Metabolism Flashcards
What can entropy measure?
Wasted energy, degree of disorder, how widely energy is distributed
How can you cheat second law?
Couple reactions
What does ATP energy of hydrolysis depend on?
Concentrations of reaction and products, Mg2+, Ca2+ and H2O
Entropy change = ?
Standard entropy change + RTln ([products]/[reactants])
Why is ATP hydrolysis so exothermic?
Phosphate and ADP have more resonance stabilisation than ATP so can spread out -ve charge, electrostatic repulsion weakens POP bridge, stabilisation due to hydration because more water can bind to ADP and Pi than ATP
What is the phosphorylation potential?
Free energy of ATP hydrolysis is its phosphorylation potential
Which reactions is ATP coupled to?
Phosphorylates glucose to prime its breakdown to pyruvate, peptides more unstable than amino acids so use ATP to build them, joining two nucleic acids
What carries CO2?
Biotin
What carries glucose?
Uridine diphosphate glucose (ADP)
What is the redox system for energy reducing pathways?
NAD/NADH
What is the redox system for biosynthesis? Why?
NADP/NADPH - phosphate group allows enzymes to recognise this redox system
Why does acetyl CoA have lower delta G than ATP and why is it more exothermic?
Less stabilised by resonance
How is the acetyl group bonded in acetyl CoA?
As thioester (activated acetyl group)
What can the heart metabolise?
Fat, glucose, ketone bodies, lactate
How many ATP does glycolysis make?
2
How many ATP does Krebs make?
30
What controls entry of glucose into fat and muscle?
GluT4
What controls entry of glucose into liver cell?
GluT2
What are the three phases of glycolysis?
Chemical priming, cleavage, energy yielding
What is delta G like in glycolysis?
Only just favourable
What are interconvertible isomers during glycolysis?
G3P and DHAP
Why is glucose > G6P irreversible?
Large free energy change
What happens to the aldehyde in glyceraldehyde? Where does the oxygen come from and what is the reducing agent?
Converted to a carboxylic acid - oxygen from water, reducing agent is NAD
Where does the energy for the reaction catalysed by pyruvate kinase come from? What does the reaction need?
Replacing C=O and C=O to 2 x C=O, needs pyruvate kinase, Mg2+ and K+
What is a summary of glycolysis?
glucose + 2Pi + 2ADP + 2NAD > 2 pyruvate + 2ATP + 2NADH + 2H+ + 2H2O
What is the usual conc of lactate? Above what conc do you get hyperlactaemia?
1mM, 5mM
Why do you get hyperlactaemia?
Buffering capacity overpowered
How much ATP is produced in the liver? What does it produce instead?
Little, net producer of glucose
What is hexokinase inhibited by? Does this affect glucokinase?
G6P, no
What is a substrate and allosteric inhibitor of PFK-1? What assists with this?
ATP, citrate
What is inhibited in the inactive state of PFK-1? What overcomes this?
Glycolysis, AMP
Where is PFK-2 found? WHich hormone controls it?
Muscle, insulin and glucagon
What does the futile cycle between F6P and F-1,6-BisP control?
Balance between glycolysis and gluconeogenesis
WHat maintains the futile cycle between F6P and F-1,6-BisP in muscle?
AMP
WHat maintains the futile cycle between F6P and F-1,6-BisP in the liver?
F-2,6-BisP
Why does glucagon cause gluconeogenesis in the liver?
Decrease in F-2,6-BisP
What does fructose-1,6-bisP stimulate?
Pyruvate kinase
Why is glucose stored as glycogen?
Glucose would cause high osmotic potential which would damage cells and also avoids glycosylation of proteins
What is one end of glycogen joined to?
Glycogenin
WHy is UDP good?
Good leaving group
Why is UTP needed?
G1P not a powerful enough glucose donor for G-G bond so needs energy from UTP
Why glycogen enzymes are active during exercise?
Phosphorylase active, synthase inactive
What activates protein kinase A?
cAMP
How does PKA activate glycogen phosphorylase?
Activates phosphorylase kinase which activates glycogen phosphorylase
Which enzyme inactivates glycogen phosphorylase?
Protein phosphatase
What activates phosphorylase kinase for normal muscle action?
Ca2+ and also 5’AMP
What happens to glycogen phosphorylase once fed?
cAMP goes to 5’AMP and protein phosphatase deactivates it
How does insulin affect glycogen synthase?
Inhibits GSK3 so glycogen synthase no longer inhibited
What are the three irreversible reactions in glycolysis?
