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MCB 450 Exam 3
MCB 450
| Question | Answer |
|---|---|
| 1000s of different reactions go on in cells. | • nutrient molecules are degraded • chemical energy is conserved & transformed • macromolecules are made from simple precursors (e.g., amino acids -> protein) |
| Chemical reactions in cells | Must take place at a rate that meets a cell's needs. |
| Must be specific: | - A particular reactant should always yield a specific product - Side-reactions producing useless or toxic byproducts must be minimized |
| Reactions are | accelerated & made highly specific by enzymes. |
| Most reactions in biological systems don't take place in | the absence of enzymes |
| Highly specific enzymes: | - Provide optimal distance and orientation |
| Carbonic anhydrase action allows | transport of CO2 from the tissues to the lungs where it is exhaled |
| Tissue: | High CO2-> low pH-> low O2 binding to Hb |
| Lung: | Low CO2-> higher pH -> higher O2 binding to Hb |
| Measure of catalytic power? | rate of enzyme-catalyzed rxn/ uncatalyzed rate |
| Trypsin | Cleaves on C-terminus of LYS or ARG of basic pair (KK*X, RK*X, KR*X, RR*X |
| Thrombin | More specific than Trypsin: Only cleaves ARG-GLY bonds in a specific recognition sequence (Leu-Val-Pro-Arg-*-Gly-Ser) |
| Most enzymes | are proteins (except small group of catalytic RNAs) |
| • Catalytic activity depends on | integrity of enzyme's native conformation, and appropriate conditions (pH, temperature, Salts) |
| • Enzymes are usually present in | very small amounts (because they are not consumed in reactions) |
| • Activity of many enzymes is | regulated |
| • Many enzymes named by adding | suffix “ase” to the name of their substrate or a word describing their activity. (Ex: Glucokinase and Phosphatase) |
| • Most enzymes require | no chemical groups for activity other than their own amino acids. |
| • Some enzymes require | additional cofactors for activity |
| Higher temperatures accelerate reactions by | increasing kinetic energy and the collision frequency of the reactants in both catalyzed AND uncatalyzed reactions |
| – Too high a temperature will | denature (unfold) an enzyme pH can influence reaction rate, ionization of active site and enzyme stability |
| – Most enzymes have a characteristic | pH optimum |
| 1. Covalent catalysis: | the active site contains a reactive group (nucleophile) that is briefly covalently modified. |
| 2. General acid-base catalysis: | a molecule other than water donates or accepts a proton. |
| 3. Metal ion catalysis: | metal ions (Positive charge) can function in various ways, e.g., -stabilize a Negative charge on a reaction intermediate -generate a nucleophile by deprotonating water -bind to S, increase interactions with E, increasing binding energy |
| 4. Proximity & orientation: | Enzyme brings two substrates close together & orients the reacting parts of substrate molecules for reaction |
| 1. Oxidoreductases: | transfer electrons between molecules: catalyze oxidation-reduction reactions. |
| 2. Transferases: | transfer functional groups between separate molecules. |
| 3. Hydrolases: | cleave molecules by the addition of water. |
| 4. Lyases: | Breaks bond without hydrolysis and oxidation. |
| 5. Isomerases: | move functional groups within a molecule. |
| 6. Ligases: | join two molecules at the expense of ATP hydrolysis. |
| Catalytic activity of many enzymes depends on presence of | small molecules called cofactors (also called coenzymes) |
| • Enzyme minus its cofactor = | apoenzyme |
| • Complete catalytically active enzyme plus its cofactor = | holoenzyme |
| • Two subdivisions of cofactors: | 1. Coenzymes = small organic molecules (e.g., Coenzyme A) derived from vitamins and can serve as transient carriers of electrons or specific functional groups (e.g., CO2) 2. Metals (Mg2+, Mn2+, Zn2+ etc.) |
| • A coenzyme that is very tightly bound to an enzyme = | a prosthetic group (Heme) |
| • A coenzyme can be used by a variety of | enzymes (e.g., NAD+/NADH) |
| • Different enzymes that use a same cofactor usually carry out similar | chemical reactions |
| ACTIVATION ENERGY – | The difference in free energy between the TRANSITION state & the SUBSTRATE |
| Active site | The pocket of an enzyme lined with aa residues that bind the substrate and catalyze its chemical transformation |
| Enzyme-substrate (ES) complex -> | Lower activation energy (DG‡) |
