Bioenergetics and Metabolism Flashcards
what is enthalpy change related to?
how much energy is released by a reaction
what is Gibbs Free Energy?
enthalpy change - temperature(change in entropy)
what is needed for a spontaneous reaction?
must either be exothermic, have large increase in entropy or both, ∆G < 0
what is ∆G for a reaction at equilibrium?
∆G = 0
what conditions are needed for standard changes in Gibbs free energy?
pH 7, 1atm of pressure, 298K
how does coupling work?
endergonic reaction (won’t occur spontaneously) coupled to exergonic reaction
what reaction is one of the main driving forces for other thermodynamically unfavourable reactions?
ATP hydrolysis
why is ATP hydrolysis so exothermic?
phosphate and ADP have more resonance stabilisation than ATP. negative charge is dissipated over more of the molecule thereby stabilising the structure- ATP has 4 negative charges at pH 7 so P-O-P bonds weakened by electrostatic repulsion, more water can bind and stabilise ADP and Pi than ATP
what is the phosphorylation potential of ATP hydrolysis?
free energy of ATP hydrolysis
order of phosphorylation potentials of biologically important phosphorylated molecules, least to most?
PEP, 1,3-bisphosphoglycerate, phosphocreatine, ATP, G-6-P, 3-phosphoglycerate
what molecules will phosphorylate ADP?
PEP, 1,3-bisphosphoglycerate, phosphocreatine
examples of ATP hydrolysis coupling?
used to phosphorylate glucose to provide enough energy to prime the molecule to be broken down to pyruvate, used to stabilise peptide chains so they can be made longer, provides energy to join 2 nucleic acids at start of DNA synthesis
what group do NADH, NADPH, FADH2 and FMNH2 carry?
electrons
what group does coenzyme A carry?
acyl
what is the main redox system for energy producing pathways?
NAD+/NADH
what is the main redox system for biosynthesis?
NADP+/NADPH
when are reactions catalysed by acetyl-CoA important?
activation of fatty acids and at start of CAC
overview of liver role in bioenergetics and metabolism?
central role in glucose homeostasis. ‘fat factory’ in terms of synthesis and export of triglycerides to adipose tissue. liver partially oxidises fats to produce ketone bodies- central to N recycling and excretion/amino acid metabolism
why can heart be called ‘dustbin’ of body?
will metabolise wide variety of substrates left over from other metabolic processes
overview of what brain uses for metabolism?
largely uses glucose, can use ketone bodies during fasting
why is control needed in metabolic pathways?
to avoid uncontrolled substrate cycle, link energy production to energy usage, to respond to physiological changes
how can amount of enzyme present be changed? (2 general ways)
altering rate of synthesis or altering rate of destruction- long term changes or metabolically controlled changes
when is glucagon produced, when is insulin produced?
glucagon in response to low blood glucose, insulin in response to high blood glucose
2 broad types of metabolic pathway?
catabolic and anabolic
what happens in anabolic pathways?
more complex biomolecules synthesised from simpler smaller units. pathways consume energy
what happens in catabolic pathways?
larger molecule broken down to smaller units to generate energy- units may become building blocks for anabolic pathways
what is the overall reaction of glucose metabolism?
C6H12O6 + 6O2 -> 6CO2 + energy as ATP
what are the 2 pathways involved in carbohydrate metabolism?
glycolysis, citric acid cycle, oxidative phosphorylation
other names for the citric acid cycle?
Krebs cycle, tricarboxylic acid cycle
how is glucose transported into most cells?
GLUTs (glucose transporters)
products of CAC?
CO2, NADH, FADH2, GTP
products of oxidative phosphorylation?
ATP, NAD+, FADH
which tissues take up glucose in an insulin independent manner? why do they do this?
brain (needs constant flow of glucose), liver cells (mop up excess glucose), erythrocytes
which tissues take up glucose in an insulin dependent manner?
fat and muscle cells
what are the insulin independent GLUTs?
GLUT 1, 2, 3
what is the insulin dependent GluT?
GluT4
how do GluT4 molecules respond to insulin?
prior to cells being exposed to insulin the GluT4 proteins are trapped in intracellular vesicles, insulin recruits these vesicles to the cell membrane to allow transport of glucose
products of glycolysis?
pyruvate, 2ATP, NADH
what are the 2 fates for NADH produced in glycolysis?
can be transported into mitochondria for oxidation or used to reduce pyruvate to lactate to regenerate NAD+
difference in carbohydrate metabolism in anaerobic conditions?
glycolysis still used (important in RBCs, cells in retina and fast-twitch white muscle), oxygen debt then repaid by increasing CAC rate to oxidise lactate produced by pyruvate conversion by LDH
what are the 2 halves of glycolysis?
first half which involves chemical priming and consumes ATP, second half which involves energy (ATP) generation
what are the 3 stages in the overall pathway of glycolysis?
glucose prepared for lysis then split into 2 3C-monosaccharides, one of these (glyceraldehyde-3-phosphate) is then oxidised to produce 2ATP and 2NADH per glucose, then rearrangement to produce pyruvate
2 examples of cells with no mitochondria?
