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Title: Biochemistry: The energy of metabolism
Description: Detailed notes about metabolism and metabolic pathways, their regulation and enzyme regulation. Aimed at 1st or 2nd year students undertaking any kind of science or health degree.
Description: Detailed notes about metabolism and metabolic pathways, their regulation and enzyme regulation. Aimed at 1st or 2nd year students undertaking any kind of science or health degree.
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Elements essential to life and health
• Bulk elements – Structural components of cells required in daily gram
amounts (H,C,N,O,Na,Mg,K,Ca)
• Trace elements – Required in daily mg amounts
• Cells comprise very few types of atoms
o H,C,N,O comprise 99% of an organism
o Contrasts the non-‐living, inorganic environment
A cell is made of carbon compounds
• Most molecules within cells are comprised of carbon due to its ability to
form large molecules
• C is small, has 4 electrons and 4 vacancies in outer shell à Forms 4
covalent bonds
• Highly stable C-‐C covalent bonds form linear/branched chains and rings
• C skeletons can bind functional groups (methyl, hydroxyl, carbonyl etc)
Small organic molecules are used to form macromolecules
• Sugars à Polysaccharides
• FA à Fats, lipids, membranes
• AA à Proteins
• Nucleotides à Nucleic acids (DNA, RNA)
Water is the most abundant substance in cells
• Most cellular interactions occur in an aqueous environment
• Water is a polar molecule à Unequal sharing of electrons results in
partial positive charge on H and partial negative charge on O à Polarity
and cohesiveness due to H bonding
• Water is a good solvent for charged and polar substances
o AA and peptides
o Small alcohols
o CHO
• Water is a poor solvent for nonpolar substances
o Nonpolar gases
o Aromatic moieties
o Aliphatic chains
Water can participate directly in chemical reactions (act as a reactant)
• Condensation reaction à Formation of a bond between OH and OH
expelling water
o New bond formed between OH groups on 2 different molecules
o Water generated
• Hydrolysis reaction à Cleavage of this bond accompanied by the addition
of water
o 2 molecules cleaved to form OH groups on the 2 monomers
• Cage formation of water around hydrophobic regions
Revision
• H, O, C, N are the 4 main elements in the human body
•
•
•
•
•
Carbon is well suited to life because it is small and can form 4 covalent
bonds and C skeletons can bind functional groups
Hydrophilic – Polar or charged molecules that dissolve readily in water
Hydrophobic – Non-‐polar molecules or groups that do not dissolve
readily in water
Hydrolysis reaction cleaves bond in a molecule and adds water molecules
Condensation reaction forms a bond between hydroxyl groups of 2
molecules and expels water
Enzymes promote reactions
• Cellular chemical reactions require higher temperatures than that found
in cells; overcome by use of specialised proteins (enzymes)
• Enzymes function as catalysts to speed up the reaction rate of a specific
substrate by lowering activation energies
• Do not affect equilibrium of a reaction and thus can be bidirectional
(substrate à Product and product à substrate)
• Often conjugated to a cofactor (inorganic ion) or coenzyme (complex
molecule) to function
• Lowers amount of total energy required for a forward reaction
Factors affecting the activity of enzymes and metabolic processes (enzyme
regulation)
• Amount of enzyme à Influenced by rate of transcription of enzyme genes
o Extracellular signals (down or up regulation)
o Protein degradation (ubiquitin)
o mRNA degradation
• Catalytic activities of enzymes à Allosteric control, feedback inhibition,
covalent modification (phosphorylation) or proteolytic cleavage
• Accessibility of substrates à Compartmentalisation/compartmentation
o Reactions occur in certain/specific parts of the cell/body
Enzyme regulation
• Factors affecting amount of enzyme
o Extracellular signals à down or up-‐regulation
o Transcription of specific enzyme gene
o mRNA degradation
o mRNA translation on ribosome
o Protein degradation (ubiquitin, proteasome)
o Enzyme sequestered in subcellular organelle
(compartmentalisation)
• Factors affecting enzyme activity
o Enzyme binds substrate
o Enzyme binds ligand (allosteric effector)
o Enzyme undergoes phosphorylation or de-‐phosphorylation
o Enzyme combines with regulatory protein
Allosteric control of enzymes
•
•
Allosteric control is the regulation of an enzyme by a regulatory molecule
that interacts at a site other than the active site (allosteric site)
Allosteric modulators can be either stimulatory (positive) or inhibitory
(negative)
Feedback inhibition of enzymes
• Inhibition of the first irreversible reaction (committed step) in linear
pathways
• Branched pathways
o Inhibition of common initial step by own product and activation by
product of another pathway
o Enzyme multiplicity à Inhibition of the committed step catalysed
by isoenzymes
o Cumulative feed inhibition à Partial inhibition of a common step
by each of the final products
Covalent modification of enzymes à Phosphorylation
• Tyr, Ser, Thr, His
• Protein kinase (ATP à ADP) phosphorylates
• Protein phosphatase dephosphorylates
Some enzymes (zymogens) are regulated by proteolytic cleavage
• Zymogens à Inactive precursors that become active following cleavage
• Trypsinogen + enteropeptidase à Trypsin (active)
• Chymotrypsinogen + trypsin à Pi-‐Chymotrypsin (active)
• Pi-‐chymotrypsin à Alpha-‐chymotrypsin
Living organisms require energy from the environment
• Energy comes in the form of chemical fuels (food) or light from
environmental sources
• Energy is required for 3 main purposes for living organisms
o Mechanical work (muscle contraction and cellular movement)
o Active transport
o Synthesis of macromolecules from simple precursors
Basic principles underlying energy flow
• Fuels are degraded and large molecules are constructed in a series of
reactions (metabolic pathways)
• ATP links energy releasing pathways with energy requiring pathways
(crucial carrier used for formation of complex molecules)
• Oxidation of carbon fuels powers ATP formation
o Carbon fuels are highly reduced elements that when broken down
yield electrons for ATP formation
• A limited number of reaction types and intermediates are common to
many pathways
• Metabolic pathways are highly regulated and compartmentalised
o Compartmentalisation and allosteric modulation regulate
metabolic pathways
Metabolism
• Highly coordinated cellular activity in which multi-‐enzyme systems
cooperate to
o Obtain energy by degrading energy rich nutrients or by capturing
solar energy
o Convert nutrient molecules to cell’s own molecules
o Polymerise monomers to polymers (polysaccharides, lipids,
proteins, membranes, nucleic acids)
o Synthesise and degrade biomolecules
o Maintain distinctive composition of different cell compartments
Catabolic and anabolic metabolism
• Catabolism and anabolism are the major metabolic pathways
• Catabolism is the breakdown food molecules into useful forms of energy +
heat lost
• Anabolism uses the energy produced from catabolism to produce new
molecules from small molecules
• Enzymes catalyse these two pathways which constitute the metabolism of
a cell
Metabolism
• Where the processes occur can be cell specific
• Centres around generation of pyruvic acid and acetyl CoA
• CHO stored as glycogen which provides energy between meals (glucose
à Glycolysis à Pyruvic acid à Acetyl CoA à CA cycle à Electron energy
carriers à Oxidative phosphorylation à ATP generation)
• Pyruvic acid à Glucose can generate glycogen during gluconeogenesis
• Protein not stored, only broken down during starvation state
• Proteins à AA à CA cycle à Pyruvic or acetyl CoA and some may enter
TCA cycle directly
• Lipids stored as TAG à FA + glycerol à Glycerol can enter glycolysis but
the bulk of the energy comes from the FA chains which break down to
acetyl CoA à CA cycle
• Production of energy from the oxidation of food molecules (either
digested or stored) which are highly reduced molecules (rich in
electrons)
• Energy carriers carry electrons to the pathways which produce ATP
Three forms of energy stores in biology
• Energy carriers à Contain 1 or more energy rich covalent bonds
• Macromolecules
o Highly reduced molecules
o Large branched polysaccharides (glycogen/starch)
o Fatty acids (Animal cells derive energy from between meals)
o Sugars and fats both degraded to acetyl CoA in the mitochondria to
drive ATP production
• Electrochemical gradients also a source of energy
o Electrical force (drive by differences in membrane potential)
o Chemical force (ion concentration)
Energy carriers are essential for biosynthesis
• Food molecules (digested or stored) are highly reduced and rich in energy
à Through catabolism they are oxidised to yield their electrons à
Electrons carried by activated carrier molecules and used to drive
metabolic pathways in the production of new molecules required by the
cell for storage or use
• About the flow of electrons
Different forms of energy carriers
• ATP à Energy release following transfer of groups
o Used to drive metabolic processes
• NADPH, NADH and FADH2 release high energy electrons and H+
o Important in driving the processes through the oxidation of food
sources
• Coenzyme (CoA) à Energy release from high energy bond of the acyl
(acetyl) group
ATP is the most abundant and widely used energy carrier
• Formed by oxidation of carbon fuels
• Breakdown is exergonic (thermodynamically favourable)
• Cells maintain ATP concentrations far above concentration equilibrium in
order to drive chemical reactions
• Participates in the enzyme catalysed reaction and supplies it with free
energy
• Provides energy by transfer of groups
o Transfer or addition of one or more phosphate or AMP provides
energy
• ATP hydrolysis is not the source of energy
• ATP structure
o Ribose group
o Adenine group
o 3 phosphate groups which yield the energy
NADH and NADPH are activated carriers of electrons
• Act as cofactors for many enzymes
• Shuttle electrons and H+ between anabolic and catabolic processes
o Reduced metabolites (food) become oxidised through catabolism
o During this reaction pathway, NAD becomes reduced (becomes
electron rich) and gives up the electrons to drive pathways to build
new molecules (anabolism)
• Extra phosphate group on NADPH (reduced form)
o No effect on the electron transfer properties (no extra energy
value)
o Allows NADPH to bind different substrates compared to NADH
•
o NADPH mainly used in synthesis of molecules and NADH used
mainly in ATP generation
NADH and NADPH derived from Vitamin B3 (niacin)
Coenzyme A is an activated carrier of acyl groups
• Ribose and adenine group, 2 phosphates, chain of various groups
• Acetyl group is transferable and held by a high energy bond
• Majority of coenzyme A structure facilitates recognition by specific
enzymes
Oxidation of organic molecules is an important fuel source
• Oxidation and reduction both apply when there is a shift in electrons
between atoms linked by a covalent bond
• Oxidation is the removal of electrons from a molecule
• Reduction is the addition of electrons to a molecule
Carbon oxidation is paired with reduction
• Oxidation of organic molecules à Loss of electron and proton (H+)
• In organic molecules
o Oxidation occurs if the number of C-‐H bonds decreases
o Reduction occurs if the number of C-‐H bonds increases
Flow of electrons in metabolism
• Responsible for all work done in living organisms
• Source of electrons is food or stored molecules (reduced compounds) in
non-‐photosynthetic organisms à Catabolism à Electron carriers and
electrons à ATP
• Electrons move from a range of metabolic intermediates to specialised
electron carriers
• Glucose à Glycolysis à Pyruvate à Acetyl CoA
• FA à Acetyl CoA
• AA à Acetyl CoA
Compartmentation
• Affects accessibility of substrates
• Particular metabolic pathways need to occur in different parts of the cell
• Breakdown of FA à ATP in mitochondrion; Lipids made in ER
• Glycolysis, CA cycle à Brain
• Beta oxidation, CA cycle à Heart
• Beta oxidation, TAG synthesis à Adipose tissue
• Beta oxidation, glycolysis, proteolysis à Muscle
Revision
• Anabolic metabolic pathways are biosynthetic
• Energy carriers, macromolecules and electrochemical gradients are the
three forms of energy stores in biology
• ATP is the most abundant energy carrier
•
•
•
•
NADH, NADPH and FADH2 are energy carriers which release high energy
electrons and H+
Oxidation of organic molecules is important for energy production
A loss of a C-‐H bond from an organic molecule is indicative of oxidation
Compartmentation is the concept used to describe metabolic reactions
that occur in separate compartments of the cell or organism
Glucose
• Important energy carrying molecule
• The oxidation of glucose via glycolysis produces pyruvate
• Can be stored as glycogen and starch
Cellular respiration
• Primarily responsible for ATP synthesis which is used for everything
• Process in which cells consume O2 and produce CO2
• Provides energy in the form of ATP
• Occurs in three major stages
o Acetyl CoA production à Cytoplasm
o Acetyl CoA oxidation à Mitochondrial matrix
o Electron transfer and oxidative phosphorylation à Inner
mitochondrial membrane
• Pyruvate is the precursor to acetyl CoA
• Sources of acetyl CoA include pyruvate generated from glycolysis, amino
acids and fatty acids
• Glucose à Oxidation via glycolysis à Pyruvate
Glycolysis
• Chemical pathway common to almost every cell
• Occurs in the cytosol (complex eukaryotic cells are compartmentalised)*
• Energy is released and captured as ATP
• One glucose molecule à 2 pyruvate molecules
• Stereoisomers (D and L) of sugars à D sugars are most common
• 6C in glucose, 3C in pyruvate
• Reduce NAD+ à NADH
• ADP + PO43-‐ à 2ATP
• Glycolysis produces 2 ATP
NAD and NADP (common redox cofactors)
• NAD is electron carrier molecules (source of reduction potential)
• Vitamin B3 (Niacin) is needed to form the nicotinamide group
• Chronic niacin deficiency leads to a disease called pellagra
Significance of glycolysis
• For some tissues (such as brain, kidney medulla and contracting skeletal
muscle) and some cells (erythrocytes, sperm cells) glucose is the only
source of metabolic energy (critically dependent on oxidation of glucose)
• The product of glycolysis (pyruvate) is a versatile metabolite used in
many ways and can have multiple fates
o Glycolysis provides precursors
o In most tissues when oxygen is plentiful (aerobic conditions),
pyruvate is oxidised (loses carboxyl group as CO2) and the
remaining 2 carbon unit becomes the acetyl group of acetyl CoA
...
