SDH lab: Subcellular fractionation Glucokinase – regulation : by sequestration in the nucleus. The protein inhibitor of Devices for assessing cellular respiration: clark ele
Marker enzyme: an enzyme that is unique, only found in a specific cellular localisation. • Pellet and supernatant, homogenate vs fractions hexokinase IV is a nuclear binding protein that draws hexokinase IV into the nucleus monitoring of ETC complexes (fluorimetry)
A powerful tool used when assess, for example, subcellular function experiments. Typical animal cell (lymphocyte): 10um. when the fructose 6-phosphate concentration in liver is high and releases it to the cytosol OCR= gradients calculated from the conc of oxygen af
Localisation: Preparation of homogenate: physical shear: Teflon pastle, glass homogeniser. Potter when glucose conc is high TCA cycle: to generate NADH and FADH2
Elvehjem. Hepatocarcinoma: overexpression of other isoforms. Overview:
Considerations: low temp, neutral pH, isotonic solution. Reaction 2: conversion of Gluc-6P to Fru-6P by phosphoglucose isomerase. The Energy is stored
Narrow gap between glass and pestle. Suspension passes through gap. Creates shear isomerisation of an aldose to a ketose. Readily reversible reaction. In molecules
force. Cell balloon analogy: squeezed and bursts. Organelles smaller and therefore not Reaction 3: phosphorylation of Fruc-6P to fructose 1,6-bisphophate. Like NADH and
damaged. Phosphofructokinase (PFK à central role In control of glycolysis: rate determining step) ATP. This chem
Low temp + pH: ideal conditions needed for when cells bursts so that proteins requires Mg2+-ATP complex. Ical energy is
Specific enzyme activity: activity of an enzyme (nmoles of product produced per unit of organelles not altered. Lysosomes could split and release proteases. pH and temp Reaction 4: catalysed by aldolase, cleavage of F-1,6BP to form 2 trioses. GA3P, DHAP. Used to do
time and per mg of total protein). A measurement often used to assess the purity of minimises their potential damaging action. Only GA3P continues along glycolytic pathway: ‘suction effect’. Chemical work.
cellular functions. Solubilisation, latency, low recovery of enzyme. Aim of homogenisation – maximum cell breakage with minimum effect on cell Reaction 5: GA3P and DHAP are interconverted by an isomerisation reaction. Triose Metabolism:
In the lab: Lyse some cells (chronic myeloid leukaemia). Intracellular proteins will be organelles. phosphate isomerase interconverts GA3P & DHAP efficiently. Under steady state, as Cells use enzym
released into the lysis buffer. Quantify succinate dehydrogenase activity. Hyper (cells/organelles shrink) , iso (otherwise can affect cells/organelles unless GA3P consumed, more DHAP converted to GA3P. To capture
Enzyme activity: succinate + FAD à fumerate + FADHs. controlled. This solution is needed), hypotonic (cell/organelles swell). If needed to Energy investment in glycolysis: glucose is phosphorylated (HK & PFK-1) consuming 2 Chemical energy
Calculate change in absorbance measurement from T (initial) to T (final) for the separate OMM from mitochondria use hypertonic sol to shrink organelles (inner ATPs. Yield: 2 x glyceraldehyde-3-phosphate From nutrients
samples. membrane shrinks) then into water (hypotonic) OMM swells and bursts à separated. Energy harvesting phase: generates 4 molecules of ATP (each reaction is performed 2x In a gradual
Fractionation of homogenate: differential centrifugation, density gradient since there are 2 molecules of GA3P. Products at the end of this phase: 2 molecules of Manner.
centrifugation pyruvate, 4ATP, 2NADH. Cell greatly redu
Centrifugation and centrifugal force. Centrifugation & relation of sedimentation Reaction 6: oxidation and phosphorylation of GA-3P. Requires coenzyme NAD+ and Pi ce wasting
catalysed by GAPDH. 1,3 BPG (acyl phosphate). First high energy intermediate of Energy in form
glycolysis. of heat.
