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Biochemistry and Metabolism
Week 1 - Enzymes
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Cholesterol - excessive leads to heart disease
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LDL and HDL
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Metabolic syndrome - obesity, elevated blood pressure, elevated level of triglycerides and low
level HDL, resistance to insulin
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Complex interplay
between glucose/fat metabolism
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Drug targets
to relieve pain - inhibit enzymes
needed for prostaglandin synthesis
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Detection
antibody has enzyme attached to it
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Obesity - linked to lifestyle, but also
environmental chemicals? - known
as obesogens, increase levels of fat
cells, increase size of fat cells, disrupt hormone action
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Control of metabolism depends on control
of enzyme activity
Glycogen breakdown
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Rate controlled by
hormones and metabolites
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Can often make mistakes, some
enzymes, such as DNA polymerase 1
have sections that check no mistake is
made
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Needed for
reactions that cannot be catalysed by
amino acid residues of the enzyme
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)
Proof enzyme binds substrate/product - shown at constant enzyme concentration, reaction
rate increased with increasing substrate concentration but a maximal velocity was reached
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Spectroscopic characteristics of substrate or enzyme may change
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Cleft or crevice in the protein structure, Only a small part of the total protein,
3-D shape is important
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May be formed by amino
acids from different parts of the protein, Specific binding depends on precise arrangement of
atoms in the active site
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Lock and key or induced fit model
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Mix enzyme and substrate, follow loss of substrate or
production of product with time in a spectrophotometer
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Tails off over time due to product inhibition, reverse reaction and substrate
depletion
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Measure rate of enzyme reaction - Beer lambert law (umol/min)
Specific activity (umol/min/mg protein)
Effect of different substrate concentrations - mix enzyme and substrate and determine initial
rate at different substrate concentrations
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Maximal rate at high [S]
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Michaels/menten model - assumes enzyme binds substrate, formation of complex, conversion
of substrate to product
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At high concentrations of substrate – reaction rate is maximal and
cannot be increased further
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Vmax is the maximal rate of enzyme reaction (mmol/min)
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Vmax = k2[ET]
k2 = Vmax/[ET]

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k2, turnover number ( kcat)
Importance of Vmax - in regulation of metabolic pathways - Determine Vmax for enzymes in a
pathway
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May identify the regulatory step
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Km will vary With each substrate (substrate specificity of enzymes), With pH, With
temperature, With ionic strength
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May be able to infer nature of these groups
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Week 3 - Enzyme inhibition
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Enzyme inhibition - The inhibition of enzyme activity is a mechanism used for control of
metabolism, Drugs act by inhibiting enzymes, Used to study mechanism of enzyme action
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g
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g
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Aspirin inhibits cyclooxygenase activity of
prostaglandin H synthase irreversibly
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Penicillin inhibition - modifies enzyme
transpeptidase in bacteria
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Reversible inhibition - can be competitive and non-competitive
Competitive - Substrate and inhibitor compete for same active site,
dependent on the concentration of the 2
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g
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Non-competitive - substrate binds to active site, inhibitor binds to separate
site and alters the structure of the enzyme so that the substrate no longer
fits
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e
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fructose 1,6-bisphosphatase is inhibited by AMP, Enzyme of
gluconeogenesis
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Uncompetitive inhibition - inhibitor binds only to enzyme substrate complex, not just to the
enzyme alone, in presence of inhibitor, Km and Vmax are reduced
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Transfer of a functional
group or electrons between substrates
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Sequential reactions - ternary complex formed - sequential displacement
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g
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NADH binds first then pyruvate, lactate
released first followed by NAD
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Substituted enzyme intermediate
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Uncatalysed reactions are slow - Charges develop, Molecules
have to be brought together – unfavourable
entropically, High activation energy (DG‡)