When animal tissues cannot be supplied with
sufficient oxygen to support aerobic oxidation of pyruvate and NADH
produced in glycolysis, NAD+ is regenerated by reduction of pyruvate to
lactate
• Some tissues and cell types that have no mitochondria cannot oxidise
pyruvate to CO2 and produce lactate from glucose even under aerobic
conditions
• Reaction 1 – Phosphorylation of glucose à G6P
o Biochemical pathways often regulated at the first step
o Hexokinase attaches a P from the ATP to the 6th carbon of glucose
to make G6P
o The activity of hexokinase is regulated
o This chemical reaction must be shielded from water to keep the
phosphate from being cleaved off ATP by a water molecule
o Hexokinase performs an induced fit, closing around ATP and
glucose once they are bound
•
•
Reaction 2 – Conversion of glucose-‐6-‐phosphate à fructose-‐6-‐phosphate
o Isomerization – A reaction that changes the shape of a single
molecule but doesn’t permanently add or remove any atoms
o Isomer – Same number of atoms, different arrangement
o Fructose has a 5 atom ring structure, glucose has 6 atom ring
§ No atoms gained/lost
Reaction 3 – Phosphorylation of fructose-‐6-‐phosphate à fructose-‐1,6-‐
bisphosphate (F-‐1,6-‐BP)*** EXAM QUESTION
o Pi from ATP transferred to sugar à F-‐1,6-‐BP
§ 2 phosphate molecules attached to two different points (1st
and 6th carbons)
o Phosphofructokinase-‐1 (PFK1) is the gatekeeper of glycolysis à
Catalyses the committed step of the glycolytic pathway
o PFK1 proposed to have important roles in metabolic
reprogramming in cancer
o Somatic cancer mutations of PFK alter enzymatic activity and
allosteric regulation
o Regulated allosterically
o PFK1 is a bacterial enzyme with 4 identical subunits
...
The enzyme also has regulatory
binding sites at the top and bottom
•
•
•
•
•
•
•
Reaction 4 – Cleavage of fructose-‐1,6-‐biphosphate (F-‐1,6-‐BP)
o Aldolase splits sugar into two 3 carbon molecules
§ Dihydroxyacetone phosphate
§ Glyceraldehyde 3-‐phosphate
Reaction 5 – Interconversion of the triose phosphates
o Triose phosphate isomerase (enzyme) rearranges atoms of
dihydroxyacetone phosphate into glyceraldehyde 3-‐phosphate
(end up with 2 equivalents of glyceraldehyde 3-‐phosphate)
o The TIM barrel (structure that occurs widely in nature) is a
conserved protein fold of triose phosphate isomerase
...
This is the pasteur effect
...
Therefore, cancer cells
that are more than 100-‐200um from capillaries depend on anaerobic
glycolysis for their ATP production
• F-‐deoxyglucose is taken up by brain cells in direct proportion to the
amount of glycolysis occurring
...
• Locations in the body where a lot of glycolysis is occurring à Solid
tumours
Summary
• Glycolysis
o Occurs in the cytosol
o 1 glucose à 2 pyruvate molecules
o 2NADH + 2H+ + 2ATP generated
o Does not require O2
o Regulated to maintain constant cellular ATP concentrations
o Is the only source of ATP for some cell types
o Solid tumours have very high rates of glycolysis
• Pyruvate
o Can be converted to lactate under anaerobic conditions
o Can be converted to ethanol by yeast
o Can be converted to acetyl CoA to feed into TCA cycle
Only a small amount of energy available in glucose is captured in glycolysis
• Glycolysis = Glucose à 2x pyruvate + 146kJ/mol energy
• Full oxidation (+6O2) à 6CO2 + 6H2O + 2840kJ/mol energy
Cellular respiration
• Process in which cells consume O2 and produce CO2
• Provides more energy (ATP) from glucose than glycolysis
• Also captures energy stored in lipids and AA
• Occurs in three major stages
o Acetyl CoA production à Cytoplasm (pyruvate à Acetyl CoA)
§ Glucose à Pyruvate (glycolysis)
Pyruvate à Acetyl CoA via pyruvate dehydrogenase
complex
o Acetyl CoA oxidation via TCA cycle à Mitochondrial matrix
o Electron transfer and oxidative phosphorylation à Inner
mitochondrial membrane
§
Cellular respiration occurs in 3 major stages
• Glucose, FA and some AA are oxidised to yield two carbon fragments in
the form of the acetyl group of acetyl CoA (CH3CO-‐)
o Coenzyme A carries functional groups around the body (such as
acetyl groups)
o Fatty acid metabolism also produces acetyl groups
• The acetyl groups are fed into TCA cycle which enzymatically oxidises
them to CO2; the energy released is conserved in the electron carriers
NADH and FADH2
o TCA cycle produces reducing factors
• These reduced coenzymes are themselves oxidised, giving up protons
(H+) and electrons
...
In the course of electron transfer, the large amount of
energy released is conserved in the form of ATP, by a process called
oxidative phosphorylation
Coenzyme A
• Coenzymes are not a permanent part of the enzymes’ structure, they
associate, fulfil a function and dissociate (not part of the chemistry)
• ADP attached to pantothenic acid (B5) and SH group which is important
to the chemistry of acetyl CoA
• Chemical intermediates attach to SH group
• The function of CoA is to accept and carry acetyl groups
• Coenzyme A contains vitamin B5 (pantothenic acid) for which deficiency
is exceptionally rare
Conversion of pyruvate to acetyl CoA
• Occurs in mitochondrial matrix of eukaryotes
• Reducing factors produced in mitochondria and used to produce ATP in
oxidative phosphorylation
• Required to enter into TCA cycle
• Net reaction
o Oxidative decarboxylation of pyruvate (lose CO2)
o Acetyl group attaches to coenzyme A
o First carbons of glucose to be fully oxidised
• Catalysed by large pyruvate dehydrogenase complex
o Requires 5 coenzymes
o Thiamine pyrophosphate (TPP), lipoyllysine and FAD are
prosthetic groups
o NAD+ and CoA-‐SH are co-‐substrates
Pyruvate dehydrogenase
• The complex is organised in cubic symmetry in Gram-‐negative bacteria,
having 60 subunits in three functional proteins
• E2 subunit carries acetyl groups
• Multiple processes happen
• In eukaryotes and gram-‐positive bacteria it is organised in dodecahedral
symmetry and consists of 96 subunits, organised into three functional
proteins in the human enzyme
• Lipoic acid is covalently linked to a lysine residue to give lipoyllysine
• The pyruvate dehydrogenase complex uses substrate channelling so that
o The intermediates of the multistep reaction sequence never leave
the surface
o The local concentration of substrates is kept high
o It prevents the loss (theft) of the activated acetyl group by other
enzymes that use this group as a substrate
• The PDC is also an example of an enzyme that uses a vitamin as a cofactor
o TPP is thiamine pyrophosphate (a vitamin B1 derivative)
o Thiamin deficient animals are unable to oxidize pyruvate normally
which leads to Beri beri (characterised by a loss of neural function)
o An elevated blood pyruvate level is often an indicator of defects in
pyruvate oxidation
Pyruvate dehydrogenase reaction
• Loss of CO2 from pyruvate à Acetyl
• Acetyl attached to E1 subunit and carried along to lipoyllysine
• Coenzyme A attaches to acetyl group (formation of acetyl CoA)
• Reoxidation of lipoamide group (recycled) à Produce reducing
equivalent FADH2 à Passes electrons to NADH
Overall reaction of PDC
• Enzyme 1
o Decarboxylation of pyruvate to an aldehyde
o Oxidation of aldehyde to a carboxylic acid
• Enzyme 2
o Formation of acetyl CoA
• Enzyme 3
o Reoxidation of the lipoamide cofactor
o Regeneration of the oxidised FAD cofactor, generation of NADH
The TCA cycle summary
• Glycolysis is not the only source of acetyl CoA (Break down of AA and
fatty acids)
• Acetyl CoA à Citrate
• Citrate is progressively oxidised to form the end product, oxaloacetate
• Oxaloacetate is then recycled to form citrate
• CO2 also produced in the oxidation of citrate
• Pyruvate is 3C à Lose one CO2 in the formation of acetyl CoA and 2 CO2
molecules in the oxidation of citrate
• The oxidation of various intermediates throughout the cycle leads to
electrons being stripped at key points; these electrons are then used to
produce NADH and FADH2
• Loss of CO2 and production of reducing factors (reduce electron carriers)
• Generates**
o 3 NADH
o 1 FADH2
o 1 GTP
FAD – A redox cofactor
• Vitamin B2 (riboflavin) is the precursor of FMN and FAD
• Riboflavin deficiency symptoms à Sore throat, lesions of the lips and
mucosa of the mouth, glossitis, conjunctivitis, dermatitis
Mitochondrion
• TCA cycle and pyruvate dehydrogenase complex found in mitochondrial
matrix (surrounded by inner mitochondrial membrane)
• Glycolysis occurs in the cytoplasm
• TCA cycle occurs in mitochondrial matrix (except succinate
dehydrogenase which is located in the inner membrane)
• Oxidative phosphorylation occurs in inner membrane
TCA
• Acetyl CoA from pyruvate (via pyruvate dehydrogenase complex)
• Acetyl CoA feeds into the cycle
o Acetyl CoA + oxaloacetate à Citrate
o Reaction catalysed by citrate synthase
• Citrate rearranged into iso-‐citrate
• CO2 lost at step 3 and step 4 (produce 2 CO2 molecules)
• Produce reducing equivalent (NAD à NADH2 at step 3,4 and 8)
• FADH2 produced from FAD at step 6
• GTP produced at step 5 (energy from the molecule transferred to GDP à
GTP)
• Oxaloacetate is the end product and is recycled back to citrate
In eukaryotes, CA cycle occurs in mitochondria
• Reduced substrate (fuel) donates electrons
•
•
•
Electron carriers pump H+ out as electrons flow to O2
Energy of electron flow stored as electrochemical potential
ATP synthase uses electrochemical potential to synthesise ATP
Sequence of events in the CA cycle
1
...
