Metabolism S3 - Energy Production in Carbohydrates Flashcards
What is the equation for glycerol phosphate formation?
Dihydroxyacetone phosphate (DHAP) –> glycerol phosphate Glycerol 3-phosphate dehydrogenase enzyme NADH -> NAD+
What is 2,3-BPG?
Important glycolysis intermediate. Produced from 1,3-BPG in RBC. Important regulator of O2 affinity of haemoglobin (tense state). Present in RBCs at same molar concentration as haemoglobin (~5mM)
What is the formula for 2,3-BPG formation?
1,3-bisphosphoglycerate 2,3-bisphosphoglycerate Bisphospoglycerate mutase enzyme
Describe the metabolic regulation of glycolysis
- High NADH concentration signals high energy levels i.e. low [NAD+] - Causes product inhibition of step 6 (1,3-BPG produced) - Inhibition of glycolysis due to availability of substrates
How may enzymes be regulated?
- Flux through pathway regulated in response to the need - In metabolic pathways, enzymes catalysing essentially irreversible steps are potential sites of control 1. Allostery - activator binds at another site. Proteins with 2 sites: a. Catalytic site: substrate -> product b. Regulatory sites: binding of specific regulatory molecule. Affects catalytic activity. Can produce activation or inhibition. 2. Covalent modification (phosphorylation/dephosphorylation)
Describe allosteric regulation of glucose
- Step 1: Hexokinase decreased by G 6-P (product). Allosteric inhibitory site on hexokinase. - Step 3: Phosphofructokinase-1. Muscle: PFK-1 decreased by high ATP:AMP ratio. Allosteric. ATP binds to PFK-1 and reduces amount of substrate. Liver: PFK-1 increased by high insulin:glucagon. Dephosphorylation of enzyme by hormonal signals. - Step 10: Pyruvate kinase increased by high insulin:glucagon. Dephosphorylation
What would happen if NAD+ wasn’t regenerated from NADH produced in glycolysis?
Glycolysis would stop due to product inhibition of step 6
Describe the oxidation/reduction of step 6
- NAD+ linked, 2 moles of NADH produced per mole of glucose - Pathway needs NAD+ - Total NAD+ and NADH in cell is constant, therefore glycolysis would stop when all NAD+ is converted to NADH - Normally NAD+ regenerated from NADH in stage 4 of metabolism BUT - RBC have no stage 3 or 4 of metabolism - Stage 4 needs O2 - supply to muscles and gut often reduced - Therefore need to regenerate NAD+ by some other route: lactate dehydrogenase (LDH)
What is the equation of the lactate dehydrogenase reaction?
NADH + H+ + pyruvate (CH3CO.COOH) NAD+ + lactate (CH3CHOH.COOH) LDH enzyme High levels of NADH and pyruvate NAD+ is oxidised
Describe the lactate dehydrogenase reaction
- Lactate produced by RBC and skeletal muscle (skin, brain, GI) - Released into blood and normally metabolised by liver and heart via LDH (highly oxygenated tissues) - Lactate acidifies cells - Liver and heart need NAD+ to be regenerated efficiently, usually well supplied with oxygen
Describe lactate utilisation
- Via pyruvate: NAD+ + lactate –> NADH + H+ + pyruvate LDH enzyme - Heart muscle -> CO2 - Liver -> glucose (gluconeogenesis): impaired in liver disease, thiamine vitamin deficiency, alcohol NAD+ -> NADH, enzyme deficiencies
What is glycerol phosphate?
An important intermediate in glycolysis, to triglyceride and phospholipid biosynthesis. Produced from DHAP in adipose tissue and liver. Therefore lipid synthesis in liver requires glycolysis. N.B: liver can phosphorylate glycerol directly
Describe lactate production
Produced from glucose and alanine via pyruvate. - Without major exercise: 40-50g / 24hrs. RBC, skin, brain, skeletal muscle, GI tract. - Strenuous exercise (including hearty eating): 30g/5 min. Plasma levels double in 2-5 min. Back to normal by 90 min. Pathological situations e.g. shock, congestive heart disease
How is the plasma concentration of lactate determined?
By relative rates of: - Production - Utilisation (liver, heart, muscle) - Disposal (kidney)
What are the consequences of elevations of plasma lactate concentration?