RBCs and cells within retina
what happens in the first stage of glycolysis?
glucose-> glucose-6-phosphate -> fructose-6-phosphate -> fructose 1,6-phosphate-> glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. consumes 2 ATP
what happens in the second stage of glycolysis?
glyceraldehyde-3-phosphate oxidised to produce 2 ATP and 2 NADH per glucose. aldehyde in the glyceraldehyde converted to a carboxylic acid. NAD+ and inorganic phosphate incorporated to form 1,3-bisphosphoglycerate- has high energy acyl bond which supplies phosphate to convert ADP to ATP. 2ATP and 2NADH produced overall
what happens in the 3rd stage of glycolysis?
rearrangement, dehydration and loss of phosphate to produce pyruvate and 2 ATP
what are the ways NAD+ are regenerated in mammalian tissues?
NADH can be oxidised in mitochondria, NADH can be oxidised by lactate dehydrogenase during conversion of pyruvate to lactate
fates of lactate produced in anaerobic glycolysis?
exported to bloodstream (Cori cycle) or converted back to pyruvate for oxidation of carbon backbone in CAC
effect of excess lactate in blood?
overpowers buffering capacity of blood, makes blood more acidic
what is the pentose phosphate pathway?
operates alongside glycolysis, ensures supply of reducing potential in form of NADPH and important intermediates such as ribose 6-phosphate for anabolic pathways
where does the pentose phosphate pathway operate?
in liver and other cell types heavily involved in biosynthesis of fats and other biomolecules such as mammary glands, adipose tissue and adrenal cortex
overall product of pentose phosphate pathway?
for every 3 molecules of glucose 6-phosphate diverted from glycolysis into PPP 2 molecules of fructose 6-phosphate and 1 molecule of glyceraldehyde 3-phosphate are returned back to the glycolysis pathway
why is gluconeogenesis important?
brain always requires glucose as fuel even if part of requirement can be met by other fuels. some organs in body have little oxidative capacity so need to recover the lactate produced by anaerobic glycolysis in these tissues in for other organs can use or re-cycle carbon chain back to anaerobic organs such as glucose
which 3 reactions in glycolysis aren’t readily reversible?
glucose -> glucose 6-phosphate; fructose 6-phosphate ->fructose 1,6-bisphosphate; phosphoenolpyruvate->oxaloacetate->pyruvate
reaction converting pyruvate -> phosphoenolpyruvate?
2 steps. first= pyruvate carboxylase catalyses reaction of pyruvate, ATP, bicarbonate to form oxaloacetate, second= PEP carboxykinase catalyses conversion of oxaloacetate to PEP using GTP
how is fructose 1,6-bisphosphate converted back to fructose 6-phosphate?
hydrolysis of a phosphate group by fructose 1,6-bisphosphatase
how is glucose 6-phosphate converted back to glucose?
hydrolysis by glucose 6-phosphatase
what GluTs does the liver use?
GluT
which has a higher Km. glucokinase or hexokinase?
glucokinase
effect of the different Kms of glucokinase and hexokinase?
liver can’t take up glucose at low blood glucose levels due to higher Km of glucokinase but can deal with high glucose concentrations. when glucose 6-phosphate builds up muscle tissue can still produce glucose 6-phosphate for glycogen or lipid synthesis
what is the substrate/allosteric inhibitor of PFK-1? what potentiates this?
ATP, potentiated by citrate
where is hexokinase?
muscle
where is glucokinase?
liver
effect of PFK-1 in inactive state? (high citrate and ATP)
glycolysis in muscle
effect of exercise on [AMP]?
causes large rise in [AMP] as adenylate kinase catalyses the reaction 2ADP <-> ATP + AMP
why does [AMP] rise rapidly as [ADP] rises during muscle contraction?
[AMP] is only around 2% [ATP] so 10% decrease in [ATP] will result in 400% increase in [AMP]
how is PFK-1 controlled in muscle?
by [AMP] (AMP increases, glycolysis increases)
how is PFK-1 controlled in the liver?
fructose 2,6-bisphosphate causes decreased gluconeogenesis and increased glycolysis. potent activator of PFK-1
how is fructose 2,6-bisphosphate formed?
phosphorylation of fructose 6-phosphate by separate kinase PFK-2
why do futile cycles such as the one between fructose 6-phosphate and fructose 1,6-bisphosphate (via PFK-1 and fructose 1,6-bisphosphatase) exist?
serve important regulatory process of signal amplification in tissues such as skeletal muscles, at cost of expending some ATP the system is made more sensitive to small changes in concentration of regulatory muscles
what enzyme controls the conversion of fructose-6-phosphate to fructose-2,6-bisphosphate to control the flux through glycolysis and gluconeogenesis?
bifunctional enzyme with domain containing PFK-2 and domain containing fructose 2,6-bisphosphatase
how is hormonal control of balance between glycolysis and gluconeogenesis exercised in the liver?
by controlling concentration of fructose 2,6-bisphosphate by controlling production of PFK-2 and fructose 2,6-bisphosphatase (recycles it to fructose 6-phosphate)
action of glucagon in liver?
acts when [glucose] is low, activates PKA which phosphorylates the bifunctional enzyme so that simultaneously PFK-2 decreases, fructose 2,6-bisphosphatase increases- so gluconeogenesis favoured over glycolysis
how is hormonal control of balance between glycolysis and gluconeogenesis exercised in cardiac muscle?
hormonal action of adrenaline causes phosphorylationn of PFK-2 via PKA on different site increasing its rate so fructose-2,6-bisphosphate increases, glycolysis increases
how is hormonal control of balance between glycolysis and gluconeogenesis exercised in skeletal muscle?