Reaction 7: phosphoglycerate kinase: first ATP generated. Reactions 6&7 example of an Enzymes store
unfavourable reaction coupled to a favourite reaction so both reactions proceed. 1,3 Chemical energy
BPG is a common intermediate (consumption by PGK pulls GAPDH reaction forward). Into NADH
Measuring SDH activity: Use a 96 well plate and decide which wells will be used in the Production of ATP does not require O2 à example of substrate level phosphorylation (high energy
assay. Add DCIP. Add SDH buffer. BCD assay could be used to measure the amount of (SLP). Since 2-GA-3-P enter 2nd phase of glycolysis, 2 ATP produced at this point. Electrons)
protein present in the supernatant (homogenate) Reaction 8: phosphoglycerate mutase (isomerase enzyme) interconverts 3- And ATP
Regulation of Metabolism Balance tubes in centrifuge phosphoglycerate. Mutase forms a 3PG-phosphoenzyme complex. Complex (chemical energy)
Studying metabolism à purification of proteins Analysis of marker enzymes: decomposes to form 2PG and regenerate free mutase. In reversible reactions, the differenced in free energy b
• Analysis of marker enzymes in sub-fractions is necessary to confirm resolution and Reaction 9: enolase forms the second high energy intermediate PEP. 2 PG is dehydrated is small. The opposite is true. A + B ß à C reverse dire
Methods to study metabolism: recovery of subcellular organelles. Quantitative data can be used to assess cross (H2O removed) to PEP by enolase. products concentration (thermodynamic control). A +
• Subcellular fractionation contamination of fractions due to the protein itself or activity. Reaction 10: pyruvate kinase generates the second ATP by coupling PEP cleavage to ATP by increasing products concentration.
• In vivo: use of radioisotopes Conclusions: determination of marker enzymes in fractions can show the effectiveness synthesis. PK requires Mg2+, K+. Example of SLP. Free energy (delta G) changes during glucose meta
• Purification (solubility, charge, of the fractionation method used à purity. The method can then be used to identify the Overall pay-off: 2 ATP + 2NADH chemical reaction in which the standard change in free
size, binding abilities) location of enzymes in subcellular organelles à purity + percentage cover. Relevance of oxygen: this pathway has not used O2. Glycolysis can occur under absorbed, indicating a non-spontaneous, unfavourabl
• Location Glycolysis à splitting sugars: catabolism of glucose to yield 2 molecules of pyruvate. anaerobic conditions. Pyruvate produced is the molecule that enters citric acid cycle. In chemical reaction where the change in the free energy
• 3D structure: X-ray crystallography ATP is confused and produced at different steps. ATP is consumed in the early steps of absence of oxygen, pyruvate can get converted into lactate. energy),indicating a spontaneous reaction.
à active site glycolysis to initiate the breakdown of glucose. ATP is produced through substrate-level- Connections with other metabolic pathways: phosphoglucomutase converts it to TCA cycle: redox reactions: the complete citric acid
Proteome: reflects the biological phosphorylation. NAD+ is reduced to NADH during the conversion of glyceraldehyde-3- glucose-1-phosphate, which is then used in the formation of glycogen. Liver and kidney Reaction 1: Citrate synthase initiates cycle: attaches
processes occurring in a cell or phosphate to 1,3-bisphosphoglyverate in glycolysis. This reduction reactions helps in only: glucose-6-phosphatase removes the phosphate group, liberating free glucose into Reaction 3: loading first NAD+ with two electrons. Isoc
organism and provides a comprehensive carrying electrons to the ETC. the blood. Can be used in pentose phosphate pathway à nucleotide synthesis NAD+. As a consequence of this electron transfer, a co
understanding of how genes translate • Glycolysis provides Cellular respiration release. Carbon skeleton 4 à 6 à 5
into functional entities. Energy and intermediates for other pathways. It takes place in the cytosol. For RBC, it is Chemiosmotic theory: reduce substate (fuel) donates e-. Electron carriers pump H+ out Reaction 4: loading second NAD+ with two electrons.