| Electrostatic Good: | Lys+ -><- Glu- |
| (Electrostatic Bad: | Arg+ <--> Lys+ or Glu- <--> Asp- ) |
| Hydrophobic Good: | Val -><- Leu |
| (Hydrophobic Bad: | Arg+ <--> Val or Ser <--> Phe) |
| Hydrogen bond Good: | Ser -><- Glu |
| Disulfide: | Only Cys-Cys |
| Pi-Pi: | Phe/Tyr -><- Phe/Tyr |
| Pi-Cation: | Phe -><- Lys/Arg |
| Common features of active sites: | 1. Three-dimensional cleft 2. Takes up a small part of the total volume of the enzyme 3. Bind substrates, products, and transition states via multiple weak attractions 4. Structural complementarity (complementary shapes after binding) |
| The shapes of active sites are modified when | S binds |
| • Binding that is too tight makes it harder for S to | reach the transition state |
| Induced fit model of substrate binding permits formation of | additional weak binding interactions in transition state |
| • Induced fit model of substrate binding brings specific functional groups into | proper position for catalysis |
| In induced fit model of substrate binding E itself undergoes a change in | conformation upon binding S |
| 1. Many weak, non-covalent interactions are formed between | E and S (e.g., H-bonds, hydrophobic, and ionic interactions) |
| 2. Formation of each weak interaction is accompanied by | small release of free energy, contributing to stability of the interaction |
| 3. Total energy derived from ES interactions = | binding energy DeltaGB |
| 4. Binding energy | • Major source of free energy used by enzymes to lower the activation energy and so increase the rate of a reaction • Gives Enzyme its specificity for Substrate |
| 5. Weak interactions optimized when | S is in its TRANSITION STATE |
| TRANSITION STATE: | • “ACTIVATED” FORM OF MOLECULE • HAS UNDERGONE PARTIAL CHEMICAL REACTION • HIGHEST POINT ON REACTION COORDINATE |
| When S first binds, only a subset of binding interactions is used to form | ES. |
| • Bound S must still undergo an increase in | free energy to reach transition state. |
| • But now, the increase in free energy required to “bend” stick into transition state is offset by | the increased interactions (i.e., binding energy) that form between E and S in the transition state. |
| • Many interactions involve parts of S that are distant from the point of reaction, so interactions between E and the non-reacting part of S provide | some of the energy needed to catalyze “stick” breakage. |
| • The binding energy contributed by the formation of weak interactions between E and S in the transition state provides | much of the energy needed to lower activation energy. |
| An enzyme lowers the | free energy of activation for the reaction (∆G‡). (i.e., enzyme increases the reaction velocity) |
| • Lower ∆G‡ = | more molecules able to get into transition state and be converted into products |
| • An enzyme does not affect the | overall free energy change (∆G) of the reaction (and therefore, does not change the equilibrium). |
| • An enzyme accelerates the approach to equilibrium by | lowering ∆G‡. |
| Substrate concentration hyperbolic curves: | reaction rate versus substrate concentration. (– evidence of saturation of enzyme by substrate) |
| -Cooperative/Allosteric Enzymes have | a sigmoidal curve with respect to the level of substrate. |
| S = | substrate |
| E = | enzyme |
| P = | product |
| k1, k-1 and k2 are | rate constants |
| v0 = | initial reaction velocity |
| Km = | Michaelis constant (k-1+k2)/k1 |
| Vmax = | maximum velocity (k2Etot) |
| [S] = | substrate concentration |
| 1. [S] >> [E] | This means that [S]tot ≈ [S]free |
| 2. Steady-state | [ES] does not change with time |
| 3. Initial velocity (vo) is measured | Before substrate has been depleted OR product has accumulated, making the rate of back-reaction (k-2) negligible. |
| Michaelis-Menten Equation describes a | rectangular hyperbola when velocity is plotted as a function of substrate concentration |
| Km is the substrate concentration giving | half-maximal reaction velocity |
| • Km is expressed in units of | concentration (e.g., mM, μM) |
| Km describes the affinity of enzyme for | substrate |
| • Small Km means | high affinity for substrate |
| • Large Km means | low affinity for substrate |
| Enzyme Inhibitionn: | An inhibitor is a compound that interacts with an enzyme to slow the rate of an enzyme-catalyzed reaction. |
| • Reversible inhibitors bind to enzymes by | noncovalent interactions. |