Isomerization to isocitrate via cis-‐aconitate
3 & 4
...
Substrate level phosphorylation to give GTP
6
...
Hydration (addition of H2O)
8
...
5 ATP are
produced
• For each FADH2 oxidised in oxidative phosphorylation, 1
...
The enzyme selects glucose over water as a
result of the conformational change that occurs when the correct
substrate (glucose) binds
Hexokinase I, II and III
• Tissue specific differences in hexokinase
• Different tissues express different hexokinases (different genes)
• There are four different isozymes of hexokinase
o Hexokinase I, II and III are all found in muscle cells
o Hexokinase II is the predominant isozyme
§ High affinity for glucose (half saturated at 0
...
1 (faster initial rate of activity; more active)
o Relative activity varies very little with fluctuations of glucose
o Main function is to catabolise glucose and provide pyruvate for
TCA cycle
o Glucose taken up by muscle cells is rapidly phosphorylated to trap
glucose in the cell
• Hexokinase IV (liver)
o Km 10mM
o Main function to regulate blood glucose levels
o Activity is regulated by glucose concentration (a small change in
concentration can result in a large change in activity)
o Rate of hexokinase activity increases with glucose concentration
(regulatory control) à More subtle regulation of activity
Hormonal regulation
• Important hormones that control metabolism include the peptide
hormones glucagon and insulin
• Peptide hormones act via cell surface receptor (eg insulin)
o Mediate effects inside the cell
o Cascade of reactions which may involve phosphorylation of
proteins (form of regulation)
o Production of secondary messengers (cAMP) which send signals
à Changes in gene expression via regulation of transcription
factors
•
•
•
Hormones do not act directly with enzymes in a metabolic pathway, but
bind to cell membranes and cause a series of reactions that eventually
lead to modification of pathway enzyme activity
o Cell surface receptors (peptide/amine hormones)
o Nuclear receptors (Steroid/thyroid hormones enter the cell and
the hormone-‐receptor complex acts in the nucleus)
A major problem in glucose metabolism is diabetes
o Type I à No insulin production
o Type II à Insulin resistance
§ Cells become insensitive to insulin
When insulin combines with its receptor on the cell surface, a series of
biochemical events (downstream events) stimulate uptake of glucose into
cells by the GLUT4 transporter
o GLUT4 is muscle specific glucose transporter
o Number of transporters on cell surface increases (regulation of
rate that glucose is imported)
The second and most important control point in glycolysis is reaction 3,
catalysed by phosphofructokinase I (PFK1)
• Fructose-‐6-‐phosphate à Fructose-‐1,6-‐bisphosphate is the commitment
step in glycolysis
• While ATP is a substrate, ATP is also a negative effector
o Glycolysis is down regulated if there is plenty of ATP
• Regulation of PFK-‐1
o Allosteric inhibitors
§ ATP is both a substrate and eventual product of glycolysis
§ Citrate (TCA cycle intermediate/product) is an indicator of
if the cell is meeting its energy needs à Increases inhibition
by ATP
o Allosteric activators
§ ADP and AMP increase in concentration when ATP
utilisation outpaces production
§ Fructose-‐2,6-‐bisphosphate increases in concentration when
blood glucose concentrations decrease
Allosteric regulation of PFK1 by ATP
• PFK-‐1 has allosteric sites that control rate of reaction
• ATP binds to an allosteric site, lowering the affinity of the active site of
fructose-‐6-‐phosphate
• AMP/ADP act allosterically to relieve this inhibition
• Increased ATP à Lower PFK1 activity
• Increased ADP/AMP (low ATP) à Higher PFK1 activity
Allosteric regulation of PFK1 by fructose-‐2,6-‐bisphosphate
• Not a glycolytic intermediate
• A regulator specifically produced to regulate glycolysis and
gluconeogenesis (creation of new glucose)
• F26BP is synthesised from F6P by PFK-‐2
•
•
•
•
o Takes fructose-‐6-‐phosphate and makes fructose-‐2,6-‐bisphosphate
in a phosphorylation reaction
o F26BP is not a glycolytic intermediate
o PFK2 à F26BP à Stimulates PFK1
Broken down by fructose-‐2,6-‐bisphosphatase (FBPase-‐2)
o Removes the phosphate group à Fructose-‐6-‐phosphate
o This cycle synthesises and removes F26BP
o Fine regulatory control of PFK1 via F26BP à Important for
regulating glycolysis
o During gluconeogenesis we must shutdown glycolysis via this
system
Regulation of F26BP levels
o Process regulated by hormones (eg insulin à Tells cells to take up
glucose and glycolysis begins)
o Both PFK2 and FBPase-‐2 activities are on the same protein
o Process regulated by phosphorylation à Regulates levels of F26BP
which is required for the activity of PFK1
o If the protein is phosphorylated then FBPase-‐2 becomes active and
glycolysis shuts down
When F26BP binds to the allosteric site on PFK1
o Increases the enzymes affinity for F6P
o Decreases the enzymes affinity for ATP
PFK-‐1 is almost inactive in the absence of F26BP
Pyruvate kinase is the third control point of glycolysis (at reaction 10)
• Pyruvate kinase controls outflow from glycolysis
• Phosphoenolpyruvate transfers a phosphate group to ADP à ATP and we
also generate a molecule of pyruvate (which then has multiple fates)
• Tissue specific isozymes
• Allosterically activated by fructose-‐1,6-‐bisphosphate
o High flow through from glycolysis
• Allosterically inhibited by
o ATP
o Acetyl-‐CoA and long chain fatty acids
o Alanine (enough amino acids)
o Signs of abundant energy supply
Hormonal regulation of pyruvate kinase
• The L (liver) isozyme, but not the M (muscle) isozyme, is subject to
covalent modification (phosphorylation)
o Liver isozyme phosphorylated by PKA
o Muscle isozyme does not get phosphorylated
• Low BG à Glucagon release à Activates cAMP dependent protein kinase
(PKA) à Phosphorylates pyruvate kinase (liver isozyme) à Glucose
metabolism in the liver is slowed; glucose is conserved
• MUST BE ABLE TO WRITE REACTION CATALYSED BY PYRUVATE KINASE
AND KNOW STRUCTURES OF PHOSPHOENOLPYRUVATE AND PYRUVATE
Conversion of pyruvate to acetyl-‐CoA
Pyruvate dehydrogenase has 3 subunits (3 different enzymes)
Pyruvate gets attached to TPP à Hydroxyethyl group gets passed to
lipoyllysine à Acetyl CoA (which feeds into TCA)
• Production of 1 NADH
Regulation of the pyruvate dehydrogenase complex
• Bridges glycolysis and TCA cycle so important regulatory enzyme (takes
pyruvate à Acetyl CoA)
• Regulated by phosphorylation/dephosphorylation
• PDH incorporates pyruvate into CoA-‐SH to give acetyl CoA for entry into
the TCA cycle
• Regulated allosterically
o ATP, acetyl CoA and NADH are inhibitors (presence of the
metabolites indicate sufficient amounts)
o AMP, CoA, NAD+ and Ca2+ are activators
§ Ca2+ is a second messenger inside the cell
• Also regulated by covalent modification
o Reversible phosphorylation of a serine residue in one of the two
subunits of E1
o PDH kinase and PDH phosphatase add and remove phosphate
§ Kinase is regulated by ATP
• High [ATP] à Kinase more active à Phosphorylated
PDH à Less acetyl CoA
• Low [ATP] à Kinase less active/phosphatase
removes phosphate à More acetyl CoA
§ Regulation of PDH complex via
phosphorylation/dephosphorylation (covalent
modifications)
§ E1 has Ser-‐OH group which is where phosphorylation takes
place on the subunit
§ When the PDH complex is phosphorylated it is down
regulated (less acetyl CoA; less active)
Regulation of the TCA cycle
• Tightly regulated at several points
• 3 steps where we produce NADH, 1 equivalent of FADH2
• Rates of glycolysis and the TCA cycle are integrated so that wasteful
consumption of glucose does not occur
o Regulate entry of pyruvate (limit amount available for pyruvate
dehydrogenase complex)
o Supplies of pyruvate match demand for acetyl CoA
• Indicators of plentiful energy supply (acetyl CoA, ATP, NADH) inhibit key
reactions
• Indicators of energy depletion (AMP, CoA, NAD+) stimulate key reactions
• Coenzyme A is also an allosteric regulator (More CoA = Depletion à
Stimulatory)
• Citrate synthase (1) is also inhibited by succinyl CoA
o Citrate synthase is where acetyl CoA enters the TCA cycle
•
•
•
•
o Succinyl-‐CoA communicates flow at this point back to the start of
the cycle
§ Presence of the intermediate communicates back to the
entry point of the cycle
o Alpha-‐ketoglutarate is an important branch for amino acid
metabolism
Regulated at highly thermodynamically favourable and irreversible steps
o Irreversible steps regulated (same as in glycolysis)
o PDH complex, citrate synthase, isocitrate dehydrogenase and
alpha-‐ketoglutarate dehydrogenase are the enzymes regulated
General regulatory mechanisms
o Substrate availability
o Inhibited by product accumulation
o Overall products (NADH and ATP) affect all regulated enzymes in
the cycle
§ NADH and ATP are inhibitors
§ NAD+ and AMP/ADP are activators
Respiratory control
• Tightly controlled
• An adult woman requires approximately 6000kJ of metabolic energy per
day
• ATP à ADP + Pi (-‐30
...