the only energy source. as electrons flow to O2. Energy of e- flow stored as electrochemical potentials. ATP (hydride ion) to NAD+. Because of this electron transfe
Homeostasis: maintenance of internal stability --> deregulation: disease or even death. Glucose à monosaccharide formed under prebiotic conditions. Exist in the ring synthase uses electrochemical potential to synthesise ATP. a second CO2 release. Produces high energy intermed
The activity of enzymes has to be regulated so metabolic pathways functions when formation. Diet: amylase, maltase, lactase. Some on the surface of the intestinal cells, Rotenone (I): Isoflavone compounds used as pesticide (rodents à rotenone). Link to Carbon skeleton 4à6à5à4. Activator Ca2+
required, avoiding a waste of energy. monosaccharides transported into the cells lining the intestine and then into the Parkinson’s disease. Acts as an inhibitor of the electron transfer from iron-sulfur centres Reaction 6: loading 2 electrons into FAD+. Succinate d
Regulatory strategies bloodstream. in Complex I to the ubiquinone. Decreases mitochondrial oxygen consumption FADH2 (analogue to NADH: an electron carrier with ele
Extracellular signal à transcription of specific genes à mRNA degradation à mRNA GLUT 1-12: facilitated diffusion of glucose. 1: mammalian tissue. 2: liver, pancreatic Malonate (II) : competitive inhibitor of the succinate dehydrogenase. The enzyme is part dehydrogenase is bound to inner mitochondrial memb
translation on ribosomes à protein degradation (ubiquitin; proteasome) à Enzyme cells, intestine. 3: brain (neuronal). 4: muscle, heart and fat cells. 5: small intestine, of the complex II in the ETC (p side= intermembrane space. n side= matrix). Prevents mitochondria-marker during the subcellular fractionat
sequestered in subcellular organelle (endoplasmic reticulum) à enzyme binds to testis, kidney, sperm. Isoform specificity (different affinities for glucose). Regulation by oxidation of succinate to fumerate, blocking entry of electrons from FADH2 into the ETC Reaction 8: leading third NAD+ with 2 electrons Malat
substrate à enzyme binds ligand (allosteric effector) à enzyme undergoes hormones: link blood sugar levels to cellular metabolic reactions à e.g. high insulin leading to a reduction in the electrons flow resulting in decreased oxygen consumption. oxaloacetate. OH group of malate donates electrons (
phosphorylation/dephosphorylation. levels results in increased rate of glycolysis. Complex I (NADH oxidase): hydrophilic, hydrophobic and peripheral arm. produced. The 4-carbon skeleton is converted into the
• Control the amount of enzyme present (Microarray and RNA sequencing) Energy-investment phase: requires use of 2 ATP molecules. Effect of ETC on Mitochondrial oxygen consumption: Inhibition of ETC complexes I-IV, further recycling.
• Allosteric control, multiple forms of enzymes, reversible covalent decreases O2 consumption by blocking electron flow. Uncoupling agents: increase O2 Hydride name given to a negative ion of hydrogen. The
modifications, proteolytic activation. consumption by dissipating proton gradient causing ETC to work harder. Stimulatory consisting of 2 electrons and a proton. Electrons trans
• Microarray: relative intensity = expression levels. Low sensitivity, low dynamic compounds increases O2 consumption by enhancing electron flow through ETC. a single atom in NADH. NAD+ to NADH is reduction, op
range known transcript only, no alternative splicing information, lower cost. Antimycin A ( III) : generated by secondary metabolism streptomyces ( bacteria ). Used TCA regulation: controlling activity of, Pyruvate dehyd
• RNA-seq: sequencing reads = expression levels. High sensitivity, high dynamic as pesticide. Binds to Qi site of Cytochrome C reductase à inhibition of oxidation of NADH) , Isocitrate dehydrogenase (activated by ADP a
range, novel transcripts sequences identified structural variation and alternative ubiquinol ketoglutarate dehydrogenase (inhibited by NADH and
splicing revealed, unlimited sample comparisons. Products at the end of this phase: 2 molecules of glyceraldehyde-3-phosphate Q cycle complex III: antimycin A is an inhibitor of Complex III in ETC. Decreased oxygen activate while ATP, NADH, citrate (ADP activate while
Allosteric control: distinct regulatory sites + multiple functional sites. Glucose à glucose6-phosphate: couples the phosphorylation of glucose to the consumption. Allosteric regulation refers to the modulation of the ac
Cooperativity: activity at one site affects others hydrolysis of ATP. Enzyme: hexokinase (1-3), or in liver glucokinase (hexokinase IV). Cyanide(IV): C double bond N. Sodium or potassium cyanide salts. Highly toxic – potent ligand to a site different to the one bound by the subst
Information transducers à feed-back mechanisms. Glucose + phosphate à glucose-6-phosphate + H2O irreversible inhibitor of the cytochrome C oxidase (complex IV). promote the activity of the enzyme ( activator) or inhib