PFK-2 isoform not phosphorylated, enzyme responds to increase in [fructose-6-phosphate] and therefore fructose-2,6-bisphosphate increases reinforcing effect of AMP increase, increasing glycolysis
what does fructose 1,6-bisphosphate stimulate?
pyruvate kinase
how is glucose stored?
as glycogen
structure of glycogen?
polymer of glucose predominantly joined at α(1->6), one end joined to protein glycogenin
when is the CAC active, fed or fasted state?
in the fed state
effect of products of CAC- citrate and ATP- on glycolysis?
act as allosteric inhibitors of glycolysis so allow conversion of glucose to glycogen in fed state when CAC is active
where is there more glycogen, the liver or muscle?
more in muscle
why is UTP needed to produce glycogen?
glucose-1-phosphate isn’t a powerful enough donor to form a glucose-glucose bond so needs energy input from UTP
enzyme required for glucose 6-phosphate -> glycogen?
glycogen synthase
enzyme and cofactor required for glycogen -> glucose 6-phosphate?
glycogen phosphorylase and AMP
how is glycogen metabolism controlled?
hormonal and electrical stimulation- stimulated by adrenaline which binds to receptor to activate adenylate cyclase to make cAMP which activates PKA which activates phosphorylase kinase and inhibits glycogen synthase. phosphorylase kinase activates glycogen phosphorylase b to make glycogen phosphorylase a.
what opposes the action of AMP stimulating phosphorylase b?
ATP
during exercise is glycogen being produced or broken down to glucose 1-phosphate? so is glycogen synthase or glycogen phosphorylase active?
being broken down, so glycogen phosphorylase is active
effect of glucose 6-phosphate on glycogen metabolism?
inhibits conversion of glycogen to glucose 1-phosphate
what enzyme breaks down cAMP to AMP?
cAMP phosphodiesterase
what stimulates cAMP phosphodiesterase to convert cAMP to AMP?
insulin
what inhibits cAMP phosphodiesterase conversion of cAMP to AMP?
caffeine
effect of Ca2+ on glycogen metabolism in muscle?
activates phosphorylase kinase which activates glycogen phosphorylase b to make glycogen phosphorylase a which is used to convert glycogen to glucose 1-phosphate
how is signal to break down glycogen turned off in well fed state?
cAMP hydrolysed to 5’AMP and protein phosphatases remove phosphates from proteins, insulin acts through glycogen synthase kinase 3 (GSK3) which is inhibited and turns on glycogen synthase thus glycogen formed
what is the Cori cycle?
muscle tissue generates lactate during explosive exercise, would cause acidosis if not exported into blood. lactate converted back to glucose via gluconeogenesis in liver, after exercise glucose transported back to muscle tissue and stored as glycogen
importance of gluconeogenesis?
maintaining normal function in brain where glucose is the primary fuel
what can the brain use as fuel?
glucose and ketone bodies
what does the body do to proteins in long term starvation?
converts them to glucose via amino acids and citric acid cycle
what are the excess products from adipose tissue and skeletal muscle during type 2 diabetes? what happens to them?
lactate, alanine, glycerol. serve as substrates for gluconeogenesis with energy required for ATP coming from beta-oxidation of FAs
role of PEP-CK under normal circumstances?
partial control (stimulation) of gluconeogenesis- negatively regulated by insulin
effect of type 2 diabetes on PEP-CK?
expression of PEP-CK rises as negative regulation by insulin lost, increased production of glucose adds to hyperglycaemia
function of metformin?
suppresses liver gluconeogenesis, treatment for type 2 diabetes
what is the CAC involved in?
generation of energy from metabolic fuels that are broken down to acetyl-CoA, provision of building blocks for metabolic processes, co-ordination of fuel use to physiological demands, control of PDH to ensure glucose supplies to brain, connection to oxidative phosphorylation
overall is the CAC a reduction or oxidation reaction?
oxidation
where does the CAC take place?
in the matrix of the mitochondria
what is produced by each turn of the CAC?
3 NADH, 1 FADH2, GTP (readily converted to ATP), CO2 (2 for each acetyl group entering cycle)
what conditions are required for the CAC?
oxidative
how is pyruvate converted to acetyl-CoA to enter the CAC?
using CoASH and NAD+ and pyruvate dehydrogenase to produce acetyl-CoA, CO2 and NADH
what happens to acetyl-CoA in the CAC?
combines with oxaloacetate to form citrate, uses citrate synthase, releases CoASH. high energy sulphur bond broken
what happens to citrate in the CAC?
uses aconitase to form isocitrate. rearrangement reaction
what happens to isocitrate in the CAC?
uses isocitrate dehydrogenase and NAD+ to form oxoglutarate, releases CO2 and NADH. oxidation and decarboxylation.
what happens to oxoglutarate in the CAC?
uses α-ketoglutarate dehydrogenase, NAD+ and CoASH to form succinyl CoA, CO2, NADH.
what happens to succinyl CoA in the CAC?
uses succinyl CoA synthetase, GDP, phosphate to form succinate, CoASH, GTP. GTP can then be converted to ATP (GTP + ADP -> ATP + GDP)
what happens to succinate in the CAC?
uses FAD to form fumarate and FADH2
what happens to fumarate in the CAC?
uses fumarase and H2O to form malate. hyddration reaction
what happens to malate in the CAC?
uses malate dehydrogenase and NAD+ to form oxaloacetate and NADH
what happens to oxaloacetate in the CAC?
uses acetyl CoA and citrate synthase to form citrate and CoASH
order of intermediates in the CAC?
oxaloacetate + acetyl-CoA -> citrate -> isocitrate -> oxoglutarate -> succinyl CoA -> succinate -> fumarate -> malate -> oxaloacetate
where do glycolysis, the PPP and FA synthesis take place? (have their enzymes)
in the cytosol
where do the CAC, beta-oxidation and the respiratory chain take place? (have their enzymes)
in the mitochondria
overall stoichiometry of CAC?