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Substrate binds non-covalently, binds by
induced fit, this allows binding of the transition state as
well, accelerates reaction, provides specificity
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Cleaves specific peptide bonds
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Chymotrypsin treated with DIPF
(diisopropylphosphofluoridate), Complete irreversible inhibition, Ser 195 modified
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Week 4 - Control of metabolism
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Metabolism is regulated in 3 ways:
Control of enzymes present- regulation of gene expression, total level of an enzyme, level of
different forms of an enzyme, half life of enzyme
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This
is chronic control - slow response, long term control mechanism
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Component in the pathway can feedback to inhibit the pathway
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Usually is the end product that feeds back
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Acute control
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Catabolic reactions - break down large molecules into small molecules, generating energy
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Energy in form of
ATP
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Feedback control of metabolism - E
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- Aspartate transcarbamyolase, aspartate + carbamoyl
phosphate -> carbamoyl aspartate and orthophosphate, first committed step in biosynthesis of
pyrimidine nucleotides, inhibited by end product of pathway
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Structure ATCase - 12 polypeptide chains, 6 catalytic subunits that perform reaction but
unaffected by CTP, 6 regulatory subunits that bind CTP
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Model for ATCase - Different conformational states, Without substrate, T state predominates
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Adding substrate - concentrated shift and relative increase in unbound active
catalytic subunits
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Allosteric enzymes - Multi-subunit, Regulated by metabolites, Sigmoidal V/S relationshipsCooperative effects, Model as conformational shifts, Separate regulatory and catalytic
subunits (sometimes)
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g
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Phosphorylation on Ser, Thr, Tye - adds charged group, favourable reaction - some enzymes
switched on, some switched off by phosphorylation
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Phosphorylation allows for amplification of a
signal, One activated kinase can phosphorylate hundreds of target proteins
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Glucose transport into cells - Concentration gradient across cell membrane (high outside),
Passive transport (no energy needed) Family of glucose transporters (GLUT) each with unique
properties, Integral membrane proteins capable of facilitating glucose transport
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Basal uptake in most tissues including brain - Km below blood glucose
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Blood glucose varies between 3-8mM
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5-6 mM, GLUT4 normally held in vesicles inside cell, Insulin causes vesicles to move to cell
surface delivering GLUT4 and switching on glucose transport in to cell
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5-6 mM (GLUT4)

Week 5 - Glycolysis
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Carbohydrate metabolism - Glycogen - carbohydrate store in animals, in muscle and liver,
glucose units - energy production in muscle, blood glucose for brain in the liver
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Multiple branches, glycogen granules - glycogen
and metabolic enzymes
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In muscle - G-6-P --> Glycolysis, In liver G-6-P --> Glucose --> Blood glucose
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Control of glycogen metabolism - Breakdown
controlled at level of the enzyme glycogen
phosphorylase, Synthesis controlled at the level of
the enzyme glycogen synthase, Control by
metabolites and hormones
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Low blood glucose - Glucagon secreted, liver glycogen breakdown, blood glucose released
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Blood glucose high - Insulin high, Glucagon low
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Glycogen phosphorylase activity low
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Insulin promotes activation
Glycolysis - glucose degradation to pyruvate, Anaerobic ATP production, biosynthetic
intermediates, conservation of glucose needs to be regulated, influence of glucose
transporters
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ATP
inhibits - Lowers F-6-P affinity,
Opposed by AMP, Signals
abundance of ATP, Allosteric
site
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e
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Fructose 2,6 bisphosphate
(F26P2) -Stimulates
phosphofructokinase, F26B2 has
allosteric effects on enzyme,
works in liver
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F6P + ATP --> F26P2 + ADP - phosphofructokinase 2
Liver enzyme inhibited by cAMP-dependent phosphorylation
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Hexokinase - Inhibited by G6P, If PFK inhibited then G6P will rise and feedback, Prevents
unnecessary conversion of glucose to G6P
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Pyruvate kinase - Allosteric, ATP inhibits - Signals abundance of energy, F1,6P2 stimulates Keeps glycolysis going (feed forward activation)
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Chronic control of glycolysis - insulin promotes synthesis
of some glycolytic enzymes, glucagon promotes
synthesis of some gluconeogenic enzymes
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Entry in to TCA cycle,
Effectively irreversible, Commits pyruvate, Multiple
control