Multiple forms of enzymes: isoenzymes: slightly different in structure. Different Km and ATP + H2O à ADP +Pi . Coupled reaction: glucose + ATP à glucose-6-phosphate + ADP Carbon monoxide & Nitric oxide(IV): competitive inhibitor of O2 at the active site of the Electron Transport Chain – deoxidises coenzymes lea
Vmax. Different tissue or organelle or at different development. Fine tuning of Reaction 1: transfer of phosphoryl group from ATP to glucose. Heoxkinase cytochrome C oxidase. Electron transport chain (ETC) refers to a series of prot
metabolism. phosphorylates kexoses. Requires Mg2+-ATP complex. Mg2+ essential for kinase activity. Oligomycin(V): Macrolides antibiotics, synthetised by Streptomyces Potent. Inhibitors of transfer electrons from electron donors to electron ac
Covalent modifications: most of them are reversible. Uncompleted ATP is a inhibitor of hexokinase. Mg2+ shields the negative charges the mitochondrial ATP Synthase (ATPase, Complex V). Decreases oxygen consumption NADH(reductant) + H+ + ½O2(oxidan
Activation/inhibiton: phosphorylation/dephosphorylation: irreversible reaction --> Ser/Thr making the g-phosphorus atom more accessible for nucleophilic attack. FCCP (Proton gradient): 2-[2-[4-(trifluoromethoxy) phenyl] hydrazinylidene]- Coenzyme oxidation and redox potentials
or Tyr, localisation, degradation Hexokinase: glucose induces a conformational change in hexokinase. Conformation propanedinitrile. Liposoluble compound à cross biological membranes Ionophore
Proteolytic activation: zymogen or proenzyme. Occurs just one. Examples: blood places ATP close to the group in glucose C6 and excludes water from active site. Allows (Protons H+). Uncoupler. Increases oxygen consumption
clotting, digestive enzymes, programmed cell death. phosphoryl group transfer.
, Molecules along the ETC have As a result of its strong exergonic nature of the citric acid cycle (energetically downwards, The rationale behind pyruvate kinase allosteric control :upstream regulation by ATP hydrolysis: The human brain
reduction potentials between the favourable negative free-energy, DG-), it is not possible to regulate it through changes in fructose 1,6-biphosphate. Reaction 3 controls reaction 10. ATP: electrical repulsion due to multiple negative
values of the NAD+/NADH couple and intermediates concentration (i.e. main reactions are far from equilibrium). • Therefore, Another allosteric regulator: L-alanine: Conversion of pyruvate into L-alanine allows the molecule.
the oxygen/H2O couple. so electrons direct regulation of the enzymes is required. • This is achieved by allosteric control of the cell to use pyruvate as the raw material to synthetize amino acids needed for protein Glycogen metabolism:
move down the energy scale towards enzymes involved in the most important steps (i.e. NADH and CO2 producing steps, synthesis Glycogen: Readily mobilised storage form of gluc
progressively more +ve reduction succinyl-coenzyme step). ATP synthesis: ATP synthesis: ADP + phosphate (Pi) Present in the cytoplasm in the form of hydrated
potentials - electrons move through 4 Ethylene: 2 carbon atoms showing sp2 hybridisation come together. Covalent bond made ATP: electrical repulsion due to multiple negative charges. Unstable, high-energy polymer of glucose residues Most glucose units a
redox protein complexes. possible by sharing 2 electrons. Double bond C=C = 1 pi + 1 sigma bond. molecule. ATP hydrolysis produces free energy. ATP synthesis requires free energy. Branches are created by a(1-6) glycosidic bonds
Electrons pass from lower to higher Another way of harnessing high energy electrons: fatty acid oxidation. Beta oxidation of ATP synthase: a molecular machine. Peripheral stalks (stator) where Pi + ADP à ATP = F1 Degradation of glucose: Requires: 1. Release of
reduction potentials. fatty acids Bring Pi and ADP into a planar transition state Remodelling of glycogen to allow further degrada
4 complexes of the ETC: Redox nature of the citric acid cycle: The citric acid cycle is an elaborated redox reaction 1. Proton transfer causes central stalk to rotate due to torque effect. glucose -6P.
Components of the ETC can be carried out in several steps. The net result is acetyl oxidation and the concomitant NAD+ 2. Central stalk rotates while peripheral stalks remain static. As a result, Requires 4 enzymatic activities.Glycogen phosph
purified from the mitochondrial inner reduction to produce NADH and CO2. Losing electrons to reduce NAD+ leads to less conformational changes take place in alpha and beta subunits. In this way, kinetic Transferase activity. Glucosidase activity. Phosp
membrane bonding capability in C atoms. This results in C atoms leaving the cycle as CO2 , causing energy (rotation) is used to produce conformational work. involved are regulated by hormones (reversible p
the shortening of the C skeleton. 3. Conformational work brings ADP and phosphate close, poising them in an optimal interactions.