2 carbons enter (as acetyl-CoA) and 2 carbons leave as CO2
why is an anaplerotic pathway needed alongside the CAC, what is this pathway?
to return carbon to the cycle. pyruvate carboxylase converts pyruvate + CO2 + H2O + ATP to oxaloacetate + ADP + Pi + 2H+
how many ATP molecules are generated from 1 NADH?
2.5
how many ATP molecules are generated from 1 FADH2?
1.5
overall ATP generated from oxidative glycolysis (including from NADH entering ETC)?
5
total number of ATP molecules generated from CAC (including NADH and FADH2 entering ETC)?
8NADH + 2FADH2 + 2GTP = 25 ATP
total number of ATP molecules generated from aerobic glycolysis + the CAC?
30
structure of PDH?
multienzyme complex of 3 enzymes
function of PDH kinase?
phosphorylates PDH and deactivates it
what are the regulatory enzymes for PDH?
PDH kinase and PDH phosphatase
function of PDH phosphatase?
dephosphorylates PDH which activates it
what inhibits PDH kinase? what does this ensure?
pyruvate- ensures PDH converts pyruvate to acetyl CoA when lots of pyruvate present
what activates PDH phosphatase?
Ca2+, and insulin in adipocytes
why does Ca2+ activate PDH phosphatase?
stimulates PDH during exercise
why does insulin activate PDH phosphatase in adipocytes?
stimulates PDH during feeding for lipid synthesis
ratio of what substances is used to regulate PDH?
ratio of NADH/NAD+ and acetyl CoA/CoA so PDH turns off if lots of NADH and acetyl CoA (products of PDH catalysed reaction)
inhibitor of citrate synthase? when is this important?
allosterically inhibited by ATP. important for gluconeogenesis and ketogenesis during starvation to supply brain
effects of citrate synthase inhibition by ATP?
oxaloacetate used in gluconeogenesis,
acetyl-CoA used to generate ketone bodies instead of being used to produce citrate in the CAC
what inhibits isocitrate dehydrogenase (ICDH)
high NADH/NAD+ ratio and ATP - conditions typical of fed state
what stimulates isocitrate dehydrogenase?
ADP
what inhibits α-ketoglutarate dehydrogenase?
its products succinyl-CoA and NADH
what stimulates α-ketoglutarate dehydrogenase?
Ca2+
why do tumours have a high oxidative glycolytic rate even when oxygen available?
running glycolysis at higher flux helps promote flux through PPP which produces ribose for nucleotide synthesis, NADPH for FA synthesis and glutathione reduction- products used for making more DNA and lipids in cell membranes- give cancer competitive replicative advantage
where and how are fats stored in the body?
stored in adipocytes + less healthily in liver,
as triacylglycerols (also called neutral fats/triglycerides)
why are fats an efficient way to store energy?
require less water than glycogen and produce more energy following complete oxidation
how are TAGs mobilised?
converted into glycerol and FFAs by lipases which progressively hydrolyse ester bonds via diacylglyceride (DAG) and monoacylglyceride (MAG)
role of hormone sensitive lipase?
converts TAG to DAG to MAG to FFA
what is adipose TAG hydrolysis highly dependent on?
adipose triglyceride lipase (ATGL)
effect of insulin on HSL and ATGL?
inhibitory
effect of PKA on HSL?
activates it by phosphorylating it
how does insulin act in opposition of cAMP raising hormones?
activates phosphodiesterase enzyme to break down cAMP
things that raise fatty acid levels?
fasting, prolonged exercise, stress
how does cAMP activate HSL?
activates protein kinase, PKA then involves ATP to activate HSL
where are FFAs released and where are they taken up? where are most taken up in exercise?
released from adipose tissue, taken up by liver and muscle to be oxidised. in exercise most taken up by cardiac and skeletal muscle
what acts as a carrier molecules of FAs? what does this binding to the FA result in?
CoA-SH. binding results in ATP conversion to AMP
what happens in β-oxidation? where does this take place?
takes place in mitochondria. converts aliphatic fat into set of activated acetyl units (acetyl CoA) that can be used in the CAC
stages of β-oxidation?
FAs activated using Coenzyme A to form acyl-CoA by fatty acyl CoA synthase. AMP produced by CoA-SH binding to a FA. overall reaction favourable as PPi formed is hydrolysed to Pi
does all β-oxidation use the same enzyme?
no, different enzymes for short, medium and long chain FAs
where does activation of β-oxidation occur?
at outer mitochondrial membrane
how does the fatty acyl CoA produced in β-oxidation get across the inner mitochondrial membrane?
modified by carnitine acyltransferase I, carried across attached to carnitine, transferred back to CoA-SH once inside by carnitine acyltransferase II
how is a fatty acid converted into acetyl CoA units?
oxidised to introduce double bond, double bond hydrated to introduce an oxygen. alcohol from the water added is hydrolysed to ketone. 4 carbon fragment cleaved by CoA to yield acetyl CoA, process repeated on FA chain.
products generated by β-oxidation?