Blood concentration normally constant below 1mM. - Hyperlactaemia: 2-5mM. Below renal threshold (not in urine). No change in blood pH (buffering capacity). - Lactic acidosis: above 5mM. Above renal threshold (in urine). Blood pH lowered
Give an overview of the metabolism of galactose and fructose
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What is sucrose?
Fructose and glucose
Where does fructose metabolism occur?
In the liver (soluble enzymes)
Give an outline of fructose metabolism
Fructose –> Fructose-1-P –> 2-glyceraldehyde-3-P (enters glycolysis) 1st step: fructokinase enzyme, ATP -> ADP 2nd step: aldolase enzyme
What is the clinical importance of fructose metabolism?
- Essential fructosuria: fructokinase missing. Fructose in urine, no clinical signs. - Fructose intolerance: aldolase missing. Fructose and fructose-1-P accumulate in liver -> liver damage. Treatment = remove fructose from diet
What is fructose?
Cane/beet sugar
What is galactose?
Milk sugar
What is lactose?
Glucose and galactose
Where does galactose metabolism occur?
In the liver (major tissue) - soluble enzymes
Outline galactose metabolism
Galactose –> galactose 1-P –> glucose 1-P –> glycolysis 1st step: galactokinase enzyme. ATP -> ADP 2nd step: galactose-1-P uridyl transferase enzyme. UDP glucose UDP galactose (reverse reaction uses UDP-galactose 4’-epimerase enzyme) UDP glucose acts catalytically
What is UDP-glucose?
Activated glucose. High energy bond to glucose
What is the clinical importance of galactose metabolism?
Galactosaemia - 1 in 30,000 births
Give a brief overview of galactosaemia
Milk rich diet in infancy. Unable to utilise galactose. Problem as galactose enters other pathways. Depletes lens of NADPH -> structure damaged -> cataracts. Accumulation of galactose-1-P affects liver, kidney, brain. Treatment is a lactose-free diet
What are the two forms of galactosaemia?
Galactokinase deficiency (rare) - galactose accumulates Transferase deficiency (common) - galactose and galactose-1-P accumulate
What is the formula for the reaction that occurs in galactosaemia?
Galactose –> Galactitol Aldose reductase enzyme NADPH –> NADP+
Describe galactosaemia in detail
Raised galactose concentration enters new pathways. Depletes NADPH levels in lens. Prevents maintenance of free sulphydryl groups on protein-free cysteine R state - NADPH keeps them in reduced form. Inappropriate disulphide bond formation - cross-linking of proteins leads to precipitation out of solution. Loss of structural and functional integrity of some proteins that depend on free -SH groups e.g. lens of eye
Outline the pentose phosphate pathway
Glucose -> G-6-P -> 5C sugar phosphatases (NADP+ -> NADPH, Co2 released - irreversible decarboxylation) —-> F-6-P OR G-3-P –glycolysis–> pyruvate lactate All enzymes in cytosol Two stage cytoplasmic pathway
Give the two stages of the pentose phosphate pathway
A) Oxidative phosphorylation: Glucose-6-P —> C5 sugar + CO2. NADP+ –> NADPH. Enzyme is glucose-6-P dehydrogenase. B) Rearrangement to glycolysis intermediates. 3 C5 sugars —> 2 Fructose-6-P + glyceraldehyde-3-P. 1. No ATP produced. 2. Loss of CO2, so irreversible. 3. Controlled by NADP+/NADPH ratio at G-6-P dehydrogenase
What are the functions of the pentose phosphate pathway?
1) Produces NADPH in cytoplasm a) Biosynthetic reducing power e.g. lipid synthesis therefore high activity in liver and adipose tissue. b) Maintain free -SH (cysteine) groups on certain proteins. Prevents oxidation to -S-S- (disulphide bonds), maintains structural integrity of proteins 2) Produce C5 sugar for nucleotides needed for nucleic acid synthesis. Therefore, high activity in dividing tissues e.g. bone marrow
Describe what occurs in glucose-6-phosphate dehydrogenase (G6PDH) deficiency
- Pentose phosphate pathway has an important role in providing NADPH to maintain SH group of proteins in a reduced state - Structural integrity and hence, functional activity of some proteins depends on free -SH groups. -G6PDH deficiency is a very common inherited defect - e.g. in RBC, decreased NADPH leads to disulphide bond formation, which leads to aggregated proteins - Heinz bodies - causing haemolysis (RBC breakdown) causing anaemia - In lens of eye, causes cataracts
What happens at the end of stage 2?