Isolation of 4 protein complexes NADH-coenzyme Q reductase (I) Succinate-coenzyme Q The rationale begin being a cycle: recycling carbon atoms and its electrons. Replenishing configuration for reaction to occur Synthesis of glycogen: requires 1. Activation of
reductase(II) Coenzyme Q-cytochrome c reductase (III) Cytochrome c oxidase (IV). of the C-skeleton allows oxaloacetate to be further recycled. Energy conversion: using the H+ gradient to make chemical energy (ATP). Creation of branches
Molecular components of complexes – electron carrier molecules: Flavoproteins Steps 6-8: configuration is everything --> regenerating oxaloacetate Hydrolysis: ATP à ADP + Pi. High ATP conc, low H+ In outermembrane (for reverse Glycogenin: 2 identical subunits. Each catalysin
Tightly bound FMN or FAD (prosthetic groups) may participate in 1 or 2 e- transfer events Down regulation inhibitors : increases in the levels of key regulatory intermediates act to reaction aka hydrolysis) Glycogen synthesis: Uses UDP-Glucose as the
Coenzyme Q (ubiquinone; CoQ; UQ) 1 or 2 e- transfer reactions. Cytochromes (b, c, c1, decrease citric acid cycle activity: Inhibitory intermediates: citrate, NADH, succinyl co-A. Oxygen provides the main driving force for building proton gradient. UDP-glucose pyrophosphorylase. Glucogenin. G
a & a3) Haem (prosthetic groups) 1 e- transfer agents (Haem iron converted from Fe2+ → Buildup of NADH leads to feedback inhibition of several steps. Excess NADH = too much ATP yield from oxidative phosphorylation (oxygen). NET ATP yield per 1 glucose molecule: is regulated by hormones
Fe3+ → Fe2+). Fe-S proteins 1 e- transfer (Fe2+ and Fe3+ states). Protein-bound Cu2+ 1 pyruvate & glucose. Substrate level: 4. Plus oxidative 26-32. Total = 30-36 ATP molecules. Regulation of carbohydrate metabolism:
e- transfer sites (Cu+ → Cu2+). Up regulation activators: ADP is the precursor for ATP. Too much ADP = little ATP. ADP Proton gradient created in mitochondria is used for sperm propulsion Glycolysis – gluconeogenesis regulation: regu
The complexes: Complex I - Accepts 2e- from NADH (link between glycolysis, TCA, β- levels to signal for activation. E.g. step 3 & 4 ATP and Brain function reactions for each pathway
oxidation and ETC). Complex II Includes succinate dehydrogenase (direct link between Activation by calcium: Calcium activates pyruvate dehydrogenase phosphatase which in ADP: adenosine triphosphate. Electrical repulsion due to multiple negative charges. Glucose to Glucose 6-P : Hexokinase activity de
TCA and ETC). As 2e- flow from NADH to CoQ, 4H+ are pumped out across IMM. turn activates the pyruvate dehydrogenase complex. Calcium also activates isocitrate Unstable, high-energy molecule. Hexokinase IV has less affinity for glucose: is satu
NADH-coenzyme Q reductase: more than 30 polypeptide chains. 7 Fe-S clusters. dehydrogenase and α-ketoglutarate dehydrogenase. This increases the reaction rate of Testing reverse rotation caused by ATP hydrolysis: replacing F2 by a fluorescent protein inhibited allosterically by Glucose 6-P.