FADH2, NADH, acetyl-CoA, AMP, PPi
how many ATPs are produced from palmitate?
around 106- 8 acetyl CoA for CAC, 7FADH2, 7NADH, 7H+ = 108, and 2 molecules of ATP consumed in palmitate activation
what happens to the fat-derived acetyl CoA produced for the CAC?
can’t be used to synthesise glucose, is completely oxidised to CO2
what happens when the liver produces mor acetyl-CoA than can be metabolised via the CAC? when might this happen?
ketone bodies formed in liver and released into blood. happens in starvation or diabetes when oxaloacetate levels drop during gluconeogenesis
what are the FAD dependent acyl-CoA dehydrogenases? (4)
very long chain (VLCDH), long chain (LCDH), medium chain (MCDH/MCAD) and short chain acyl-CoA dehydrogenase (SCAD)
effects of MCAD (medium chain acyl-CoA dehydrogenase) deficiency?
associated with cot death as babies can’t oxidise FAs as readily so die at night when glycogen depleted.
when is FA oxidation needed, fed or fasted state?
fasted state when glycogen depleted
what causes Jamaican vomiting sickness?
unripe ackee contains inhibitors of acyl-CoA dehydrogenases, depletes glycogen reserves
effect of insulin of fatty acid oxidation/metabolism?
inhibits it
what are the 3 ketone bodies?
acetoacetate, β-hydroxybutyrate and acetone
which ketone body is exhaled on the breath?
acetone
what happens to acetoacetate in the muscle?
cleaved to 2 acetyl-CoA which enters CAC
why can’t liver cleave acetoacetate to acetyl-CoA?
doesn’t have the tranferase required to transfer CoA from succinyl-CoA to form acetoacetyl-CoA
what is the issue with ketoacidosis?
the low pH- not the increase in ketone bodies itself
effect of diabetes on ketogenesis?
increases it
what happens to the glycerol produced from TAGs?
circulated to liver for recycling (since adipose tissue doesn’t have glycerol kinase)
what is an important source of gluconeogenic precursors in ruminants?
conversion of propionate into succinyl-CoA
how is fatty acid oxidation regulated?
lipolysis of TAG, re-esterification of FAs, transport into mitochondria, availability of NAD+ and FAD
why aren’t FAs re-esterified under fasting conditions?
insulin concentration low so Glut4 isn’t recruited to membrane of adipose cells so little glucose uptake, depletes glycerol and prevents FFAs from re-esterification, so FAs released from adipose cells
what prevents FA synthesis and degradation occurring alongside each other?
malonyl-CoA produced during FA synthesis, inhibits carnitine shuttle in liver (and maybe skeletal muscle + pancreas)
where are very long chain FAs oxidised?
in peroxisomes
what are peroxisomes?
rounded oxidising organelles found in the cell (especially liver cells) which chew up fats to shorter chain FAs
where does fatty acid synthesis take place?
cytoplasm
steps of fatty acid synthesis?
acetyl-CoA + CO2 + ATP -> malonyl CoA + ADP + Pi, catalysed by acetyl-CoA carboxylase
why does fatty acid synthesis use NADPH not NADH?
allows 2 different reducing potentials at the same time in the cell- high NADPH/NADP ratio to favour reduction in FA synthesis at same time as high NAD/NADH ration to favour oxidation in glycolysis
what process produces the majority of NADPH in cells?
pentose phosphate pathway
how is the acetyl group from Acetyl-CoA generated in the mitochondria exported to the cytosol for FA synthesis?
carried out as citrate (produced by CAC)
reaction converting citrate back to oxaloacetate and acetyl-CoA in cytoplasm after export from mitochondria?
ATP + citrate + CoA -> oxaloacetate + acetyl-CoA + ADP + Pi, catalysed by ATP citrate lyase
what enzyme adds acetyl-CoA to the growing FA chain?
fatty acid synthase
what conditions favour fat synthesis/lipogenesis?
high carbohydrate diet and insulin
3 key steps of regulation of FA synthesis?
control of malonyl-CoA synthesis, control by AMP and citrate, uptake of glucose controlled by insulin, control of TAG synthesis
how do AMP and citrate control FA synthesis?
AMP increases phosphorylation of ACC via AMPK. citrate activates ACC allosterically. feed forwards signal that acetyl-CoA is abundant- inhibited by acyl-CoA
how is TAG produced?
3 fatty acyl-CoA + glycerol 3-phosphate -> TAG. glycerol 3-phosphate is produced from glyceron-phosphate + NADH
where is TAG produced?
liver, adipose, lactating mammary glands
how does insulin stimulate TAG production?
stimulates the enzymes that add acyl groups to glycerol- possibly by altering the phosphorylation of them. stimulates glucose metabolism to provide glycerol backbone for TAG synthesis
what are the 2 ‘halves’ of amino acid metabolism and what are their fates?
nitrogen metabolism and the carbon backbone metabolism, N collected in liver for excretion as urea, C backbone enters CAC and is metabolised
what are the ketogenic amino acids and what does this mean?
leucine and lysine- must be metabolised as ketone bodies, can’t enter CAC directly
what does glucogenic amino acid mean?
can enter the CAC directly so could be used to make glucose via gluconeogenesis
where is amino acid metabolism most intensive? where else does it take place?
liver, also takes place in muscle
where is the urea cycle focused?
liver
what happens to ammonia generated by amino acid metabolism in the muscle?
captured as glutamate to form glutamine for export to the liver
how is N excreted in mammals?
as urea from the liver into the blood, then transported to kidney for disposal
why is urea ideal for N excretion?
water soluble, not basic or acidic, ideal for detoxification
what are responsible for transfer of amino groups between amino acids? what amino acids are they transferred to?