(which carries an unpaired electron and is therefore a radical)
o Production of O2 is related to the concentration of potential
electron donors, the local concentration of O2
• Regulation via hypoxia inducible transcription factor 1 (HIF1)
o Regulation of PDH via PDH-‐kinase
§ HIF-‐1 increases synthesis of PDH kinase à
Phosphorylation of PDH à Inactive
§ Regulates entry into ETC during hypoxia
§ Slows NADH and FADH2 production, slowing supply of
electrons to the respiratory chain
o Regulation of complex IV (cytochrome oxidase)
§ HIF-‐1 increases synthesis of a protease that degrades a
subunit of complex IV (COX4-‐1)
§ Triggers synthesis of alternate subunit (COX4-‐2) which is
specifically suited to hypoxic conditions
o Also up-‐regulates glucose transporters and glycolytic enzymes
Summary
• BE ABLE TO DRAW GLUCOSE, G6P, PYRUVATE AND LACTIC ACID
• PDH complex regulated allosterically and by phosphorylation
• Products of the TCA cycle (NADH and ATP) affect all regulated enzymes in
the cycle
• Coupling can be observed using an oxygen electrode and by measuring
ATP production
• Oxidative phosphorylation can be uncoupled in vitro by various
uncoupling reagents and in vivo by uncoupling proteins
• Uncoupling protein 1 (UCP1) is found in some animals, human infants
• In hypoxia there is an imbalance between electron supply and transfer to
O2 leading to ROS production
Gluconeogenesis
• Glucose can be synthesised from non-‐carbohydrate precursors via
gluconeogenesis, using steps shared with glycolysis and alternate
enzymes to bypass the irreversible reactions
o 3 irreversible steps in glycolysis which must be bypassed in
gluconeogenesis
• Different tissues will utilise gluconeogenesis differently
• Gluconeogenesis is coordinated with the regulation of glycolysis
o To avoid futile cycling
• Occurs when
o Glucose supplies are limited (during meals, fasts, exercise)
o Tissues that almost completely rely upon glucose for metabolic
energy
§ Brain, nervous system, erythrocytes, testes, renal medulla,
embryonic tissues
§ Brain alone requires 120g glucose per day (60% of the
glucose stored as glycogen in muscle and liver)
• Supply of glucose from glycogen stores is not always sufficient à Need a
way to synthesise glucose
o Glycogen stores depleted during fasts/vigorous exercise
Gluconeogenesis
• Formation of new sugar
• Process that converts pyruvate (and related 3&4C compounds) to glucose
• Other intermediates may also serve as precursors
o Glycolytic intermediates involved in gluconeogenesis
• Occurs mainly in the liver (kidney and small intestine too)
o Liver can make glucose under certain conditions
• Shares many steps with glycolysis
• The synthesis of glucose from non-‐carbohydrate precursors
o Occurs in all plants, animals, fungi and microorganisms
• Important precursors of glucose in animals are
o Pyruvate
o Lactate (During vigorous exercise, the lactate produced is
converted in the liver back to glucose via TCA cycle)
o Certain AA (Feed into TCA cycle)
o Glycerol
Glycolysis vs
...
g regulation by insulin
o Insulin released in the fed state à Glucose taken up and stored
o Insulin also regulates several pathways transcriptionally
(participates in gene regulation)
o Up-‐regulate glycolytic enzymes
• Enzymes involved in glycolysis/gluconeogenesis regulated by insulin
o Hexokinases II & IV
o PFK-‐1
o Pyruvate kinase
o PFK-‐2/FBPase-‐2
Glycolysis and gluconeogenesis are reciprocally regulated by allosteric
modulators
• F6Pà F16BP (glycolysis)
• F16BP à F6P (gluconeogenesis via FBPase-‐1)
o AMP is inhibitory of FBPase-‐1 in gluconeogenesis (ensures the
pathways do not occur at the same time)
o ATP is a negative allosteric regulator of glycolysis
o ADP/AMP are positive regulators of glycolysis
• Not opposite reactions
Glycolysis and gluconeogenesis are reciprocally regulated by fructose-‐2,6-‐
bisphosphate (F26BP)
• Allosteric regulation of F6P (F6P à F26BP via PFK2)
•
•
•
•
•
F26BP not a glycolytic intermediate but is essential for regulation
A regulator specifically produced to regulate glycolysis and
gluconeogenesis
Levels of F26BP important for regulating glycolysis
o Inhibit FBPase-‐1
o Upregulates PFK1
F26BP is synthesised from F6P by PFK-‐2
Broken down by fructose-‐2,6-‐bisphosphate (FBPase-‐2)
Regulation by F26BP
• Has opposite regulatory effects on glycolysis and gluconeogenesis
• F26BP allosterically regulates fructose-‐1,6-‐bisphosphatase in a similar
manner
o Activates phosphofructokinase (glycolysis)
o Inhibits fructose-‐1,6-‐bisphosphatase (gluconeogenesis)
• When F26BP binds to its allosteric site on FBPase-‐1
o Decreases the enzymes affinity for F6P
o Inhibited by as little as 1uM F26BP
o Increases FBPase-‐1 sensitivity to AMP
• When F26BP binds to its allosteric site on PFK-‐1
o Increases the enzymes affinity for F6P
o Decreases the enzymes affinity for ATP
Regulation of F26BP levels
• PFK2 and FBPase-‐2 are enzymes on the same protein
o Make the same non-‐glycolytic intermediate F26BP
• High BG à Insulin binds to cell surface à De phosphorylation à
Activates PFK2 à F26BP à Activates PFK-‐1 à Glycolysis
• Low BG (want to shut down glycolysis) à Glucagon à Signalling cascade
resulting in phosphorylation of cAMP à FBPase-‐2
• Increased F26BP stimulates glycolysis inhibits glucongeogenesis
• Decreased F26BP inhibits glycolysis and stimulates gluconeogenesis
Summary
• Gluconeogenesis is the synthesis of glucose from non-‐carbohydrate
precursors
o Pyruvate, lactate, amino acids and glycerol (but not fatty acids in
animals)
• Gluconeogenesis and glycolysis share 7 common steps, but not those that
are irreversible
o Hexokinase (step 1)
o PFK-‐1 (step 3)
o Pyruvate kinase (step 10)
§ These reactions are bypassed by 4 different enzymes
• Pyruvate carboxylase
• PEP carboxykinase
• Glucose-‐6-‐phosphatase
• Frcutose-‐1,6-‐bisphosphatase
Conversion of pyruvate to PEP has two alternate pathways,
pyruvate or lactate, depending on tissue
Gluconeogenesis is energetically expensive but necessary for specific
tissues
Regulation of gluconeogenesis is coordinated with glycolysis to prevent
wasteful cycling of ATP
Glycolysis and gluconeogenesis are reciprocally regulated by fructose-‐2,6-‐
bisphosphate
§
•
•
•
Glycogen
• Glycogen is a glucose storage molecule
• Cells triggered by insulin to take up glucose à Stimulates synthesis of
glycogen
• Polymer of alpha(1-‐4) linked subunits of glucose with alpha(1-‐6) linked
branches
• Each chain has 12-‐14 glucose residues
• Glycogenin protein in the centre
o Glycogenin has sugars attached
Glycogen as a source of fuel in animals
• Source of fuel in animals (starch in plants)
• Skeletal muscle
o <2% wet weight
o Major source of glucose for contraction
• Liver
o <10% wet weight
o Plays a role in maintaining blood glucose levels
o Provides glucose to other tissues between meals or fasting
(especially the brain)
• The total amount of energy stored is less than that of triacylglycerols
(fatty acids)
• Muscle glycogen can be exhausted in less than 1 hour during vigorous
activity (depleted relatively quickly; the liver is richer in glycogen)
• Liver glycogen can be depleted in 12-‐24 hours
o After 24 hours when glycogen stores are depleted you begin to
break down proteins and eventually fatty acids (which cannot be
converted to glucose, which is required for nervous function)
• Fatty acids cannot be
o Converted to glucose in mammals
o Metabolised anaerobically
o Used as fuel for the neurons of the brain
Why not store monomeric glucose?
• Glycogen stored in cells at 0
...