Glucose to G6P, F6P to F-1,6-BisP, and PEP to pyruvate
How many ATP does pyrivate kinase forwards produce?
1
How many ATP does pyruvate to PEP produce?
Two
What does increased AMP cause in muscle?
Increased glycolysis
What increases glycolysis and decreases gluconeogenesis in the liver?
F-2,6-BisP
What controls PFK-2 in cardiac muscle and liver?
Adrenaline in cardiac muscle, glucagon and insulin in liver
What regulates PFK-2 in skeletal muscle?
No hormonal control, regulated by F6P availability
How much glucose is produced from fat?
None
How does pyruvate get into the matrix?
Symported with a proton
How many NADH, FADH2, GTP and CO2 per Krebs turn?
3NADH, 1FADH2, GTP, 2CO2
Which TCA intermediates are symmetrical?
Fumarate and succinate
What is link reaction coupled to?
Losing CO2 means acetyl CoA is generated
What does lipoamide do?
Flexible arm, tethers acetyl group between two sites and moves reducing potential to a third site
Where is succinate dehydrogenase?
Inner mitochondrial membrane
What takes place in the cytosol?
Glycolysis, pentose phosphate, FA synthesis
What takes place in the mitochondria?
Krebs, beta oxidation, ETC
How much oxaloacetate is usually in the cell?
Small amounts
Why is an anapleurotic pathway needed?
If you keep removing compounds you lose carbon so need a pathway with intermediates
How many ATP from NADH?
2.5
How many ATP from FADH2?
1.5
In cytosol what is ratio of [NAD]:[NADH]?
1000
In mitochondrion what is ratio of [NAD]:[NADH]?
8
What does PDH phosphatase deficiency cause?
Lactic acidosis
What does the PDH complex contain?
PDH kinase and PDH phosphatase
What do PDH kinase and PDH phosphatase do?
PDH kinase deactivates PDH and PDH phosphatase activates it
What inhibits PDH kinase?
Inhibited by pyruvate
What activates PDH phosphatase?
Ca2+ for exercise and insulin in adipocytes for lipid synthesis
What else regulates PDH?
What inhibits isocitrate dehydrogenase?
The NADH/NAD ratio found in the fed state and ATP
What stimulates isocitrate DH?
ADP
What stimulates and inhibits alphaKG DH?
Inhibited by its own products, stimulated by Ca2+
What is limiting when there’s lots of acetyl CoA?
Oxaloacetate
What does the purine nucleotide cycle do?
ATP breakdown can produce fumarate to produce more oxaloacetate
What do purine nucelotide cycle deficiencies cause?
Muscle cramps
How can you measure the Krebs cycle?
Oxygen or carbon “chase the label”
How can you use fMRI in the Krebs cycle?
Deoxyhaem is paramagentic and haem is diamagnetic so alters signal
What converts triacylglycerols/triglycerides into glycerol and fatty acids?
Lipases
What activates lipases?
Phosphorylation by PKA
What activates protein kinase?
cAMP from glucagon, adrenaline, noradrenaline
What breaks down the cAMP which activates protein kinase? How?
Insulin, activates phosphodiesterase
What does beta oxidation do?
COnverts aliphatic fat to acetyl CoA units
What happens to the fatty acids to make acyl-CoA?
Activated by CoA
Why is beta oxidation bond favourable?
High energy bond converting ATP to AMP, the PPi formed makes 2 x Pi
Where does beta oxidation occur?
Inner membrane
What are the enzymes for short, medium and long chain beta oxidation?
Very long chain acyl CoA dehydrogenase = VLCDH, long chain = LCDH, medium = MCDH, short = SCAD
What catalyses beta oxidation?
Carnitine acyltransferase I
How does carnitine acyltransferase I travel?
Attached to carnitine
What happens once enzyme is inside the cell in beta oxidation?
Passed back to CoA again by carnitine acyltransferase II
Why are fats good for storage?
Don’t need hydration, unlimited storage, highly reduced, bound to albumin in blood to prevent capillary damage
What is the process of beta oxidation?
Oxidise to introduce double bond, hydrate to introduce an alcohol, oxidise the alcohol to a ketone, add CoA and you get acetyl CoA and a chain two carbons shorter
What does beta oxidation produce?
FADH2, NADH and acetyl CoA
Can you get glucose from fat derived acetyl CoA?
No
What happens if the liver produces more acetyl CoA than it’s possible to use during starvation or diabetes?