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Week 6 - Pentose pathway/Lipoproteins
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Pentose phosphate pathway - synthesis of NADPH, Sugars other than hexoses, e
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Pentose for
nucleic acid synthesis
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Active in adipose tissue - fatty
acid synthesis
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G6PDH
deficiency causes haemolytic anaemia - in red blood cells and harms metabolism causing lysis
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Non-oxidative stage - 2 reaction chains to make different lengths sugars
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Glyceraldehyde-3-phophate used in glycolysis
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o NADPH required - adipose tissue synthesising fatty acids
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Sugars recycled via glycolysis
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o Pentoses required - Rapidly dividing cells
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 Gluconeogenesis - a way of making
glucose, maintain blood glucose level,
recycling products of glucose metabolism,
anabolic pathway, synthesis of
carbohydrate from non-carbohydrate
precursors
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Body needs 160g/day
glucose
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Active
skeletal muscle produces lactate and
alanine, Lactate produced from pyruvate
by anaerobic glycolysis, Alanine produced
from pyruvate
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Substrates are lactate, amino acids and glycerol
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Liver
gluconeogenesis provides glucose for other tissues - brain and muscles
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Lots of energy needed - for gluconeogenesis - 6 ATP/GTP used
per glucose synthesised
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Substrates
enter pathway - Lactate converted to pyruvate for entry (lactate dehydrogenase)
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Glycerol enters at dihydroxyacetone
phosphate
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Fructose-1,6-bisphophatase - key
control point
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AMP inhibits, ATP
and citrate stimulate
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 Cori cycle - see picture
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Precursor of steroid hormones carried in plasma in lipoproteins
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Gut absorption variable - Mean is 50% (range 30-80%)
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Cholesterol homeostasis - regulate diet, endogenous synthesis, tissues, excretion, use and
metabolism
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 3-Hydroxy-3-methylglutaryl coenzyme A reductase - rate
controlling step in cholestrol synthesis
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Translation of mRNA can be inhibited
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Phosphorylation by AMPactivated protein kinase inhibits acutely, Inhibited by
drugs (statins)

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Cholesterol transport - cholesterol not readily soluble in plasma - transported in lipoproteins
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g
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VDLD - e
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endogenous
triacylglycerol
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g
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HDL - phospholipid/Cholesteryl ester
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g
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95 g/ml
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0631
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Hydrolyses triagylycerols to two fatty acids and
one monoacylglycerol, Products taken up by skeletal muscle or heart for energy production by
β-oxidation or adipose tissue for conversion to triacylglycerol for energy storage
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Important for regulating plasma LDL and plasma cholesterol levels
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Cholesterol excretion - converted to bile acids in the liver
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Bile acids are excreted into the bile and then into small intestine and faeces,
Solubilise dietary lipids
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Eventual block
due to thrombosis, High levels of LDL a risk factor, Uptake of modified LDL by macrophages,
Cholesterol loading (foam cells) in arteries
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Deficiency in
LDL receptors - leads to reduced rate of LDL clearance so high LDL numbers in blood
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Fatty acid breakdown - Fat as a major fuel store
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Highly concentrated, anhydrous, highly reduced, higher energy per gram than carbohydrate
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Saturated, monounsaturated and
polyunsaturated fatty acids
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Hormone-sensitive lipase
(triacylglycerol lipase), Stimulated by hormones e
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Fatty acids used as fuel,
Glycerol used in glycolysis/gluconeogenesis
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Therefore bind to albumin in plasma for
circulation around the body, Released from adipose
tissue and circulate to skeletal muscle or heart for boxidation
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2 carbon units removed in each cycle
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Fatty acyl CoA in cytosol
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Cross mitochondrial membrane as fatty acyl carnitine
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Each round yields acetyl CoA, NADH, FADH2
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Fixes ~40% of energy
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Unsaturated fatty acids - Move double bonds from one position to another on the fatty