4M (places osmotic stress on cells)
• Glucose would dominate the osmotic properties of the cell leading to
osmotic entry of water into cell and subsequent cell lysis
•
Prohibits glucose uptake (as extracellular glucose is 5mM)
Glycogen beta-‐particles
• Elementary glycogen particles (or granules)
• Often associated with endoplasmic reticulum tubules
• Each glycogen molecule consist of
o 55000 glucose molecules
o 2000 non-‐reducing ends
Glycogen degradation
• Requires 4 enzyme activities
• Glycogen phosphorylase à Degrades glycogen
• Glycogen debranching enzyme
o Catalyses two reactions
o Remodels glycogen for further degradation
• Phosphoglucomutase à Converts the breakdown product into a form
suitable for further degradation
Reducing sugars
• Interconversion between alpha and beta
• Glucose is an aldose (aldehyde group)
• Have a free aldehyde or ketone in the open chain form
• Undergo oxidation by oxidising substances
• Non-‐reducing sugars lack a free aldehyde or ketone and are unable to
undergo oxidation (sucrose)
Water as a reactant
• Water can participate directly in chemical reactions
• Condensation reaction à Formation of a bond between OH and OH,
expelling water
• Hydrolysis reaction à Cleavage of this bond accompanied by addition of
water
Glycogen phosphorylase
• Catalyses the breakage of the alpha(1-‐4) linkages between glucose
subunits at a non-‐reducing end
• Uses Pi and incorporates phosphate into the glucose molecule so it is
liberated from the molecule (G1P released)
• Phosphorylase stops when within 4 subunits of an alpha(1-‐6) link due to
hindrance
Dealing with branch points
• Glycogen phosphorylase acts repeatedly to remove glucose subunits à
Produces glucose-‐1-‐phosphate
• Stops when within 4 subunits of an alpha(1-‐6) linkage
• Debranching enzyme catalyses two successive reactions
o Transfer three glucose subunits to a nearby non-‐reducing end
(move a branch point along the chain)
o Cleaves the remaining alpha(1-‐6) linked glucose
Glucose-‐1-‐phosphate must be isomerised to glucose-‐6-‐phosphate for metabolism
• Phosphoglucomutase
o Catalyses the reversible conversion of G6P à G1P
o Last step of glycogen breakdown (G1P à G6P which is the 2nd
intermediate in glycolysis and can be used to make pyruvate)
o First step of glycogen synthesis (G6P à G1P)
Metabolism of glucose-‐6-‐phosphate
• Muscle glucose-‐6-‐phosphate enters glycolysis to serve as an energy
source
• In a liver cell, G6P à Glucose (can export glucose by hydrolysing the Pi on
G6P to make glucose)
• Liver and kidney glucose-‐6-‐phosphate is dephosphorylated to glucose
and released into the blood
Glycogen synthesis requires three enzyme activities (not including G6P à G1P
conversion by mutase)
• UDP-‐glucose phosphorylase à Forms activated glucose (UDP glucose)
o Activated glucose used to make glycogen
• Glycogen synthase à Adds activated glucose to growing glycogen
polymer
o Grows a chain of alpha (1-‐4) molecules
• Glycogen branching enzyme à Introduces alpha (1-‐6) branch points
Glycogen synthesis
• Glycogen degradation and synthesis uses different pathways
o Different enzymes allows us to regulate
• Glycogen synthesis uses the glucose of UDP glucose, the activated form of
glucose
o UDP also seen in RNA
o Contains a ribose molecule
o UDP acts as a handle on glucose
Activated sugar nucleotides
• Most reactions in which hexoses are transformed/polymerised involve
sugar nucleotides
o The anomeric carbon of sugar is activated by attachment to a
nucleotide
• Sugar nucleotides are the substrates for the polymerisation of
monosaccharides into many different polysaccharides
• Suitability for biosynthetic reactions
o Formation metabolically irreversible
o Reactivity of the nucleotide group
o Nucleotidyl group is a good leaving group
o Tagging of sugars allows for dedication to specific purposes
Formation of activated sugars
•
•
G1P made from G6P via phosphoglucomutase
G1P + UTP à UDP-‐glucose + PPi (via UDP-‐glucose phosphorylase)
Glycogen synthase
• Sugar unit transferred onto non-‐reducing end of growing glucose polymer
to make a new alpha 1-‐4 linkage
• UDP used as carrier for glucose and can be recycled
• Catalyses the addition of UDP glucose to a non-‐reducing end of glycogen
to make a new alpha (1-‐4) linkage
Glycogen branching enzyme
• Takes alpha 1-‐4 units and shifts them to generate branch points
• Branch points useful for quick breakdown (lots of non-‐reducing ends
available)
• Catalyses the transfer of a terminal fragment of glucose residues from the
non-‐reducing end of a chain to the C6 OH group of a more interior glucose
residue of the same or another glycogen chain to form a new branch
Glycogenin facilitates the formation of new glycogen chains
• Glycogen synthase cannot initiate a new chain
o Requires a preformed alpha (1-‐4) polyglucose chain (>8 residues)
• Glycogenin is both an enzyme and a primer
o Catalyses addition of glucose residues onto itself (used by glycogen
synthase)
• New glycogen chains begin with the autocatalytic transfer of UDP-‐glucose
to glycogenin, followed by several additions of glucose residues to form a
primer that can be acted upon by glycogen synthase
• Each chain has 12-‐14 glucose residues
• Primer à Short oligomer of sugars (or nucleotides) to which an enzyme
adds additional monomeric subunits
Glycogen synthesis summary
• Glycogenin forms a primer of 8-‐12 alpha (1-‐4) linked glucose units
• UDP glucose pyrophosphorylase synthesises UDP glucose from UTP and
G1P
• Glycogen synthase catalyses the addition of UDP glucose to a non-‐
reducing end of glycogen to make a new alpha (1-‐4) linkage
• Glycogen branching enzyme catalyses the transfer of a terminal fragment
of glucose to a non reducing end of a chain to the C6 OH group of a more
interior glucose residue of the same or another glycogen chain to form a
new branch (alpha 1-‐6)
Glycogen breakdown summary
• Glycogen phosphorylase breaks alpha (1-‐4) linked glucose units to
produce G1P
o Important for regulation
o Phosphorylates the alpha 1-‐4 residues
• De-‐branching enzyme catalyses two successive reactions
•
o Transfer three glucose subunits to a nearby non-‐reducing end
o Cleaves off the remaining alpha (1-‐6) linked glucose
Phosphoglucomutase converts G1P to G6P
Glycogen phosphorylase is regulated allosterically and hormonally
• Phosphorylase a and b (different forms)
o Phosphorylase b is less active
o Phosphorylase b kinase phosphorylates phosphorylase b into
phosphorylase a (more active form)
o Phosphorylase a phosphatase dephosphorylates phosphorylase a
into phosphorylase b
o Glucagon/epinephrine signals glycogen breakdown
o Regulate glycogen synthesis and breakdown hormonally
• Glucagon/epinephrine signalling pathway
o Starts phosphorylation cascade via cAMP
o Activates glycogen phosphorylase
• Glycogen phosphorylase cleaves glucose residues off glycogen, generating
G1P (which is then converted to G6P and fed through glycolysis)
Epinephrine (adrenaline) and glucagon induce glycogen degradation
(glycogenolysis)
• Epinephrine/glucagon initiates a signalling cascade
• Adenylate cyclase à Cyclic AMP à Activates protein kinase A à
Phosphorylates phosphorylase kinase à Phosphorylase kinase
phosphorylates phosphorylase b à a (active)
Glycogen phosphorylase in liver as a glucose sensor
• Regulated allosterically by glucose
• Binding of glucose to an allosteric site on phosphorylase a induces a
conformational change
o Exposes phosphorylated serine side chains to the action of
phosphorylase a phosphatase (PP1)
• Phosphorylase a phosphatase dephosphorylates phosphorylase a à
phosphorylase b (less active)
Carbohydrate metabolism differs between tissues
• Liver
o Liver has different proteins for glycolysis (specific glucokinase
isozyme)
o Liver able to release glucose into bloodstream (breaks down G6P
à glucose) whereas other tissues use G6P into glycolysis and
make pyruvate
Epinephrine (adrenaline) and glucagon induce glycogen degradation and inhibit
glycogen synthesis
• Protein kinase A phosphorylates glycogen synthase which inactivates it
Insulin induced glycogen synthesis
•
•
•
•
When BG levels are high insulin stimulates glycogen synthesis by
inactivation of glycogen synthase kinase 3
Glycogen synthase kinase phosphorylates glycogen synthase (inactive)
Phosphatase activates glycogen synthase
Inactivation of glycogen synthase kinase allows PP1 to dephosphorylate
and activate glycogen synthase
Phosphoprotein phosphatase 1 is central to glycogen metabolism
• PP1 can remove phosphoryl groups from all three enzymes
phosphorylated in response to glucagon/epinephrine
o Phosphorylase kinase
o Glycogen phosphorylase
o Glycogen synthase
• PP1 Is tightly bound to its targets via glycogen targeting protein (Gm)
o Binds glycogen and above 3 enzymes
• PP1 is itself subject to covalent and allosteric regulation
These allosteric and hormonal signals coordinate carbohydrate metabolism
globally
Summary
• Glycogen is a polymer of alpha (1-‐4) linked subunits of glucose, with
alpha (1-‐6) linked branches constructed around a core primer based on
the enzyme glycogenin
• Glucose 6 phosphate is converted to glucose-‐1-‐phosphate by
phosphoglucomutase (reversibly), before activation via the addition of a
nucleotide, forming UDP glucose – the building block of glycogen
• Glycogen branching and debranching enzymes are responsible for
forming and degrading/remodelling the alpha (1-‐6) linked branches
• Epinephrine and glucagon induce glycogen breakdown (via
phosphorylase kinase which phosphorylates/activates phosphorylase)
and impair glycogen synthesis (via phosphorylating/inactivating glycogen
synthase)
• Insulin and the availability of glucose induces glycogen synthesis, with
phosphoprotein phosphatase 1 (PP1) dephosphorylating/activating
glycogen synthase, and dephosphorylating/inactivating phosphorylase
Type I diabetes
• Results from the autoimmune destruction of the insulin producing beta
cells in the pancreas
o Cells destroyed by autoimmune processes
o Glucose metabolism limited
• Manifests itself in childhood (juvenile diabetes)
• Metabolism of glucose is limited by the rate of uptake; glucose uptake is
deficient in type I diabetes
• Classical symptoms
o Polyuria (frequent urination)
o Polydipsia (increased thirst)
•
•
o Polyphagia (increased hunger)
o Weight loss
BG builds up to high levels à Increased urination à Increased thirst
Cells unable to take up glucose à Increased hunger
Glucose uptake
• Mediated by GLUT transporters
• In some cells GLUT transporters are always present
o GLUT1 (ubiquitous; in all cell types)
o GLUT2 (liver)
o GLUT3 (brain)
§ Always expressed à Brain reliant on glucose as an energy
source
§ Other transporters are regulated
• In skeletal muscle, cardiac muscle and adipose tissue, GLUT4 is
sequestered in vesicles and moves to the plasma membrane in response
to insulin
o Recruited into membrane under insulin signal
Effects of type I diabetes on CHO and fat metabolism
• Pancreas fails to secrete insulin