Complexes I and II produce a common product: Reduce coenzyme Q – substrate for many of the steps in the cycle, and therefore increases flux throughout the pathway. ATP needed in a single day: 50 to 75kg. High ATP consumption is a not matter of human Hexokinase IV and glucose 6-
complex III. Flavoprotein 1: NADH dehydrogenase FMN, Fe-S centres. Flavoprotein 2: Regulation of the citric acid cycle: Indirect control of enzyme activity by shifting the metabolism being especially fast. phosphatase Regulated by
succinate dehydrogenase, FAD (covalent), Fe-S centres, b-type haem.Only TCA enzyme equilibrium between reactants and products’ concentrations is not feasible. Therefore, Human brain: Glucose is the main energy source for the brain. Glucose is used to produce localisation Regulated by transcriptional regulat
is an integral membrane protein in IMM. 3 Fe-S clusters, 2 small subunits, with haem b direct allosteric regulation is required. ATP, through oxidative phosphorylation. This explains its high level of oxygen consumption Glucagon increases glucose 6-phosphatase
and bind UQ Electron transport chain as well. Human brain: energy consumption. Human brain is the most energy-demanding Hexokinase in liver:
Complex III: Complex III Oxidises UQH2 while reducing cytochrome c (Cytochrome c is Chemical work à required to drive reactions which are non-spontaneous i.e. organ known in nature. Its energy requirement is out of proportion with its size in relation to The protein inhibitor of hexokinase IV
the substrate for Complex IV). Cytochrome bc1 complex 2 b-type haems, Rieske Fe-S unfavourable, positive free energy delta G the entire human body. is a nuclear binding protein that draws hexokina
centre c-type haem (cyt c1). Cristae: existence of suitable compartment in mitochondria enables the accumulation Energy consumption: The high-maintenance nature of human brain increases its 6-phosphate concentration in liver is high and rel
Complex IV: reduces molecular oxygen. Cytochrome aa3 complex 2 a-type haems Cu and use of a H+ gradient to drive ATP. vulnerability. If the energy supply is cut off for 10 minutes, there is permanent brain concentration is high.
ions. Consists of 13 subunits Redox potential: Is the tendency of a molecule to acquire electrons and thus be reduced. damage. There is no other organ nearly as sensitive to changes in its energy supply. Linear Regulation of production of F2, 6bp: single enz
UQ/UQH2 and cytochrome c are mobile electron carriers à efflux of protons driven by A molecule’s redox potential value depends on having one or more electron orbitals able correlation between brain weight and body weight by hormones. Both enzymes' activities are part o
complex I,III and IV to accept or donate electrons The more positive a redox potential is, the greater the Where is the ATP used up in the brain? Active K+ transport, Glutamate signalling reciprocally regulated by insulin and glucagon.
Other enzymes also supply e- to UQ: -sn-glycerophosphate dehydrogenase (IMM- orbitals affinity for electrons (tendency to become reduced) The more negative a redox 1. Active K+ transport into neurons: Enzyme called Na+K+ ATPase is an ATP Fru 6-P to Fru 1,6-bisP: Regulation by allosteric
bound shuttle enzyme) .Fatty acyl-CoA dehydrogenases (3 soluble matrix enzymes potential is, the greater the orbitals propensity to donate electrons (tendency to become synthase working in reverse gear. (citrate, xylulose) In liver: Clear example of horm
involved in β-oxidation oxidized). Conformational changes caused by ATP hydrolysis results in ATPase changing its affinity gluconeogenesis by using F2,6BP as the allosteri
Coenzyme Q: mobile electron carrier, highly hydrophobic, diffuses freely in the Oxygen is the most electropositive electron acceptor in biology. Electrons flow towards towards Na and K ions. This mechanism is similar to the one operating in the protein Pyruvate kinase regulation: Abundance of ener
hydrophobic core of IMM. Can take part in 1 or 2 e- reactions. positive potentials. As a result of electron flowing, H+ (protons) are pumped. complexes of the electron transport chain energy supply (glucagon signalling): Activation of
Cytochrome C: mobile e- carrier, H2O soluble. Globular; haem group lies in the centre 2. Glutamate signalling: Glutamate mediates most of the excitatory signalling in human kinase isozyme L Isozyme M (prevalent in muscle
of the protein. Associates along the membrane surface in its reduced state. Carries e- to Ubiquinone: a brain. Glutamate opens cation channels, thus, activating the postsynaptic membrane. converted to glucose and glycogen via gluconeog
the 4th complex (cytochrome c oxidase). Reduction of O2 requires passage of 4e- protein-free electron Glutamate role as a neurotransmitter includes its recycling into Glutamine. Glutamate energy production. The first enzyme in each path