Ketone bodies will be formed
What does the feta heart use for fuel?
Uses glucose then switches to fatty acids after birth
What is MCAD deficiency?
Babies can’t oxidise fatty acids and die when glycogen is depleted
What causes Jamaican vomiting sickness?
Unripe ackee contains acetyl CoA dehydrogenase inhibitor so glycogen reserves are depleted
What are the three ketone bodies?
acetoacetate, B hydroxybutyrate and acetone
What happens to acetoacetate in muscle mitochondria?
Cleaved to make 2 x acetyl CoA to enter Krebs
Why can’t liver do succinyl-CoA to acetoacetate CoA?
Doesn’t have transferases needed
How does acetoacetate make acetyl CoA in mitochondria?
Two acetyl CoAs form acetoacetyl CoA, add another acetyl CoA then get something complicated but then produces acetoacetate and acetyl CoA which starts again
How can the glycerol from fatty acids produce glucose?
Goes to DHAP using glycerol kinase and then G3P dehydrogenase
Why must glycerol > glucose be done in the liver?
Adipose tissue doesn’t have the enzymes
What happens to long chain fatty acids in mitochondria?
Oxidised in peroxisomes, to supply mitochondria with shorter chains
What controls peroxisome synthesis?
A transcription factor
What inhibits the carnitine shuttle? Why?
Malonyl CoA so synthesis and degradation can’t occur together
What regulates fatty acid oxidation?
Lipolysis of triglycerides, carnitine huttle inhibition, competition with Krebs for NAD and FAD
If there’s no GluT4 why are more fatty caids released from adipose cells?
No GluT4 so no glucose in fat cell so free fatty acids can’t reform triglycerides so more fatty acids released
Why is fat easier to store than glycogen?
Needs less water adn more energy rich
Where does fat synthesis and oxidation happen?
Synthesis in cytoplasm, oxidation in mitochondria
Process of malonyl CoA production? What is the enzyme?
acetyl CoA + CO2 + ATP > malonyl CoA + ADP + Pi using acetyl CoA carboxylase
How does acetyl CoA get to the cytosol?
Carried there as citrate
What provides the reducing potential for fatty acid synthesis?
NADPH
What supplies NADPH?
G6P oxidation
What does fatty acid synthase do?
Adds acetyl CoA to a fatty acid strand
What does malonyl CoA do?
Removes CO2 to drive the reaction
What residue of fatty acid synthase does the fatty acid attach to?
Cysteine
After 16 carbons what happens to the fatty acid?
Detached by hydrolysis, either elongates further or made into triglyceride or phospholipid
How does citrate make neutral fat?
citrate to acyl-CoA to malonyl CoA to free fatty acid to neutral fat
What is the acetyl CoA carboxylase in liver and adipose?
Acc1
What is the acetyl CoA carboxylase in heart and skeletal muscle?
Acc2
Where does the acetyl CoA attach to the membrane?
Near carnitine acyltransferase so the malonyl CoA will inhibit FA oxidation
What does insulin activate to make more acetyl CoA?
Pyruvate DH
What does insulin activate to increase malonyl-CoA supply?
acetyl CoA carboxylase
What does a high carb diet do?
Lots insulin so lots of fat synthesis enzymes, directly stimulates PFK and ACC and indirectly increases NAPDH production and acetyl CoA transport into cytoplasm
Where do TAGs go after production in the liver?
Into a VLDL and exported into blood then adipose
How is TAG removed from blood?
Lipoprotein lipase binds VLDL and chylomicrons by releasing apoproteins, hydrolyses at tissue surface and the fatty acids are released into cells
In adipose tissue, what does PKA do?
TAG lipase activate phosphorylation
What increase cAMP concentration?
Glucagon, adrenaline and noradrenaline
What breaks down cAMP?
Insulin
How do glucogenic amino acids enter the Krebs cycle?
Directly
What happens to ketogenic amino acids?
Must be metabolized as ketoacids
Why is amino acid metabolism most intensive in liver?
Urea cycle here, serum protein synthesis occurs here, specialist biosynthesis
What does muscle tissue do to branched amino acids?
Oxidises them and glutamate/glutamine produced in exported to liver
What do 2-oxo acids do?
Transfer amino groups between amino acids courtesy of amino transferases
AlphaKG + amino acid = ?
Glutamate + 2-oxo acid
What does B6 do?
