
CAC cycle
o Citrate synthase
Makes citrate by adding acetyl group (2C) to oxaloacetate (4C)
o Aconitase
Makes Isocitrate by removing and reading water
*Aconitase is Iron-sulphur complex that is sensitive to free radical
superoxide and damaged
o Isocitrate dehydrogenase
Makes -ketoglutarate by making NADH and removing CO2
o -ketoglutarate dehydrogenase
Makes Succinyl CoA by removing CO2 , making NADH and adding
CoA-SH
Similar to PDH
o Succinyl CoA synthetase
Makes Succinate by releasing CoA-SH and substrate level
phosphorylation to make GTP which is then made into ATP (no energy
loss)
o Succinate dehydrogenase
Makes Fumarate by making FADH2

*It is complex II of ETS, in inner mitochondrial membrane

Handles dangerous free radicals
o Fumarase
Makes malate by adding water and rearranging
o Malate dehydrogenase
Forms oxaloacetate by oxidation (make NADH)
o *This was for 1 pyruvate (2 pyruvate formed from glycolysis)
o *Each glucose: 6 CO2, 6 NADH+H+, 2FADH2, 2GTP (ATP)
*CAC is amphibolic
o CAC cycle intermediates can be made into biologically important molecules
Amino acid synthesis: -ketoglutarate, oxaloacetate
Fatty acid synthesis: citrate
Neurotransmitters: acetyl CoA (acetyl-choline), -ketoglutarate
(glutamate)
Heme (haemoglobin, cytochromes) : succinyl CoA
Gluconeogenesis: malate

How rates of key enzymes are regulated to modulate activity of CAC cycle\
Pyruvate dehydrogenase
o Inhibited by ATP, acetyl CoA, NADH
Isocitrate dehydrogenase
o Inhibited by ATP, NADH
o Activated by ADP
-ketoglutarate dehydrogenase
o Inhibited by ATP, succinyl CoA and NADH
*ATP/ADP ratio
o 2/10: goes faster
o 10/2: slow down
NADH/NAD+ ratio (redox state)
o High NADH means a lot of reducing power
-KDH and IDH highly regulated by Ca+2
How energy from the transport of electrons to O2 is transformed into high energy bonds
in ATP
Complex I
o Flavin mononucleotide on the top of complex I with greater reducing power
than NADH
o 2 Electrons from NADH passed to Flavin mononucleotide
o Electrons pass down iron sulphur complex (quantum tunnelling)
o Changes conformation of CI subunits
o Reduces Ubiquinone (Q10,Q9) to QH2 (ubiquinol)
o
o Pumps 4 protons for 1 NADH

o *Protons must form weak hydrogen bonds and pass down proton wire (can be
blocked)
o *Unbiquinone (Q10) reduced Semiquinone (1 OH) into ubiquinol (2OH)
o *Release free radicals when damaged
o Inhibited by rotenone (stop electron flowing to Q)
Complex II
o Succinate comes in fumerate is produced
FADH accepts electrons one at a time
Dangerous free radicals form
Must be within enzyme
Pass electron to Q
o *NADH accepts electrons 2 at a time
o Not pump hydrogens (FADH2 not have enough reducing power)
o *CI and CII use Q and converge on CIII
o *Not release free radicals
Complex III
o Ubiquinol from complex II donates electron to complex III
Transfer protons to make ubiquinone
2 Electrons pass to 2 cytochrome C
Travels to complex IV
4 Hydrogens transferred
2 from QH2
2 from matrix
2 reduced QH2
Not a pump!
o Antimycin a (blocks Q cycle)
Cytochrome c
o Carries one electron to complex IV from complex III
Complex IV
o Cytochrome c oxidase
o Has a heme
o Cytochrome c transfer electron to copper cytochrome (a) and another copper
cytochrome (b)
o Oxygen comes in, split as electrons are donated
o Single oxygen, 2 hydrogen come down and make water
o Electrons passing through have an effect on conformation of CIV
Move 2 proton moved into intermembrane space (similar to pumping)
o Copper ions and iron to hold cytochrome
o Electrons flowing through complex IV cause conformational change and
pump 2 protons
o Oxygen is final electron acceptor to form water
o Protons removed from matrix on water formation makes complex IV receive 1
electron per cytochrome c and pump 2 protons
o 2 Protons pumped and 2 removed from matrix (increase membrane potential)
o Cyanide inhibit
o Trigger apotosis : programmed cell death

 ETS arranged into 2 supercomplexes

o I,III,IV
10 proton per NADH
o II,III,IV
6 protons per FADH2
Why net yield of oxidative phosphorylation is weird
Some membranes can leak deliberately
o Generate heat
o Lower oxidative stress
Electron transport carriers which transfer electrons from NADH, FADH2 to O2
Understand chemiosmotic coupling hypothesis and the mechanism of action of ATP
synthase
The internal rod moves around within the catalytic knob
o Exerts pressure on subunits
o Changes their conformation
o Each rotation of internal rod makes 3 ATP
NADH from glycolysis has two pathways
o Malate aspartate shuttle
2.5 ATP per NADH
o Glycero-3-phosphate dehydrogenase shuttle
1.5 ATP per NADH
Less efficient but faster
*NADH cannot get into mitochondria by itself