Pyruvate does not enter directly into stage 3 (tricarboxylic acid cycle). Pyruvate dehydrogenase
What is the reaction for pyruvate dehydrogenase (PDH)?
Stage 2 -> 3 CH3COCOOH (pyruvate) + CoA + NAD+ –> CH3CO~CoA (acetyl CoA) + CO2 + NADH + H+ Irreversible so key regulatory step Pyruvate cannot be formed from acetyl CoA Subject to multiple regulation
Where does the PDH reaction occur?
Mitochondrial matrix - pyruvate transported from cytoplasm across mitochondrial membrane
Describe pyruvate dehydrogenase (PDH)
A large multi-enzyme complex (5 enzymes). Different enzymes require various cofactors (FAD, thiamine pyrophosphate and lipoic acid). B-vitamins provide these factors, so reaction is sensitive to vitamin B1 deficiency (can’t process products of glycolysis)
What does PDH deficiency lead to?
Lactic acidosis
What are alternative names for the tricarboxylic acid (TCA) cycle?
Krebs cycle Citric acid cycle
Where does the TCA cycle take place?
Mitochondria
What does the TCA cycle produce?
Some energy (as ATP/GTP). Also produces precursors for biosynthesis
What is the significance of oxaloacetate?
It is the start and end of the TCA cycle
What forms of phosphorylation occur in the TCA cycle?
4 oxidative phosphorylation steps 1 substrate level phosphorylation step
Give an overview of the TCA cycle
- Other pathways feed in and out of this pathway - Single pathway - Acetyl (CH3CO-) converted to 2CO2 - Oxidative (requires NAD+, FAD) - All carbons that enter as glucose leave as CO2 - Central pathway for catabolism of sugars, fatty acids, ketone bodies, amino acids, alcohol - Strategy is to produce molecules that readily lose CO2 - Breaks C-C bond in acetate (acetyl~CoA) carbons oxidised to CO2 - Intermediates act catalytically - no net synthesis or degradation of TCA cycle intermediates alone
What is the overall equation for one TCA cycle?
CH3CO~CoA + 3NAD+ + FAD + GTP + Pi + 2H2O –> 2CO2 + CoA + 3NADH + 3H+ +FADH2 + GTP
What are the products of the TCA cycle from one glucose molecule?
Glucose –> 2 x 2C into TCA –> 6NADH + 2FADH2 + 2GTP
Describe the regulation of the TCA cycle
By an irreversible step By energy availability e.g. ATP/ADP ratio and NADH/NAD+ ratio
Describe regulation of TCA cycle by the isocitrate to alpha-ketoglutarate step
- Isocitrate dehydrogenase enzyme - CO2 out - NAD+ (+ADP) –> NADH (-NADH, ATP) - Regulated by ADP when energy low
Describe the regulation of the TCA cycle by the alpha-ketoglutarate to succinyl-CoA step
- alpha-ketoglutarate dehydrogenase enzyme - CoA in - CO2 out - NAD+ –> NADH (- NADH, ATP, succinyl-CoA) - Inhibited by product
Describe the ways in which the TCA cycle supplies biosynthetic processes
Releases energy and interconverts substrate molecules
Give energy account for glycolysis and TCA cycle
- ATP from glycolysis -> (4ATP) 2ATP net (=-62kJmol^-1) TCA cycle -> 2GTP=2ATP (-62kJmol^-1) Total from substrate level phosphorylation = -124kJmol^-1 2. Still need to account for -2746kJmol^-1 3. Chemical bond energy of electrons in NADH and FADH2 4. High energy electrons in NADH and FADH2 transferred to oxygen with release of large amounts of energy - used to drive ATP synthesis
Give an overview of catabolism stage 4 (oxidative phosphorylation)
- Mitochondrial - Electron transport and ATP synthesis - NADH and FADH2 re-oxidised - O2 required (reduced to H2O) - Large amounts of energy (ATP) produced
Describe the use of reducing power in ATP synthesis
Two processes: 1. Electrons on NADH and FADH2 transferred through a series of carrier molecules to oxygen (ELECTRON TRANSPORT). Releases energy in steps 2. Free energy released to drive ATP synthesis (OXIDATIVE PHOSPHORYLATION)
Describe the membranes of a mitochondrion
Outer membrane is leaky Inner membrane is impermeable, especially to H+ ions
Describe the final electron acceptor
Oxygen. Captures electrons on O2. Use to form H2O -> 6H+ total