acid, Remove double bonds by making them saturated using NADPH if necessary
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Inhibited by malonyl CoA
(intermediate in fatty acid synthesis) when fuel molecules are abundant, Inhibits acyl carnitine
production
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Fuel source for heart and kidney (cortex)
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Synthesis increased if fatty
acids broken down (catabolic hormones)Adipose tissue releases fatty acids in starvation and
diabetes
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Acids (ketoacidosis)
Uses of acetly CoA - enters in citric acid cycle, converted to CO2, pyruvate dehydrogease
irreversible, cannot convert acetyl CoA to glucose, therefore cannot convert fatty acid to
glucose
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Prominent in oil-rich seeds, e
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sunflower, and allow growth of seedlings until
photosynthesis can begin, Occurs in organelles called
glyoxysomes in plants
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Acetyl-ACP and malonyl-ACP condense to form
acetoacetyl-ACP, Release of CO2,
Reduction/dehydration/reduction, Product is butyrylACP
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Needed - synthesis of palmitate, 8 acetyl
CoA, 7 ATP and 14 NADPH
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See picture
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Reaction with ACP:
malonyl CoA + ACP -> malonyl-ACP + CoA and Acetyl CoA + ACP -> acetyl-ACP + CoA
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Transfer of acetyl groups - from mitochondria to cytosol, carried as citrate, generates NADPH
from NADH
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Acetyl CoA carboxylase - Palmitoyl CoA
prevents polymerisation (and therefore prevents activation)
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Inhibited by phosphorylation- AMP–

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activated protein kinase, when energy stores are low, AMP is high
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Elongation of palmitic acid - Elongation system on cytosolic face of endoplasmic reticulum
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Desaturation of fatty acids - fatty acyl CoA desaturases in endoplasmic reticulum
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Therefore essential fatty acids linoleic acid (C18:2) and linolenic
acid (C18:3) must come from diet
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Anabolic state - insulin high, adrenaline and glucagon low, Triacylglycerol breakdown low
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Protein turnover - Many cellular proteins are constantly degraded and resynthesized in
response to changing needs
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Needed to remove damaged
proteins
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Fat mobilisation, muscle protein degradation, general increase in
substrate/fuel supply for survival and repair
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Excess amino acids cannot be stored, protein
breakdown to supply these if not in diet
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Amino acids as metabolic precursors - protein synthesis, as cellular fuels, as precursors for
biosynthesis; serine needed for lipid biosynthesis, glycine needed for porphyrin synthesis,
arginine needed to make nitric oxide
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Degradation
starts with removal of amino group
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Glutamate
dehydrogenase - enables release of amino acid group as ammonia - can use NAD or NADP,
inhibited by ATP and GTP, stimulated by ADP and GDP
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Carbon skeletons - degraded to metabolic
intermediates, glucogenic - can be converted to glucose,
ketogenic - can be converted to ketone bodies, mixed
glucogenic and ketogenic
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Ketogenic amino acids - converted to acetyl CoA
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Amino groups - excreted - in humans in form of urea, in
birds and reptiles as uric acid, ammonia in aquatic
animals
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The urea cycle, TCA cycle and gluconeogenesis are linked
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Regulation of the TCA cycle and oxidative
phosphorylation - final common pathway for
al fuel molecules, aerobic process, generate
energy
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Occurs in mitochondrion
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If ATP consumed, ADP increase
and so does electron transport
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Regulating reduction of oxygen - Molecular oxygen is an ideal terminal electron acceptor
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Reactive oxygen species (ROS) can damage cellular components including DNA
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Catalase can
convert hydrogen peroxide into water
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Trigylceride ( adipose tissue - fatty acids) - ketone
bodies, soluble fuel
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Fuels in different tissues - Brain - glucose, ketone
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Heart muscle - fatty acids,
Adipose tissue - fatty acids, Liver - amino acids
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Regulation by hormones - Anabolic - insulin
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Regulation within pathways - occurs by - Allosteric interactions, Covalent modification,
Adjustment of enzyme levels, Compartmentalisation within the cell, Specialisation between
organs
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Regulaton of glycolysis - key control point is phosphofructokinase
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Anabolic control points - Fatty acid – acetyl CoA carboxylase, Glycogen – glycogen synthase,
Glucose – fructose 1,6 bisphosphatase, Protein - insulin
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Opposing effects of hormones and allosteric regulators - Glucagon stimulates glycogen