PYRIDOXAL PHOSPHATE Prosthetic group of amino transferase, acts as temporary parking spot for amino group and transfers to glutamate, alanine and aspartate
What does alanine do at the interface of Cori cycle and amino acid synthesis?
Carries ammonia and carbon of pyruvate to the liver where the ammonia is excreted and the pyruvate makes glucose
What happens to excess ammonia + glutamate? Which enzyme does this?
Makes glutamine using glutamine synthase
In the liver what happens to glutamine?
Produces glutamate and ammonium ion in the mitochondria using glutaminase
In the liver what happens to glutamate?
Makes ammonia and alpha KG using glutamate dehydrogenase
How does glutamate DH work?
Oxidative deamination, either NAD or NADP cofactor, allosterically regulated by GTP and ADP
How is nitrogen introduced to the urea cycle?
In form of aspartate and ammonia
What is the connection between the Krebs and urea cycle called?
Krebs bicycle, links fate of amino groups with their carbon skeletons
What can excess ammonia do to brain?
Accumulate and cause it to swell
Which neurones relase glutamate derived from alpha KG?
Excitatory
WHich cells take uo the glutamate and what do they do with it?
Glial cells and recycle it to glutamine to replenish lost carbon
What activates methionine’s methyl group?
ATP - called SAM or AdoMEt
What are donors for pathways which only need one carbon?
Folic acid or methionine
Where does folic acid collect the 1C fragment from?
Serine
What do ubiquinone and cytochrome c do?
Shuttle electrons between complexes
Which ETC complex is irreversible?
IV - control here
What do FMN and FAD have for redox? Which complexes are they found in?
Isoalloxazine ring in complex I and II
What do Fe-S complexes do?
Transfer single electrons and carry out redox of flavins and UQ
What do cytochromes do?
Transfer electrons between Fe2+ and Fe3+
Which complex uses Cu not Fe-S?
IV
What is complex I?
NADH-Q reductase
What is complex 2?
Succinate dehydrogenase/succinate-Q reductase
What is complex III?
Q-cytochrome c oxidoreductase
What is complex IV?
Cytochrome c oxidase
Why is ubiquinone membrane-soluble?
Lipophilic side chain
How many H+ for each 2e-?
10
How many H+ for Complex I, Q and complex 4?
4, 4, 2
How many haems does cytochrome b have?
Two at opposite ends = one high potential, one low potential
What is experimental evidence for transport chain order?
Monitor redox state using spectroscopy (visible for cytochromes, UV for UQ, e- sping resonance for Fe and Cu), no O2 = all components reduced, introduce O2 and oxidation goes backwards, can electrochemically measure redox potential
What are the 4 redox centres?
Cyt a, a3, CuA and CuB
How many subunits does F0 have?
10
What are the subunits in F1?
3 alpha-beta twins and one gamma rotor
What determines how many protons are needed to make ATP in the F0?
C units
How do ATP and ADP exchange in and out of the mitochondria?
Due to charge
How does phosphate enter the mitochondria?
Due to charge
What are the three F1 states?
Loose (bind ADP and Pi), tight (H2O released) and Open (ATP produced, low affinity for ADP and Pi)
What happens if there are more electrons in than electrons out?
Get QH. making O2- making aconitase making Fe2+ which makes .OH
When and why is gluathione produced?
When you produce lots of NAD to protect against free radical damage making peroxide and H2O
Why does muscle respond only to adrenaline?
No glucagon receptors
How much fatty acid is released during exercise?
A small amount from muscle triglycerides
What happens to the Pi produced from ATP breakdown during exercise?
Makes G1P from glycogen then makes G6P then glycolysis
What forms AMP?
Adenylate kinase
What is AMP deaminated to?
IMP
How does IMP allow glycolysis use for longer?
Makes adenosin which stimulates vasodilation, also stimulates glycolysis
How does cell make more succinyl CoA during exercise?
Makes it from isoleucine and valine
What happens to GluT4 during insulin resistance?
Won’t be recruited to sarcolemma so excess glucose doesn’t enter muscle so glycogen synthesis impaired and glycogen breakdown increases
What happens when the pancreas can’t maintain increased insulin production from insulin resistance?
Type II diabetes
How does lack of insulin sensitivity affect lipoprotein lipase activity?
Decreases it, prevents VLDL upake, hormone sensitive lipase is acitvated from cAMP accumulation to produce circulating fatty acids and glycerol and further impairs glucose metabolism
What happens when muscles and adipocytes can’t take up excess glucose?