Some of unused free energy released on e- transfer from cytochrome c to O2 is carrier. Ubiquinol is conversion to glutamine require energy from ATP. produced either by fatty acid oxidation or by the p
harnessed by pumping 4H+ out through proton channel – - mechanism not fully known. the entry point for Gluconeogenesis stimulates pyruvate carboxylase and inhibits pyr
Chemiosmotic Theory: proton circuit: substrate oxidation/light energy. electrons from Glucose: energy in brain, RBC cells. Precursor. PEP carboxykinase promoter region: Enzyme a
Electron transfer & metabolism FADH2 (citric acid Gluconeogenesis: Occurs in all animals, plants, fungi and microorganisms. In mammals: Hormonal signalling
Overall chemical equation: glycolysis + pyruvate oxidation + citric acid cycle. cycle). liver (and renal cortex and epithelial cells on the inside of small intestine). Synthesis from PEP to pyruvate : Abundant energy supply (high
Electrons transfer by the hydride ion takes place just from a single atom in NADH. How is electron non-carbohydrate precursors chain fatty acids) allosterically inhibits pyruvate
Electron is 2,00 lighter than proton. Billion times lighter than NAD+. flow used to Inside mitochondrion: Pyruvate to oxaloacetate. Enzyme: pyruvate carboxylase inactivated by phosphorylation: glucose oxidatio
Main types of electron transfers: A- Transfer of a hydride ion carrying two electrons H:− achieve H+ (carboxylation of pyruvate to produce oxaloacetate). CoA is an allosteric activator for pyruvate carbox
e.g. NADH B- Concomitant transfer of an electron ē and a proton H+ , e.g. FADH2 C- pumping? All the Subunit of pyruvate carboxylase: 1. Formation of carboxyl phosphate 2. Transfer to Krebs cycle) PEP carboxykinase levels are regula
Direct transfer of electrons ē complexes exist in 3 biotin: carboxybiotinyl-enzyme 3. Transfer to pyruvate Glycogen synthesis: control of the access to su
Concomitant: happening together because of something. conformation: each How to get the oxaloacetate from the mitochondrion into the cytoplasm? (I) signalling promoters: uptake and utilisation of glu
Cytochrome C is a membrane-free electron shuttle. conformation has a Generating NADH (II) Without generating Glycogen synthesis and degradation: tight regu
High-reducing power molecules: such as antioxidant vitamins. different affinity Overall reaction: different enzymes
Iron-sulfur world theory proposes early life may have formed on the surface of iron towards H+. Glycogen synthesis and degradation: Insulin si
sulfide minerals acting as catalysts in hydrothermal vents on the oceans floor. Conformational bringing it to the glycogen molecule. Adrenalin sig
Microporous in rocks would provide the kind of organisation that cell organelles do. work is induced by PP1, removing PP1 from the glycogen molecule.
Inorganic gases: CO, CO2, HCN, H2S. electrons flow. present or removed
NADH: very negative electrode potential. Strong reducing agent. NADH binding causes a Conformational work: triggered by electrons’ kinetic energy is used to induce a Phorphorylase – regulation: allosteric inhibitor:
slowing down of isocitrate and a-ketoglutarate oxidation. conformation with low H+ affinity (C). This conformational change enables H+ to be Summary: Metabolism is formed by interconnec
Regulation of citric acid cycle released into the outer membrane space, so creating high H+ concentration. being reciprocally regulated Regulation is slightly
Harnessing glucose’s energy inside the cell: energy is wastefully released into the air Electrons flow’s kinetic energy is used to create a high concentration gradient of protons use of different enzyme isoforms
as heat. No energy is harnessed or stored. Energy is stored in molecules like NADH and (H+) across the mitochondria’s inner membrane.
ATP. This chemical energy is used to do chemical work. Pyruvate: glucose to pyruvate is reversible. Pyruvate to Acetyl CoA requires pyruvate
Open vs close form Pyruvate kinase: substrate PEP present in open form induced by dehydrogenase complex. Irreversible commitment to mitochondria (energy production or Cori cycle: lactate formed by active muscle is converted into glucose by the liver
binding of regulatory molecules (allosteric control). Upstream regulation by fructose 1,6- fatty acid synthesis). Pyruvate enters mitochondria via a controlled channel. Irreversible Precursors of glucose used in gluconeogenesis: lactate, glycerol, amino acids.
bisphosphate reaction 3 controls reaction 10. commitment to mitochondria. ATP and NADH production phase: Reaction 6-10. Summary: opposite pathway to glycolysis. Non-carb precursors. Energetically expensive,
Reaction 10: pyruvate kinase generates 2nd ATP by coupling PEP cleavage to ATP synthesis but essential.