breakdown (phosphorylase)
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Glucagon
stimulates triglyceride breakdown (lipase)
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Glycogen breakdown for muscle contraction - Glycogen breakdown can be activated by
hormones that raise cAMP, hormones or other influences that raise calcium ion concentration,
and by raised levels of AMP
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Muscle contraction - Adrenaline released, Ca2+ released, ATP consumption
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Glucagon - cAMP
raised /F2,6P2 reduced, phosphofructokinase (inhibited), fructose 1,6 bisphosphatase
(stimulated), Liver only
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Also cAMP – protein kinase
A/triglyceride lipase/stimulated
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Glucagon stimulates glycogen breakdown, Glucose uptake by tissues
will be low, Glycolysis will be suppressed by other tissues, Make glucose from noncarbohydrate precursors, Preserve glucose for brain, Mobilise alternative fuel (fatty acid)
under low blood glucose conditions
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Triglyceride breakdown - ketone bodied
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Diabetic ketosis occurs when insulin is absent
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AMP - activated protein kinase - 'cells fuel gauge' ATP usage reflected in levels of AMP
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Inhibits anabolic
enzymes - Acetyl CoA carboxylase, HMG CoA reductase, Glycogen synthase
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Starvation - withdrawal of all food intake
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Use about 7,000 KJ/day
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Fuel use - dominated by need of brain for glucose and muscle
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Need to conserve muscle
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5mM
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Increased glycogen and triglyceride breakdown,
increased gluconeogenesis and decreased glycolysis
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F2,6P2 reduced, Phosphofructokinase activity reduced
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Gluconeogenesis stimulated Reduced inhibition by F2,6P2, Amino acids and glycerol as substrates, By 36h 75% of blood
glucose supplied by this route, Protein breakdown in muscle, Alanine outflow
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Low insulin reduces glucose uptake by muscle and adipose tissue, Fatty acid metabolism by
muscle raises citrate and inhibits glycolysis
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This would deplete 50% of total body protein in 17 days
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Survival during long term starvation - Fatty acids used by muscle and liver, Ketone bodies used
by brain, Both derived ultimately from triglyceride breakdown - Survival depends on fat
reserves
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This produces a metabolic block that may have pathological
consequences, most are single gene disorders, many inherited as autosomal recessive,
although some are X-linked or dominant
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stoppage of the subsequent steps in the
metabolic path
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Types of disorder - amino acid metabolism - alkaptonuria, albinism, phenylketonuria
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Mucopolysaccharide
metabolism - hurler’s syndrome, hunter’s syndrome
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Copper metabolism - wilson’s disease
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Foreign compound metabolism - glucose-6-phosphate dehydrogenase (g6pd) deficiency
Disorders of phenylalanine and tyrosine - Alcaptonuria, Albinism, phenylketonuria
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Tyrosine
-> Homogentisic acid -X-> Further products
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Albinism - Defect in an enzyme responsible for manufacture of melanin from tyrosine
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) Defective
gene for tyrosinase
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Melanin absorbs UV light to protect keratinocytes from DNA
damage
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Phenylalanine -X-> Tyrosine -> DOPA/Homogentisic acid
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Defective gene for phenylalanine hydroxylase
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Ganglioside GM2 is broken down by removal of sugar by hexosaminidase A
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Leads to coronary heart disease
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Lesch-nyhan syndrome - Defect in an enzyme of purine metabolism, HPRT (hypoxanthineguanine-phosphoribosyl transferase, X-linked recessive disorder
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Role in gene therapy - Use of cloned HPRT gene to correct
deficiency in HPRT- cells
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Transfect - put normal HPRT gene

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into deficient cells
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Glucose-6-phosphate dehydrogenase deficiency - Common inborn error of sugar metabolism,
Originally identified as sensitivity to the drug for malaria primaquine
First enzyme of the pentose phoshate
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Slow metabolisers - Inborn errors in cytochrome P450 enzyme, Enzymes needed to metabolise
foreign compounds such as drugs - Needed to detoxify and clear from body, Present in all
living organisms, Phase 1 – a polar group is introduced into the molecule, Phase 2 –
conjugation reaction with endogenous substrates such as sugars, amino acids, Results in
water-soluble products that are readily excreted by kidney
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Treatments for inborn errors of metabolism - diagnosis - control problems with diet and
control them
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