2-oxo acids, courtesy of amino transferases. transferred to glutamate, alanine and aspartate
what is the prosthetic group of amino transferase?
vitamin B6
what is the glucose-alanine cycle?
alanine = carrier of ammonia and carbon skeleton of pyruvate from skeletal muscle to liver. in liver ammonia excreted, pyruvate used to produce glucose which is returned to muscle
what catalyses glutamate -> glutamine?
glutamine synthetase, also requires ATP hydrolysis
what happens to the glutamine produced in the muscle?
transported to liver, NH4+ liberated in mitochondria by glutaminase,
what happens to glutamate in the liver?
N in glutamate released as ammonia via oxidative deamination using glutamate dehydrogenase in mitochondria
what makes glutamate dehydrogenase unusual?
can use either NAD+ or NADPH as cofactor, allosterically regulated by GTP and ATP
what is the N input into the urea cycle in the form of?
aspartic acid and ammonia
what happens to ammonia in first step of urea cycle?
reacts with bicarbonate to form carbamoylphosphate, requires 2 ATP
what happens to carbamoylphosphate in the urea cycle?
added to ornithine to make citrulline
what happens to citrulline in the urea cycle?
condenses with aspartate and uses ATP to form arginosuccinate
what happens to argininosuccinate in the urea cycle?
cleaved to form fumarate and arginine
what happens to fumarate produced in the urea cycle?
used in the CAC
what happens to arginine in the urea cycle?
hydrolysed by arginase to release urea and ornithine ready to restart cycle
how are enzymes catalysing the urea cycle reactions distributed in the cell?
distributed between the mitochondrial matrix and the cytosol
what is the Krebs bicycle/aspartate-arginosuccinate shunt?
the interactions between the CAC and the urea cycle
effects of ammonia toxicity (large excess of ammonia)?
can produce hepatic encephalopathy whereby ammonia accumulates in brain drawing water in and damaging brain as it tries to expand. also detrimental effects on brain of depleting CAC of α-ketoglutarate by first converting it to glutamate and then glutamine
overview of the chemiosmotic hypothesis?
reducing potential generated by β-oxidation and the CAC (+ a little from glycolysis). the NADH and FADH2 generated are then oxidised by O2 to produce water- process pumps H+ across mitochondrial membrane- force generated used to make ATP from ADP and phosphate, this fuels the cell. process takes place in mitochondria
mitochondrion structure?
cristae of inner mitochondrial membrane provide very large SA. inner membrane of single liver mitochondrion has over 10000 sets of respiratory chains and ATP synthase molecules
how many mores sets of electron transfer systems in heart mitochondria than liver?
3x
what % of the protein of heart tissue is found in mitochondria?
30%
what pumps protons out of the mitochondria? what supplies the energy for this?
the PMF, energy from oxidation of NADH and FADH2
what gradients does the PMF work against?
pH gradient and charge gradient
how are ATP synthesis and the proton motive force coupled?
protons travel through ATP synthase which forms ATP by the condensation of ADP and Pi
how can oxygen consumption in mitochondria be measured?
by an oxygen electrode: mitochondria placed in buffer, oxygen electrode records decrease in oxygen level
what is state 4 respiration?
ADP not present, mitochondria respire slowly to compensate for leakage of protons across inner mitochondrial membrane
what is state 3 respiration?
oxygen consumption increases markedly when ADP present- demonstrates that system is coupled- protons can only flow across IMM when ADP present so oxygen used to oxidise reducing agents
what do uncouplers such as protein ionophores and 2,4-DNP do?
promote H+ re-entry so dismantle the PMF. when present O2 consumed even when no ATP produced
how many redox centres, different polypeptides and supramolecular complexes are there in the ETC?
20 redox centres, 70 different polypeptides, in 4 supramolecular complexes
how are electrons shuttled between complex I + II to complex III?
by ubiquinone (Q) being reduced to QH2
how are electrons shuttled from complex III to IV?
cytochrome c
what does complex 4 transfer electrons to?
O2
what is NAD?
nicotinamide adenine dinucleotide
what is FMN?
flavin mononucleotide
what is FAD?
flavin adenine dinucleotide
where is the redox centre in FAD?
the isoalloxazine ring
what prosthetic group does complex I use for oxidation?
FMN
what prosthetic group does complex II use for oxidation?
FAD
what do complexes I, II, and III use to carry out redox of flavins and Q?
iron-sulphur proteins
what makes ubiquinone membrane soluble?
long lipophilic side-chain
how is Q reduced to QH2?
2 steps: transports electrons from complex I and II to III
how do cytochromes transfer electrons?
transfer single electrons by Fe2+/Fe3+ redox
what are the cytochrome categories?
a, b and c
where are cytochromes b and c1 found?
in complex III
where are cytochromes a and a3 found?
in complex IV
how can cytochrome c be tracked?
by its spectroscopic properties
experimental evidence for order of the ETC?
redox states within proteins can be monitored by variety of spectroscopies: visible (cytochromes), UV (Q) or electron spin resonance (Fe and Cu). if deprive mitochondria of O2 all of the respiratory chain components reduced. as O2 introduced oxidation occurs in order: cyt a, cyt c, cyt b, flavins and the NADH. can also order complexes according to redox potential as measured by electrochemical methods. redox potential of individual reactions give their order, differences in energy tell us there are 3 spans with sufficient energy to synthesise ATP- correspond to proton pumping by complex I, III and IV
complex I donor and acceptor?