Non-esterified fatty acids elevated, further inibits glucose uptake, promotes VLDL-triacylglycerol secretion
What happens following low glycolysis rate from no GluT4 during diabetes?
Worsens hyperglycaemia, worsened by fatty acid oxidation, B oxidation produces acetyl CoA and NADH which inhibits PDH and turns off Krebs, muscle produces lactate and alanine so get gluceoneogenesis
How do you get lipotoxicity?
Overfeeding > lots of malonyl CoA > fatty acid synthesis inhibits carnitine acyltransferase > increased gluconeogenesis, lipid synthesis and restricted B oxidation
What does fatty acid influx promote in muscke?
B oxidation but no coordinated Krebs increasee
What do products of increased fat oxidation cause in mitochondria?
Accumulate and might activate Ser kinases that impede insulin signalling and GluT4 translocation
What does the Guthrie test look for?
Phenylketonuria - deficiency is PAH (phenylalanine hydroxylase)
What inhibits citrate synthase?
ATP (allosterically)
Why is citrate synthase regulation important for gluconeogenesis?
Oxaloacetate is using at the start of gluconeogenesis but if ATP is high then citrate synthase inhibition diverts oxaloacetate to gluconeogenesis whilst the acetyl CoA is used to generate ketone bodies
What phosphorylates ADP?
Phosphoenolpyruvate and phosphocreatine
What buffers ATP in many tissues?
Phosphocreatine
How can you measure PCr?
31P nmr spectroscopy
Where does oxygen for oxidation of G3P come from?
Water
What activates PKA?
cAMP
What inhibits gluconeogenesis?
Ethanol
What does alanine do in the glucose-alanine cycle?
Carries NH3 and the carbon skeleton of pyruvate from muscle > liver
WHat happens to excess ammonia?
Added to glutamate to form glutamine by glutamine synthetase
What happens to glutamine in the liver?
NH4+ liberated by glutaminase
How does liver release collected nitrogen from glutamate?
As NH3 via oxidative deamination using glutamate dehydrogenase in the mitochondria
What does glutamate dehydrogenase use as a cofactor? What regulates it?
NAD+ or NADP+, allosterically regulated by GTP and ADP
What happens in the urea cycle?
Nitrogen input as aspartate and ammonia, plus ATP then NH3 reacts with bicarbonate to form carbamoylphosphate, this is added to ornithine to make citrulline, aspartate condenses with it +ATP to form argininosuccinate, this is cleaved to form fumarate and arginine, this is hydrolysed by arginase to release urea and ornithine to start again
What do ost excitatory neurones release?
Glutamate from alphaKG so must be replaced in TCA
How do glial cells replace carbon lost from TCA?
Take up the glutamate and recycle it back to glutamine
Why does excess ammonia in the brain deplete TCA of alphaKG?
Converts it to glutamate then glutamine
What do folic acid and methionine do?
The main 1C donors for a range of biosynthetic pathways inc DNA and RNA synthesis
How does folic acid collect 1C fragments?
Via serine hydroxymethyltransferase
WHat is the first reserve used during a sprint?
PCr
WHat is the phosphate from ATP breakdown used for? What stimulates this?
By phosphorylase to make G1P from glycogen - stimulated by Ca2+ from muscle contraction and adrenaline
What forms AMP?
Adenylate kinase
What happens to AMP?
Deaminated to inosine monophosphate which stimulates glycogenolysis, then degraded to adenosine which stimulates vasodilation
What happens during long term exercise?
Glycogen and fatty acids oxidised, fatty acids released from adipose due to hormone sensitive lipase, some fatty acid released from muscle triglyceride breakdown, fatty acids and glycogen-derived pyruvate make acetyl CoA
What happens if run out of oxaloacetate?
Convert isoleucine and valine to succinyl-CoA
Why do raised FFA levels cause insulin sensitivity?
Prevent GluT4 from reaching cell surface because FFAs inhibit IRS-1 (part of the signalling pathway)
Why is there decreased glycolysis and Krebs cycle if no insulin signalling?
Insulin signalling would normally increase it
What does higher FFAs increasing beta oxidation do?
Produces acetyl CoA and NADH which inhibit TCA, also makes alanine and lactate which causes gluconeogenesis in the liver
How do you get metabolic overload in skeletal muscle?
Fatty acid influx during overnutrition, beta oxidation with no coordinated TCA increase, by products e.g. ROS and acetylcarnitines accumulate, may activate Ser kinases which impede insulin signalling and GluT4 translocation