• Insulin receptor not activated
• Failure to recruit GLUT4 transporters in membrane (in skeletal muscle,
cardiac muscle and adipose tissue)
• Glucose not taken up by cells
• Insufficient glycolysis (due to insufficient glucose)
• Insufficient TCA cycle and oxidative phosphorylation
• Triglyceride breakdown and fatty acid oxidation
Inborn errors of metabolism
• Rare genetic disorders that affect enzymes in metabolism
• Gene mutations à Individual enzymes affected
• Body cannot properly turn food into energy (enzymes defective)
• Usually caused by defects in specific proteins (enzymes) that help break
down (metabolise) parts of food
Glycolysis in disease
• Glycolytic mutations are relatively rare due to importance of this pathway
(produces pyruvate for TCA and precursors for other biosynthetic
pathways)
• Majority of mutations in enzymes à Cells cant respire à Cell death
Pyruvate kinase deficiency
• Final step of glycolysis (phosphoenolpyruvate + ADP à Pyruvate + ATP)
• Erythrocytes (RBCs) obtain all their ATP from glycolysis
• A deficiency in pyruvate kinase (PK) results in erythrocytes with
decreased energy
•
Build up of glycolytic intermediates can increase the level of 2,3-‐
bisphosphoglycerate (23BPG) à Decreases affinity of haemoglobin for O2
à Affects tissue oxygenation
Phosphofructokinase-‐1 (PFK1) and cancer
• Altered glycolytic flux is a hallmark of cancers
• PFK1 proposed to have important roles in metabolic reprogramming in
cancer
• Cancer mutations of PFK alter enzymatic activity and allosteric regulation
o R48C mutant has reduced citrate inhibition (citrate regulates
PFK1)
o N426S mutant partly relieves ATP inhibition
• Inhibition of glycolytic flux by loss of function mutations may confer a
selective advantage for cancer cell growth and metastasis by redirecting
carbon flow through the pentose phosphate pathway
o Directing glucose through PPP
Mitochondrial diseases
• Where Krebs and oxidative phosphorylation occurs
• Mitochondria has its own DNA
• Mutations of mitochondrial proteins à Decrease in ATP (as mitochondria
is a major ATP source)
• Mitochondrial or nuclear genetic defects involving enzymes used in
oxidative phosphorylation impair cellular respiration; decreasing the
ATP:ADP ratio
• Tissues with high energy demand are particularly vulnerable (brain,
nerves, retina, skeletal, cardiac muscle)
• Red ragged fibres à Clumps of diseased mitochondria
Point mutations in protein coding genes
• Complex I mutations à Leber’s hereditary optic neuropathy
o Not a multisystem disorder but a highly selective degeneration of
the optic nerve
• Cytochrome b (part of complex III)
o Cytochrome b is an electron carrier
o Exercise intolerance
o Myalgia
• Complex IV (cytochrome c oxidase, COX)
o Only a few patients with multisystem disorders
• Complex V (ATP synthase)
o Neuropathy
o Ataxia and retinitis pigmentosa (decreased ATP synthesis rate and
decreased stability of the F0F1 ATPase)
o More severe phenotype is called Leigh syndrome
Leber’s hereditary optic neuropathy
• Degeneration of retinal ganglion cells and their axons that leads to loss of
central vision (predominantly young adult males)
•
•
Transmitted through the mother as it is primarily due to mutations in the
mitochondrial genome (only the egg contributes mitochondria to the
embryo)
LHON is usually due to one of three pathogenic mitochondrial DNA
(mtDNA) point mutations in the ND4, ND1 and ND6 subunit genes of
complex I of the oxidative phosphorylation chain
Leigh syndrome
• About 20-‐25% of sufferers have a mutation in mtDNA
• May have the following symptoms à Loss of appetite, vomiting, failure to
thrive, irritability/continuous crying, delayed motor and language
milestones, seizures, generalised weakness, decreased muscle tone,
tremor, problems with muscle coordination, abnormal eye movements,
vision problems, heart problems
• Lactic acidosis due to TCA insufficiency à kidney problems (pyruvate
converted to lactate instead)
• Some patients have deficient activity in pyruvate dehydrogenase
o Step before entry to TCAs
o Produces acetyl CoA which is imported into TCA
• Knowledge of affected genes mean you can bypass the pathway as
treatment
MERRF syndrome (Myoclonic epilepsy with ragged red fibres)
• Symptoms à Muscle twitches (myoclonus), weakness (myopathy), and
progressive stiffness (spasticity)
• Muscle cells have red ragged fibres (diseased mitochondria)
• Caused by mutations in mitochondrial tRNA-‐lysine
Fructose bisphosphate deficiency
• Deficiency of fructose-‐1,6-‐bisphosphate, one of the enzymes in
gluconeogenesis (Opposite to PFK1 in glycolysis)
• In FBP deficiency
o There is not enough fructose bisphosphate for gluconeogenesis to
occur correctly
o As three carbon molecules cannot be used to make glucose they
will instead by made into pyruvate and lactate
o May cause hypoglycaemia and lactic acidosis
o Glycolysis will still work, as it does not use this enzyme
Glycogen storage diseases
• Due to defects in glycogen synthesis or breakdown
• Enzymes affected may include glycogen synthase, G6P, branching enzyme,
debranching enzyme, phosphorylase kinase, GLUT2 and others
• Type la (Von Gierke disease)
o Most common type of glycogen storage disease
o Caused by mutations in G6P à Enlarged liver, kidney failure,
shortage of white blood cells (neutropenia)
§ In most cell types G6P can enter glycolysis
§
Liver must be able to export glucose
Altitude sickness
• Increased altitude is coupled with decreased atmospheric pressure
therefore, for every breath inhaled there is less O2 available à Bodies are
forced to work harder to metabolise
• Respiration must increase so that our muscles and brain can get enough
O2 for electron transport, so that enough ATP can be made for energy
usage
• The body may overcompensate by sending too much blood to the brain à
swelling (cerebral oedema) à Dilation of the blood vessels leading to the
head and neck causes headaches and brain swelling leads to imbalance,
vomiting, hallucinations
• Increased blood flow/pressure can also cause leakage from the blood
vessels into the alveolar sacs of the lung causing difficulty in breathing,
gurgling sounds in the lung, coughing, exhaustion à Body compensates
by increasing HR and BP, forcing more fluid into the lungs à Drown
Oxygen deprivation in heart attack (myocardial infarction)
• Interruption of blood supply to a portion of the heart
• In the absence of O2 the cell must rely on glycolysis for ATP production à
glycolysis does not produce much ATP
• Stores of glycogen and phosphocreatine are rapidly depleted
o Phosphocreatine is a backup energy storage molecule
o Creatine kinase catalyses conversion of creatine to
phosphocreatine
• ATP becomes too low to maintain membrane ion pumps
• Osmotic balance is upset and cellular organelles begin to swell and leak
Creatine/phosphocreatine
• Phosphocreatine (creatine phosphate) is a readily available source for
replenishing ATP
• Acted on by creatine kinase to release ATP
Hypoxic/ischemic injury
• Accumulation of succinate in ischaemic cells
• Leads to loss of oxidative phosphorylation and reduction in ATP
production which causes
o Failure of ATP dependent Na/K pumps and Ca2+ pump that
normally maintains high cell K+ and low cell Na+ and Ca2+
o K+ decreases and Na+/Ca2+ increase in cell
o Na+ brings water into cell à swelling
o Ca2+ causes activation of phospholipases that disrupt the
membrane and is an important signalling molecule
• Decrease in ATP à Increased glycolysis à Decreased pH from lactic acid
build up within the cell and decreases glycogen stores
• During ischemia succinate accumulates
•
During reperfusion succinate is consumed à Too much QH2 produced à
Drives reverse electron transport at complex I à Drives ROS production
at complex I à Cell damage
Summary
• Type I diabetes results from inability to produce insulin
• Type I diabetes impacts on glucose uptake by skeletal, cardiac, muscle
and fat cells
• Inborn errors in glycolytic enzymes are rare but found
• Mitochondrial diseases are a group of diseases resulting from mutation of
proteins important in electron transport and other processes in the
mitochondria
o Leber’s hereditary optic neuropathy
o Leigh syndrome
o MERRF sundrome
• Other inborn errors in metabolism include fructose bisphosphate
deficiency and glycogen storage diseases
• At high altitude, O2 deficiency can lead to increased HR, brain swelling,
leakage of blood vessels of alveolar sacs of lung and death
• HR leads to O2 deficiency, rapid depletion of ATP and energy stores and
breakdown in osmotic balance of cells
Pentose phosphate pathway
• Produces NADPH and ribose-‐5-‐phosphate
o NADPH similar in chemical properties to NADH (source of
reducing power)
o NADH occurs in oxidative and energy metabolism, NADPH electron
donor in biosynthesis
• Required for reducing power and nucleotide precursors
• NADPH is an electron donor
o Reductive biosynthesis of fatty acids and steroids
o Repair of oxidative damage
• Ribose-‐5-‐phosphate is a biosynthetic precursor of nucleotides
o Used in DNA and RNA synthesis
o Used in synthesis of some coenzymes (eg Coenzyme A)
o We need ribose to make NAD and NADP (cofactors)
o Ribose at the heart of ATP
o Ribose linked to phosphates and base in RNA
§ Ribose in RNA has an extra OH at C2
§ Deoxyribose in DNA does not à Increased flexibility
NADPH vs
...
PPP
• Oxidative phase à 2 NADPH + loss of CO2 à C5 sugar produced
• Carbons shuffled around in non-‐oxidative steps provide intermediates for
glycolysis
• Glucose à G6P (via hexokinase)
• G6P à Glycolysis or PPP
• NADPH negatively feedsback to the first step (G6P à 6-‐
phosphogluconolactone via glucose-‐6-‐dehydrogenase)
o Product inhibits its own production
G6P dehydrogenase deficiency
• Can be fatal in cases of high oxidative stress (certain drugs, herbicides,
some foods cause oxidative damage)
• Need glutathione and NADPH for protection from oxidative stress
• Confers resistance to malaria due to high oxidative stress in RBCs (as can
sickle cell anaemia à Deficiency in haemoglobin)
o Selection pressure to maintain the deficiency in the population
• Side product of oxygen metabolism is radicals à Hydrogen peroxide
• Glutathione peroxidase uses glutathione to breakdown hydrogen
peroxide à If deficient in G6P dehydrogenase à Form hydroxyl radicals
from hydrogen peroxide à Oxidative damage to lipids, proteins, DNA
•
•
•
Primaquine and compounds found in fava beans lead to increased
amounts of peroxides and other reactive oxygen species (cause oxidative
damage)
If people with G6PD deficiency eat fava beans à Deficient in G6P
dehydrogenase à Not enough NADPH à Oxidative stress à Hydroxyl
radicals
o Erythrocytes lyse
o Haemoglobin is released into the blood
o Jaundice and kidney failure can result
G6PD deficiency impairs the ability of red blood cells to maintain their
levels of reduced glutathione and therefore their ability to deal with
oxidative stress
What happens in the erythrocytes?