Describe electron transport
- Electrons transferred through a series of carrier molecules (PTCs - protein translocating complexes, mostly within proteins), to O2, with release of energy - Approx 30% energy used to move H+ across membrane (a lot of energy is released as heat) - [H+] gradient (membrane potential) across inner mitochondrial membrane - positive relative to outside = proton motive force (pmf)
Describe the role of proton translocating ATPase
Aka F1F0-ATPase or ATP synthase/synthetase ATP + 2H+(mitochondrial matrix) ADP + Pi + 2H+(cytoplasm) Reversible - becomes ATP synthesis
Describe ATP synthesis
- Return of protons is favoured energetically by the electrochemical potential (electrical and chemical gradient) - Protons can only return across membrane via ATP synthase and this drives ATP synthesis
What is oxidative phosphorylation?
Electron transport coupled to ATP synthesis
Describe oxidative phosphorylation
- Electrons transferred from NADH to FADH2 to molecular oxygen - Energy released used to generate proton gradient: pmf - Energy from dissipation of proton motive force is coupled to synthesis of ATP from ADP
Describe the difference in energy production of oxidative phosphorylation when NADH and FADH2 are used
- Electrons in NADH have more energy than in FADH2, so NADH uses 3 PTCs, FADH2 uses only 2 - Greater pmf -> more ATP synthesised - Oxidation of 2 moles of NADH -> synthesis of 5 moles ATP - Oxidation of 2 moles of FADH2 -> synthesis of 3 moles ATP - So just NADH -> more energy
Describe the regulation of oxidative phosphorylation
- Normally oxidative phosphorylation and electron transport are tightly coupled - Both regulated by mitochondrial [ATP] - High ATP = low ADP - When [ADP] is low, no substrate for ATP synthase so inward flow of H+ stops - [H+] in intermitochondrial space increases, preventing further H+ pumping: stops electron transport - Reverses with low [ATP]
Describe inhibition of oxidative phosphorylation
- Inhibitors (prefers poisons as bind straight on) block electron transport e.g. cyanide prevents acceptance of electrons by terminal translocating oxygen - Pmf decreases, less energy for ATP synthesis -> ATP levels fall -> lethal
Describe the uncoupling of oxidative phosphorylation
- Uncouplers (e.g. DNP, fatty acids) increase permeability of membrane to H+ - H+ enters mitochondria without driving ATP synthase - Dissipates pmf - No phosphorylation of ADP - No inhibition of electron transport -> continues -> energy released as heat
Describe the effect of oxidative phosphorylation diseases
Genetic defects in proteins coded by mitochondrial DNA (some subunits of PTCs and ATP synthase) lead to decrease in electron transport and ATP synthesis
Describe the effect of oxidative phosphorylation coupling
- NADH/O: 142kJmol^-1 lost. FADH2/O: 105kJmol^-1 lost - Rest of energy lost as heat - Efficiency depends on tightness of coupling - Can be varied in some tissues e.g. brown adipose tissues
Describe brown adipose tissue
- Contains thermogenin (UCP1): naturally occurring uncoupling protein - In response to cold, noradrenaline: 1. Activates lipase which releases fatty acids from triacylglycerol 2. Fatty acid oxidation -> NADH and FADH2 -> electron transport. Fatty acids activate UCP1 3. UCP1 transports H+ back into mitochondria so electron transport is uncoupled from ATP synthesis. Energy of pmf released as extra heat - Family of UCPs: role in heat generation by uncoupling but may have other functions
Where is brown adipose tissue found?
- Newborn infants (higher proportion): to maintain heat, especially around vital organs. Small body and large surface area 2. Hibernating animals (metabolism turned down): to generate heat to maintain core body temperature
Compare and contrast oxidative phosphorylation and substrate level phosphorylation
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