NADH donor, ubiquinone acceptor
complex II donor and acceptor?
succinate donor, ubiquinone acceptor
complex III donor and acceptor?
reduced Q donor, cyt c acceptor
complex IV donor and acceptor?
reduced cyt c donor, O2 acceptor
how many protons are pumped out by complex I for every 2 electrons?
4
how many protons are pumped out by Q/complex III for every 2 electrons passing through?
4
how many protons are pumped out by complex IV for every 2 electrons it passes to O2?
2
how many protons are pumped out for each package of 2e- that passes from NADH to O2?
10
describe complex I?
uses NADH to reduce ubiquinone. largest of the protein complexes- around 40 polypeptides. NADH reduces FMN, electrons pass through 8-9 FeS centres, this reduces Q to QH2. proton pumping driven by conformational changes
describe complex II?
succinate dehydrogenase (important part of CAC). has bound FAD that is reduced by succinate in mitochondrial matrix. 3 FeS centres pass electrons to Q to produce QH2
describe complex III
uses Q to reduce cyt c. contains FeS protein, cyt c1 and cyt b so often called bc1 complex. cyt c1 receives electrons from the FeS centre, transfers them to cyt c. cyt c loosely associated with outer surface of mitochondria. cyt b in complex spans mitochondrial membrane, has 2 haems at opposite sides of protein. complex III releases 4H+ to intermembrane space when electrons transferred fro QH2 to cyt c. acts as electron wire as part of proton motive Q cycle.
what does antimycin inhibit?
complex III
describe complex IV
cytochrome oxidase. reduces O2 to H2O using cyt c. 4 redox centres: cyt a, cyt a3, CuA (2 Cu ions) and CuB, all work together to ensure O2 reduced to O2^2-, prevents formation of superoxide. 2 more e- cleave O-O, with 4H+ used to make water
reaction catalysed by ATP synthase?
ADP + Pi -> ATP + H2O
structure of ATP synthase?
2 parts, F0 and F1. F1 on its own just hydrolyses ATP and Pi together. initially ATP strongly bound to ensure it’s formed, then released by further conformational change which requires the binding change mechanism. F0 is complex of 10 subunits, which translocate H+s to the γ subunit of the F1 core. protons flow through F0, generates rotation of the subunits, in turn drives the γ subunit. as the γ subunit moves it drives the binding change mechanism. 3H+ used for each ATP produced
what are the 3 conformations that each site of the ATP synthase cycle through?
O (open- low affinity for ADP and Pi), L (loose- binds ADP and Pi loosely), T (tight- tight binding required to squeeze out the water)
steps of ATP synthesis by ATP synthase?
ADP and Pi binds to L site, energy in to convert L to T (ADP + Pi -> ATP + H2O), energy in to convert T to O, ATP released
what can be used to transport metabolites into and out of the mitochondrion?
ATP and ADP exchange courtesy of charge gradient, phosphate enters courtesy of pH gradient
danger posed by reactive oxygen species in mitochondrion?
if rate of e- entry into respiratory chain greater than rate of e- transfer through chain, partially reduced Q radical can be produced, in turn donates electron to O2. superoxide (O2-) acts on aconitase (4Fe-4S protein) to release Fe2+, Fe2+ leads to formation of hydroxyl free radical
function of reduced glutathione (GSH)?
opposes formation of hydroxyl free radical by Fe2+ caused by superoxide in mitochondria
how much of circulating glucose does brain use at rest?
25%
how much of brain fuel requirement can be met by ketone bodies?
50%
what can’t the brain use as fuel?
fat
major fuels of muscle?
glucose from glycogen, FAs, ketone bodies.
what can’t the muscle do?
store glycogen, carry out gluconeogenesis
what sort of metabolism does heart muscle use?
relies entirely on aerobic metabolism
favoured fuel of muscle at rest?
fatty acids
organs that use GluT4 transporters?
muscle, adipose tissue
role of liver in metabolism?
acts as buffer of blood glucose
metabolic processes liver can carry out?
FA synthesis, TAG metabolism (beta oxidation), ketogenesis, gluconeogenesis, glycolysis, amino acid metabolism, urea cycle
function of adipose tissue in metabolism?
stores FAs, releases them according to demand, releases hormones that regulate metabolism. no glycerol kinase so if low glucose uptake (no insulin) then low glycerol 3-phosphate concentration, free FAs not re-esterified, so will be exported
pancreas involvement in metabolism?
blood glucose level sensed by glucokinase, all G6P goes straight to oxidative phosphorylation, linked to insulin synthesis and release
role of kidney in metabolism?
disposes of urea, maintains blood pH, carries out gluconeogenesis
long term adaptive changes to fed state (high carb diet)?
insulin stimulates enzymes necessary for fat synthesis. arises directly by stimulating PFK and ACC, indirectly by increasing NADPH production and acetyl-CoA transport into cytosol
VLDL and the liver?