• Hemoglobin (Fe2+) à oxidation à Methaemoglobin (Fe3+)
o In order to carry O2, the heme iron must be 2+
o Methaemoglobin cannot carry oxygen
o If we are able to make NADPH we can reduce Fe3+ to Fe2+
• Methaemoglobin signifies damage, cells destroyed
Summary
• The PPP is a process by which cells can generate reducing power
(NADPH) that is needed for
o The biosynthesis of various compounds
o Reduction of oxidised glutathione
• Produces ribose-‐5-‐phosphate
• The non-‐oxidative phase of the PPP can convert pentose phosphates back
to G6P and other glycolytic intermediates
• A deficiency in the first enzyme in the pathway (glucose 6 phosphate
dehydrogenase) leads to a decreased amount of reduced glutathione, and
increased susceptibility to oxidative damage, particularly in erythrocytes
(red blood cells)
Revision
• GSH (glutathione)
o Gamma-‐glutamate + cysteine + glycine
o SH group from cysteine
• Peroxidase reaction (H2O2 (hydrogen peroxide) à H2O) via the reducing
power of glutathione
o H2O2 + H+ + 2e-‐ à 2H2O
• Glutathione S-‐transferase takes glutathione and conjugates it to various
toxins with nucleophilic centres
o By conjugating a toxic molecule to glutathione it becomes more
soluble and the body can then remove compounds conjugated to
glutathione from the cell
Nitrogen metabolism: Amino acids
• Roles of amino acids
o Energy production
o
o
o
o
Prophyrin synthesis
Neurotransmitter and hormone synthesis
Glutathione synthesis
Nitric oxide synthesis
Importance of Nitrogen in biochemistry
• Nitrogen (with H, O and C) is a major elemental constituent of living
organisms
• Mostly in nucleic acids and proteins
• Also found in
o Several cofactors (NAD, FAD, biotin)
o Small hormones (epinephrine)
o Neurotransmitters (serotonin)
o Pigments (chlorophyll)
o Defense chemicals (amanitin)
Biochemistry of molecular nitrogen
• N2 comprises 80% of the Earth’s atmosphere but is virtually unusable by
most organisms due to the highly resistant N-‐N bond (tightly bound)
o Resistant to modifications so needs to be extracted
o Hydrogen molecules added à Ammonium (NH3)
§ Need N2 + 3H2 à 2NH3 (NH3 + H2O à NH4+ + OH-‐)
o Nitrogen fixing bacteria and archea convert N2 to NH3
(ammonium) by a process called nitrogen fixation
o Some N fixing microbes live in symbiosis with plants
(proteobacteria with legumes such as peanuts, beans)
o A few live in symbiosis with animals (spirochaete with termites)
o Combined, represents 60% of newly fixed N2 (fixed from the air)
• A few non-‐biological processes convert N2 to biologically useful forms
o N2 and O2 à NO via lightening (represents 25% of newly fixed
N2)
o NO can be incorporated into biological system
o N2 and H2 à NH3 via the industrial Haber process
§ Requires T>400C, P>200 atm
§ Represents 15% of newly fixed N2
The Nitrogen cycle
• Chemical transformations maintain a balance between N2 and
biologically useful forms of nitrogen
o Fixation à Bacteria reduce N2 to NH3/NH4+
o Nitrification à Bacteria oxidise ammonia into nitrite (NO2-‐) and
nitrate (NO3-‐)
o Assimilation à Plants and microorganisms reduce NO2-‐ and NO3-‐
to
§ NH3 via nitrite reductases and nitrate reductases
§ NH3 (from either fixation or assimilation) is incorporated
into amino acids
§ Organisms die, returning NH3 to soil
§ Nitrifying bacteria again convert NH3 to nitrite and nitrate
o Denitrification à Nitrate (NO3-‐) is reduced to N2 under anaerobic
conditions
§ NO3-‐ is the ultimate electron acceptor instead of O2
The nitrogen cycle
• Amino acids and other reduced N-‐C compounds degraded by animals à
NH4+ (ammonia)
• NH4+ nitrification by bacteria and archea à NO2-‐ (nitrite) à Nitrifying
bacteria à NO3-‐ (nitrate)
• NO3-‐ (nitrate) can be denitrified by bacteria, archea and fungi to N2 à
Nitrogen fixing bacteria and archea à NH4+ ammonia) à NH4+
incorporated into biological system (AA etc)
Ammonia is incorporated into biomolecules via glutamate and glutamine
• Glutamate and glutamine are the crucial entry point for the NH4+
• In bacteria and plants (but not mammals), glutamate synthase catalyses
the incorporation of ammonia (NH4+) into alpha-‐ketoglutarate to
generate glutamate
• Alpha ketoglutarate + NH4+ forms glutamate (driven by NADPH)
o Reaction does not require ATP (it is part of the other coupled
reaction in the equation)
o Alpha-‐ketoglutarate + NH4+ + NADPH + ATP à Glutamate +
NADP+ + ADP + Pi
• In mammals, glutamate concentrations are maintained by the
transamination of alpha-‐ketoglutarate during amino acid metabolism
(from diet)
• Glutamate serves as a nitrogen source for biosynthesis reactions, such as
the synthesis of amino acids from alpha-‐ketoacids
• Glutamine (Gln) is made from glutamate (Glu) by glutamine synthetase in
a two step process
o This reaction is driven by ATP (phosphorylates glutamate)
o Glu + ATP à Gamma-‐glutamyl + NH4+ à Gln + Pi
o Phosphorylation of glutamate creates a good leaving group that
can be easily displaced by ammonia
Regulation of glutamine synthetase by allosteric inhibitors
• Glutamine synthetase
o Found in nearly all organisms
o Plays a central role in the conversion of toxic free NH3 to
glutamine and in the metabolism of amino acids
o Converts ammonia and glutamate to glutamine which can be used
in a variety of processes
o Undergoes cumulative regulation by six end products of glutamine
metabolism
§ AMP
§ CTP
§ Histidine
§ Tryptophan
§ Carbamoyl phosphate
§
Glucosamine-‐6-‐phosphate
Glutamine synthetase is also inhibited by adenylylation
• Adenylylation is a post translational modification where AMP binds to a
hydroxyl group of a molecule via adenylyltransferase (AT)
• Adenylylation inactivates glutamine synthetase
• Deadenylylation activates glutamine synthetase (converts glutamate to
glutamine)
• Adenylylation is indirectly
o Stimulated by glutamine and Pi
o Inhibited by alpha-‐ketoglutarate and ATP
Revision
• The process by which nitrogen fixing bacteria and archea convert N2 to
NH3 is nitrogen fixation
• The four chemical transformations that maintain a balance between N2
and biologically useful forms of nitrogen
o Nitrogen fixation
o Nitrification (conversion of ammonia to nitrite and nitrate)
o Denitrification (nitrate à N2)
o Assimilation
• The enzyme in bacteria and plants which catalyses NH4+ + alpha-‐
ketoglutarate à glutamate is glutamate synthase
• Glutamate serves a key role in biosynthesis à Provides an entry point for
ammonia into the system (N source in the synthesis of AA from alpha-‐
ketoacids)
• Glutamine synthetase is a primary regulation point in biosynthesis
(Glutamate + ATP + NH3 à Glutamine)
• Two main mechanisms by which glutamine synthetase is regulated
o Adenylylation
o Allosteric modulation
Amino acid biosynthesis
• Source of nitrogen is glutamate or glutamine
o Form key entry points for N entry
o The N can be used in various ways and can be added to C skeletons
• Carbon skeletons come from intermediates of
o Glycolysis
o Citric acid cycle
o Pentose phosphate pathway
• Bacteria can synthesise all 20 but mammals require some in diet
(essential AA)
Amino acids made from intermediates of major pathways
• Substrates (C skeletons) from major pathways can be attached to N and
form AA
• Some AA can be made from other AA (eg Glutamine formed from
glutamate via glutamate synthetase)
•
•
•
•
•
The carbon skeleton comes from three sources
o Glycolysis
o Citric acid cycle
o Pentose phosphate pathway
Nitrogen enters these pathways via transamination with glutamate and
glutamine
Non essential amino acids can be made from one of 6 precursors
produced in major biochemical pathways (TCA, glycolysis, PPP)
o Alpha-‐ketoglutarate
o 3-‐phosphoglycerate
o Oxaloacetate
o Pyruvate
o Phosphoenolpyruvate
o Ribose-‐5-‐phosphate
Essential AA must be supplied in the diet
A deficiency of one AA effects the synthesis of all proteins required for life
Amino acid biosynthesis
• Non essential AA (except Arg) are synthesised by simple reactions
involving 1-‐5 steps
• Essential AA are synthesised by more complex steps involving 5-‐16 steps
(essential AA require more steps for synthesis)
• Reactions involve either
o Transfer of the amino group from glutamate by transamination
reactions (transamination)
o Transfer of methyl groups from tetrahydrofolate (derived from
folic acid/Vitamin B9) or S-‐adenosylmethionine
• 5-‐phosphoribosyl-‐1-‐pyrophosphate (derived from ribose-‐5-‐phosphate) is
a notable intermediate in several pathways of amino acid (Try, His) and
nucleotide synthesis
Transamination
• Alpha-‐ketoglutarate + AA (glutamate/glutamine) à AA + alpha-‐keto acid
(intermediate)
o Reversible reaction
o Transfer of amino group from one AA to another by
aminotransferases
• Uses the pyridoxal phosphate (PLP) cofactor
• L-‐glutamine acts as a temporary storage of nitrogen
• L-‐glutamine can donate the amino group when needed for AA
biosynthesis, thus readily reversible
• Pyridoxal phosphate is
o A cofactor in transamination reactions
o Derived from pyridoxine (vitamin B6)
o Used to transfer an amine group, primarily from glutamate or
glutamine, to a carbon skeleton for AA synthesis
Feedback inhibition regulates AA biosynthesis
•
•
•
•
Inhibition of the first irreversible step (or committed step) in linear
pathways
Feedback inhibition and activation of branched pathways involves the
inhibition of common initial step by its own product and is activated by
the product of another pathway
Enzyme multiplicity in branched pathways
o Inhibition of the committed step catalysed by 2 or more enzymes
Cumulative feed inhibition of branched pathways
o Eg glutamine synthetase
o Partial inhibition of a common step by each of the final products
Enzyme multiplicity regulated amino acid biosynthesis
• Inhibition or activation keeps AA synthesis in balance
• Aspartate may produce 3 different products (AA)
o One directly inhibits an enzyme and another may inhibit a
different enzyme, one may have no feedback effect
Roles of AA
• Energy production
o The TCA cycle
o Gluconeogenesis (Via TCA)
o Ketogenesis (important in generation of ketone bodies)
• Synthesis
o Proteins
o Nucleotides
o Porphyrins
o Creatine
o Glutathione
o Neurotransmitters
o Rigid polymers
o Hormones
o Polyamines
o Nitric oxide
• Some AA may enter TCA (can be either glucogenic and drive
gluconeogenesis or ketogenic and generate ketone bodies)
• Alpha-‐ketoglutarate + glutamate synthase à Glutamate
• Glutamate + glutamine synthetase à Glutamine
Roles of AA à Porphyrin synthesis
• Porphyrins are large, ring like protein structures
• Porphyrin makes up the heme of haemoglobin, cytochromes and
myoglobin
• In higher animals, porphyrin arises from reactions of glycine with
succinyl-‐CoA
• In plants and bacteria, glutamate is the precursor
• Pathway generates two molecules of the important intermediate delta-‐
aminolevulinate
o Two molecules of delta-‐aminolevulinate condense to form
porphobilinogen (important intermediate)
o Four molecules porphobilinogen combine to form protoporphyrin
o Fe ion is inserted into protoporphyrin with the enzyme
ferrocheletase
o Fe ion functions as both an electron donor or acceptor
o Fe ion binds O2 in haemoglobin (erythrocytes) and myoglobin
(myocytes)
Heme is the source of bile pigments