Structure of glycogen

Storage in cytosol of many tissue driven by insulin

Using driven by glucagon
Water insoluble
Protects cell from osmotic pressure
Only liver and kidney glycogen accessible to other tissue
Liver store up to 10% wet mass of glycogen

Muscle only 2% due to space limit (has to contract)

Branching by -1,6 glycosidic linkages
Chain by -1,4 glycosidic linkages
Reducing end attached to glycogenin

Different roles of liver and muscle glycogen

Pathways of glycogen synthesis and degradation
Glycogen synthesis (glycogenesis)
o Hexokinase (glucokinase) changes glucose into glucose 6-phosphate
o Phosphoglucomutase makes G6P to G1P
o UDPG Pyrophosphorylase make UDPG from G1P (endergonic)
o Glycogen synthase associate with glycogenin to control size of granules
o *Glucose added to non-reducing ends

o Branching enzyme makes new branch with 7 glycosyl residues

Each branch must be 11 residues before transfer
New branches are 4 residues away
Branches move in toward core
Dense granule
Glycogen breakdown

o Glycogen phosphorylase
Phosphorylysis
Ensure released glucose is charged and trapped in cell
No Pi is used to keep glucose in cell (no ATP used)
Glucose 1 phosphates released
Leaves 4 glycosyl residues
Inhibited by ATP, glucose, G6P
o Debranching enzyme
Transferase that transfer 3 glycosyl units
Hydrolyse -1,6 links
o Phosphoglucomutase
Reversible enzyme
G1P to G6P
o Glucose-6-phosphatase
Make glucose from G6P
Only in liver and kidneys
Regulate the activity of glycogen synthase and glycogen phosphorylase
Glycogen metabolism stimulated by glucagon or epinephrine (adrenalin)

Metabolic pathway used by liver to synthesize glucose

Gluconeogenesis
o Inputs
Lactate
Amino acids
Glycerol
TCA intermediates

o 3 Bypass
Bypass 1
Pyruvate carboxylase in mitochondria
Phosphoenolpyruvate carboxylkinase in cytosol
Bypass 11
Fructose 1-6-bisphosphatase in cytosol

Bypass III
Glucose 6 phosphatase
Consume 12 ATP
o Gluconeogenesis and glycolysis must not run at the same time

o Futile cycles
o Phosphofructokinase + fructose 1,6-bisphosphatase
o Allow heat generation
Enzyme defects associated with glycogen storage diseases (GSD)
11 types of GSD
von Gierkes disease (type 1 GSD
o Glucose 6-phosphatase mutation
o Hypoglycaemia
o Enlarged liver and kidneys
o G6P used to make triglycerides
Mc Ardles disease
o Glycogen in muscle
o Lack of glucose release
o Little glycogen phosphorylase activity (myophosphorylase)
Roles of insulin and glucagon to regulate blood glucose levels
Insulin
o A-chain and B-chain (C-chain is cleaved)
o Quaternary structure bonded together by disulphide bridges
o 51 aa protein

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Growth hormone
Drives glucose uptake in tissues( especially skeletal muscle and liver)
Promotes fat deposition, glycogen storage, growth
Generally anabolic

How glucagon stimulates glycogen breakdown and glucose synthesis

Roles of different energy sources during exercise and starvation
Diabetes mellitus
o Normal fasted blood glucose = 5mM
o Diabetic greater than 11mM, fasted greater than 7mM
o Cardiomyopathy
o Nephropathy
o Retinopathy
o Impaired peripheral circulation
Type I
Insulin dependent,
Loss of beta cell
No insulin production
Type II
Insulin dependent
In older people
Obesity, stress
Insulin insensitivity
High fat in blood alter signalling or glucose metabolism
Oxidative stress, maternally inherited
Too much fuel too few mitochondria
Protect cell by decreasing GLUT
Increase blood glucose
Mitochondrial DNA mutation impair glucose uptake
Starvation

o Brain, heart start using ketone bodies as full source

Acetoacetate, acetone, beta-hydroxybuyrate
Brains cannot use fat
Acidic lower in pH- ketoacidosis
In untreated type I
Alcoholics (impair liver gluconeogenesis)
Exercise
o 1.ATP
o 2.Replace ATP by creatine phosphate using creatine kinase
o *If ATP stores decline cells die by necrosis
o 3.Muscle glycogen
o 4.Fatty acids
o 5.Proteins
o Anaerobic pathways
Creatine phosphate, glycogen self-contained in cell
Respiration takes time for transport of oxygen, more reactions required
Two stages of photosynthesis and the overall reaction during photosynthesis
Structure of chloroplast and molecules involved in light absorption
Reactions taking place during light reactions
Absorption of light is coupled to ATP synthesis
Process of Calvin cycle
How the absorption of light is coupled to the synthesis of glucose
Calvin cycle is regulated