TAGs made in liver, not stored there in healthy individuals. TAGs packed with Apo B-100 (apoprotein) and packaged into VLDL. VLDL then exported to blood, most TAG ends up in adipose in healthy individuals
digestion of dietary triacylglycerol?
dietary TAG churned and emulsified by bile acids from gallbladder in SI. makes small fat micelles with large total SA. lipase from pancreas hydrolyses TAG at surface of micelles. FAs and MAGs from TAG hydrolysis absorbed into brush border, converted into TAG, packaged into chylomicrons- contain Apo B-48 and phospholipids to stabilise the structure, secreted into lymph system and enter bloodstream at thoracic duct
how is TAG removed from blood?
adipose and aerobic muscle have lipoprotein lipase (LPL) on outer surface, binds VLDL and chylomicrons by recognising surface apoproteins. LPL hydrolyses TAG at tissue surface, FAs released into cells. adipose makes TAG, muscle oxidises FAs
control of lipolysis in adipose tissue?
TAG lipase activated by phosphorylation catalysed by PKA. caused by glucagon, adrenaline, noradrenaline raising cAMP. insulin opposes by breaking down cAMP with a phosphodiesterase. adipose triglyceride lipase (ATGL) antagonised by insulin (enzyme needed for TAG hydrolysis to DAG). fasting, prolonged exercise, stress raise blood FA content
metabolic changes in short term exercise?
in 100m sprint muscle phosphocreatine lasts 4 secs, glycogen would last 80s but exhaustion after 20s. adrenaline and Ca2+ stimulate phosphate produced from ATP breakdown to be used by phosphorylase to produce glucose 1-P from glycogen. middle distance runners partly use aerobic ATP production. AMP formed by adenylate kinase, deaminated to IMP which stimulates glycogenolysis. IMP degraded to adenosine, stimulates vasodilation. both allow continued glycolysis during longer bursts of anaerobic metabolism, mid term + after exercise recycle lactate as glucose (Cori cycle)
metabolic changes in long term exercise?
glycogen and FAs oxidised, FAs mainly released from adipose courtesy of HSL and ATGL. small amount of FA released from breakdown of muscle triglyceride. FAs and glycogen-derived pyruvate produce acetyl-CoA, produces ATP by oxidative phosphorylation and CAC. oxaloacetate supply can be outstripped by acetyl-CoA supply- so cell converts Iso and Val to succinyl-CoA to add into CAC.
what is ‘hitting the wall’ in long distance running?
when glycogen is depleted and start to rely on fat which is slower at generating ATP
role of AMPK?
recognises ATP depletion and limits further depletion by inhibiting synthesis of glycogen, and FA and cholesterol biosynthesis. at same time initiates compensating changes that boost or maintain ATP levels
regulatory events mediated by AMPK?
upregulates SNS, FA oxidation + glucose uptake in muscle, glycolysis in heart, downregulates lipolysis, glucose uptake, FA synthesis + FFA esterification in adipose, cholesterol, FA and glycogen synthesis in liver, insulin secretion in pancreas
what increases AMP levels activating AMPK?
nutrient or exercise-induced stress
early stages of type II diabetes?
reduced insulin secretion, subsequent insulin resistance, raised glucose
effects of insulin resistance in type 2 diabetics?
raised glucose, GluT4 not transported to membrane so excess glucose can’t enter muscle or adipose, elevated non-esterified FAs (NEFAs) further inhibits glucose uptake in these tissues, promotes VLDL-TAG secretion. activates HSL so more FAs and glycerol produced and released + acetyl-CoA and NADH produced which inhibit PDH so turns of CAC. glycogen synthesis impaired, rate of glycolysis is low. alanine and lactate produced by muscle for gluconeogenesis in liver.
how does type 2 diabetes prevent VLDL uptake?
produces low activity of adipose tissue lipoprotein lipase extracellularly
lipotoxicity?
in liver excessive malonyl-CoA levels promotes de novo FA synthesis, inhibits CPT1. long chain CoAs diverted from TCA cycle to biosynthetic enzymes that produce TAGs, DAG, ceramide. in muscle FA influx promotes beta-oxidation without increase in TCA cycle, so metabolic byproducts of incomplete fat oxidation accumulate in mitochondria
why is proline used in collagen/why is it special?
no H on N of amino acid so can’t form H bonds with anything else + backbone has slight curvature. can form sharp bends in collaged, and can form polyproline helix which doesn’t have stabilising H bonds.
what is the conformational change when a Hb subunit binds O2?
iron pulled upwards, moves the His the iron attaches to up, which moves the alpha helix this is a part of so alpha helix bond to other alpha helix breaks, forms bond with another alpha helix it is brought into proximity with initiating conformational change in beta subunits
substrate specificity for chymotrypsin?
small hydrophobic amino acids in selectivity pocket select proteins with large hydrophobic Trp and Phe
substrate specificity for trypsin?
favourable for positively charged Lys/Arg
substrate specificity for elastase?
alanine in specificity pocket interacts with Leu, Iso, Val, Phe, Trp
FMN structure?
has triple ring structure
hexokinase as example of importance of proximity in pathways?
in skeletal muscle hexokinase close to ATP/ADP pump so ATP pumped out is used in glycolysis, produced ADP which returns via pump to mitochondrial matrix
effect of ischaemic damage to tissue on metabolism?
causes hypoxia so will have same effect as oxygen deprivation
Warburg metabolism in cancer?
to start with cells are hypoxic in cancer proliferation, to synthesis new nucleotides need ribose- PPP used to synthesise ribose
where is glucose produced in liver transported to primarily?
brain and erythrocytes (RBCs)