• Bilirubin is breakdown product of heme
• Green compound seen in bruises
• Urobilin gives urine its yellow colour
• Stercobilnin gives red-‐brown colour to faeces
Defects in porphyrin synthesis
• Most animals synthesise their own heme
• Mutations or misregulation of enzymes in heme biosynthesis pathway
lead to porphyrias
o Precursors accumulate in red blood cells, body fluids and liver
o Homozygous individuals also suffer intermitted neurological
impairment, abdominal pain
Roles of AA à Neurotransmitter and hormone synthesis
• Tyrosine à Epinephrine
• Glutamate à GABA
• Tryptophan à Serotonin
Roles of amino acids à Glutathione synthesis
• 3 AA form glutathione (glutamate, cysteine, glycine form a reduced
tripeptide)
• Glutathione (GSH) is present in most cells at high amounts
• Reducing agent/antioxidant
o Keeps proteins and metal cations reduced
o Keeps redox enzymes in reduced state
o Removes toxic reactive oxygen species
• Oxidised to a dimer (GSSG)
Roles of amino acids à Nitric oxide synthesis
• Pollutant NO plays an important role in neurotransmission, BP regulation,
blood clotting and immunity
• Synthesised from Arginine
o Arginine à Citrulline + NO
o Mediated by NO synthase
o Driven by NADPH
Revision
• Two amino acids which provide a source of N in metabolism are
•
•
•
•
•
The three pathways which yield the carbon skeleton for AA synthesis
o Glycolysis
o TCA
o PPP
Essential AA in hosts must be supplied in the diet
Reactions used to transfer an amine group, primarily from glutamate or
glutamine to a carbon skeleton for AA synthesis are transamination
In addition to biosynthesis, AA are involved in energy production; the
three main energy production pathways they are involved in are
3 examples of heme proteins which contain iron-‐porphyrin (heme)
groups are haemoglobin, cytochrome,
The enzyme which converts arginine to NO is NO synthase
•
Protein synthesis and degradation
• Protein synthesis à Dehydration (or condensation) reaction where a
peptide bond is formed between amine and carboxylic acid with the loss
of a water molecule
• Protein degradation à Hydrolysis reaction where a water molecule is
inserted to break the peptide bond and reform the amine and carboxylic
acid
• Form a dipeptide (with a peptide bond) with an N and C-‐terminus
Amino acid catabolism in mammals
• AA from dietary proteins are the main source of amino groups
(ammonium)
• When metabolised AA provide carbon skeletons which can be fed into the
TCA cycle and gluconeogenesis pathways
• Most AA are metabolised in the liver
• AA are used to
o Produce glucose or lipids
o Produce energy
o Provide carbon skeletons
o Produce proteins
• Excess ammonium (NH4+) must be excreted via the urea cycle
Dietary protein is enzymatically degraded through the digestive tract
• Acute pancreatisis à Obstruction of pancreatic secretions à Proteolytic
activation of zymogens to attack the pancreatic tissue à Pain and damage
• Stomach à pepsinogen (zymogen) à Pepsin (active via proteolysis)
• Pancreas
o Chymotrypsinogen à Chymotrypsin
o Trypsinogen à Trypsin
Digestive enzymes are secreted as zymogens or proenzymes
• Zymogens often work in cascades
• Enteropeptidase breaks down trypsinogen à Trypsin
• Trypsin has many actions including chymotrypsinogen à chymotrypsin
The digestion and absorbtion of proteins
• Proteolytic enzymes break proteins down to single amino acids or
oligopeptides (short branches of AA)
• AA enter intestinal cell directly via AA transporters then transported into
blood stream
• Oligopeptides too large to enter intestinal cell (broken down by
peptidases) into tripeptides or dipeptides (which are further broken
down to single AA in the intestinal cell)
Amino acid catabolism in vertebrates
• Removal of the amino groups is the first step of degradation for all AA
• AA group transferred to alpha-‐keto glutarate à glutamate
• Glutamate, glutamine, alanine and aspartate are central in nitrogen
metabolism as they are readily converted into the TCA cycle
o Alanine from muscle
o Glutamine from muscle and other tissues
o AA from ingested proteins
• Glutamine and alanine are important in the transport of amino groups
from extra hepatic tissues and skeletal muscle respectively to the liver
• Glutamate plays a role in removing excess ammonia via the urea cycle
• Aspartate provides the second amino group in the urea cycle
Ammonia is safely transported in the bloodstream as glutamine
• In the tissues
o Excess NH4+ in tissues is added to glutamate à glutamine (via
glutamine synthetase)
• Glutamine is transported in the bloodstream
• NH4+ is released in the liver à Release glutamate
• NH4+ enters urea cycle à urea which can then be excreted
Glucose-‐alanine cycle
• Vigorously working muscles operate nearly anaerobically and rely on
glycolysis for energy
• Glycolysis yields pyruvate and if not eliminated then lactic acid build up
• This pyruvate can be converted to alanine for transport to the liver via
transamination reaction (glutamate + pyruvate à alanine + alpha keto-‐
glutarate (transamination reaction))
• This alanine in the liver can enter the urea cycle and gluconeogenesis
pathway (Alanine + alpha ketoglutarate) à glutamate + pyruvate
o Pyruvate used by liver and fed into gluconeogenesis to form
glucose which is exported and sent to tissues where required
• NH4+ is toxic to the body and must therefore be regulated (must be kept
out of the bloodstream)
Amino groups are transferred to alpha-‐ketoglutarate by aminotransferases
• This is an example of a transamination reaction
• PLP (pyridoxal phosphate) is the essential cofactor in this reaction
(derived from vitamin B)
•
•
•
Transfer of amino groups from one to another
Alpha ketoglutarate and AA (via amino transferase + PLP) à Glutamate
and alpha-‐keto acid (Amino group transferred to glutamate)
Alpha keto acids contain a carboxyl and ketone group (eg pyruvate)
AA catabolism in terrestrial vertebrates
• Removal of the amino group (transamination) is the first step of
degradation for all AA
• In most cases the amino group is transferred to alpha-‐ketoglutarate to
form glutamate
• AA in liver à NH4+ attached to glutamate à Urea cycle (excretion of
ammonium as urea)
• Glutamate is transported from the liver cytosol to the liver mitochondria
to release NH4+
• Left over carbon skeletons of AA are converted to glucose or oxidised by
the TCA cycle
Excretory forms of nitrogen
• AA not used for new AA or other products are removed as a single
excretory end product
• Animals excrete nitrogenous wastes in different forms à Ammonia, urea
or uric acid
• NH4+ is highly toxic to animal tissues
• Mammals secrete NH4+ as urea
• In ureotelic organisms (many terrestrial vertebrates; sharks and
mammals)
o The liver of mammals and most adult amphibians converts
ammonia to the less toxic urea
o The circulatory system carries urea to the kidneys where it is
excreted
o Conversion of ammonia to urea is energetically expensive;
excretion of urea requires less water than ammonia
• Uricotelic animals (birds, reptiles)
o Insects, snails and many reptiles including birds excrete uric acid
o Uric acid is relatively non-‐toxic and does not readily dissolve in
water
o Can be secreted as a paste with little water loss
o More energetically expensive to produce than urea
• Ammonotelic animals (aquatic vertebrates eg fish and the larvae of
amphibia)
o Animals that excrete nitrogenous wastes as ammonia need access
to lots of water
o They release ammonia across the whole body surface or through
gills
• Ammonia excretion requires high amounts of water (fish)
o Low energy expenditure, high water loss
• Uric acid excretion limits water loss (excreted almost dry)
o High energy expenditure, low water loss
The urea cycle à Excess glutamate is metabolised by the mitochondria of
hepatocytes
• Glutamine from muscles etc
...
A CO2 molecule is released in
photorespiration
Improving rubisco
• Rubisco is rate limiting for photosynthesis
• Improved efficiency would have a large impact on CO2 reduction in the
atmosphere and food production
o Improve specificity à Replace crop gene with rubisco from red
algae or purple photosynthetic bacteria)
o Increase catalytic activity à Introduction of altered rubisco with
higher activity
o Increase levels of expression
C3 and C4 photosynthesis
• C3 plants use the conventional pathway of CO2 uptake in which the first
intermediate produced is a C3 compound
o CO2 à 3-‐phosphoglycerate (C3)
• C4 plants use an alternative pathway of CO2 uptake in which the first
intermediate produced is a C4 compound (In order to avoid
photorespiration)
o CO2 à Oxaloacetate (C4)
o Found in tropical grasses (eg maize, sugarcane) and is more
efficient in intense sunlight and high temperatures
• Characteristics of C4 plants
o High photosynthetic rates
o High growth rates
o Low photorespiration rates (better efficiency)
o Low rates of water loss
o Specialised leaf structure
C4 photosynthesis
• PEP carboxylase, the enzyme that incorporates atmospheric CO2, has no
affinity for O2 (Replaces rubisco which has the affinity for O2)
• C4 plants have different anatomy which allows them to concentrate CO2
and overcome photorespiration
• Two types of cells
o Mesophyll cells
o Bundle sheath cell
• CO2 from air à Carbonate
•
•
•
•
•
•
•
PEP à PEP carboxylase à Oxaloacetate à Malate à CO2 à Enters
normal calvin cycle and rubisco converts it to 3-‐phosphoglycerate
Malate à Pyruvate which is used to regenerate oxaloacetate for the cycle
(requires an additional 2 ATP than the C3 pathway)
The rubisco in the bundle sheath cells is never exposed to O2 in the
atmosphere so the plant cannot undergo photorespiration
CO2 fixation by the C4 pathway requires more energy (which is why not
all plants do it)
o The C3 pathway requires 3ATPs per CO2 fixed
o The C4 pathway requires 5ATPs per CO2 fixed
Concentrates CO2 for rubisco in the calvin cycle but requires additional
energy
Used when the gains in efficiency outweigh the increased energy
requirement
o The C4 pathway requires high sunlight for energy production
(increased ATP synthesis); therefore they are usually plants in
high sunlight areas
o Rubisco’s affinity for CO2 decreases with increased temperature;
above 30 degrees the C4 pathway provides a significant gain in the
efficiency of CO2 fixation
o Concentration of CO2 allows stomata to partially close during the
day, conserving water
Therefore, photosynthesis in C4 plants is more efficient in hot, arid or
tropical climates
Summary of C4 photosynthesis
• CO2 fixed into organic acid in mesophyll cells by PEP carboxylase which
has no O2 affinity
• CO2 fixed by rubisco in bundle sheath cells; [CO2] maintained at high
levels in these cells
• C4 photosynthesis requires more energy than the C3 pathway (5ATP vs
...
5kJ/mol)
o G6P à Glucose + Pi (∆G10 = -‐13
Title: Biochemistry: The energy of metabolism
Description: Detailed notes about metabolism and metabolic pathways, their regulation and enzyme regulation. Aimed at 1st or 2nd year students undertaking any kind of science or health degree.
Description: Detailed notes about metabolism and metabolic pathways, their regulation and enzyme regulation. Aimed at 1st or 2nd year students undertaking any kind of science or health degree.