| Enzyme activity recovers when | inhibitor is removed. (-> Example: Ibuprofen and COX (cyclooxygenase) enzyme) |
| • Irreversible inhibitors (inactivators) react with enzymes through | covalent bonds. |
| Enzyme activity does not recover when | inhibitor is removed. (-> Example: aspirin (acetylsalicylic acid) and COX) |
| Competitive inhibitor binds | reversibly in the active site. |
| • Inhibitor & substrate therefore compete for | access to the enzyme. |
| • Statin drugs lower cholesterol by | competing against HMG-CoA for the active site of HMG CoA Reductase |
| • HMG-CoA is not converted to mevalonate ->->-> | cholesterol |
| Competitive inhibitor effect on Km: | Increases the apparent Km for a substrate. -> More substrate (higher Km) is needed to achieve ½ Vmax |
| • Competitive inhibitor effect on Vmax: | Not affected. Inhibition can be reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of inhibitor. |
| Non-competitive inhibitor binds | reversibly to the enzyme, but at a different site than the substrate binding site. |
| • Non-competitive inhibitors can bind to E and ES | equally well. |
| • Non-competitive inhibitors change Vmax but | does not affect Km |
| • Noncompetitive inhibitor effect on Vmax: | Lowers Vmax - cannot be overcome by increasing substrate concentration. Kcat (efficiency) is reduced. |
| • Noncompetitive inhibitor effect on Km: | No effect on substrate binding à Km does not change in the presence or absence of the noncompetitive inhibitor. |
| • irreversible inhibitors covalently modify | an enzyme, destroying its activity permanently |
| 1. Group specific irreversible inhibitors – | forms covalent bond with specific aa side chain (whole inhibitor stays bound) |
| – Sarin Gas - | Inhibits acetylcholinesterase degrades the neurotransmitter acetylcholine allowing muscle to relax after contraction – Sarin blocks muscles from relaxing -> asphyxiation |
| 2. Substrate analog irreversible inhibitors – | Looks like natural substrate. Will react with Enzyme and stay bound – Triose phosphate Isomerase (Glycolysis) |
| 3. Suicide Irreversible Inhibitor – | Unusual type of irreversible inhibitor -> Enzyme modifies the inhibitor into a reactive form in the active site. |
| – Aspirin (acetylsalicylic) acetylates | Cox1 and salicylic acid leaves the pocket |
| Allosteric means | “another site” – Bind outside of active pocket |
| • Allosteric modulators can be: | – Homotropic effectors (typically the substrate itself). – Heterotropic effectors (often a downstream metabolite that feeds back to regulate the initial key step in a pathway). |
| • Allosteric agents induce | shape changes in substrate pocket |
| – Allosteric activators | enhance binding of substrate |
| – Allosteric inhibitors | reduce binding affinity |
| • In allosteric regulation, Vmax and Km | can both be affected |
| • Allosteric enzymes often deviate from | Michaelis-Menten kinetics. – Looks like cooperative binding |
| • Allosteric activators can overcome | accumulation of inhibitory factors |
| • Allosteric inhibitors can prevent | the waste of energy in making unnecessary products |
| • Metabolism is the | chemical reactions that occur in cells and organisms that required for life |
| • Metabolism is composed of | many interconnecting reactions and pathways |
| • Metabolism consists of | energy producing and energy consuming reactions |
| • Thermodynamically unfavorable reactions can be | driven by favorable ones |
| • ∆G is the | energy of a reaction that is available (Free) to do work. |
| • If ∆G < 0, free energy is | released by the reaction, termed exothermic (favorable). |
| • If ∆G > 0, energy is | required for the reaction to proceed, termed endothermic (unfavorable). |
| • ∆G approaches 0 as the reaction | proceeds to equilibrium. |
| • ∆G predicts if a reaction will | proceed spontaneously or NOT. It says NOTHING about the kinetics or reaction rate (i.e., how long will it take to complete the reaction). |
| Change in free energy | ability of a reaction to do work |
| Change in enthalpy Reflects: | • Changes in the kinds and numbers of chemical bonds and non-covalent • Interactions broken and formed during a reaction (change in heat content) |
| Change in entropy Reflects: | • Changes in a system’s randomness. • Increase in entropy is an increase in disorder (creating order from disorder takes energy, increases ∆G) |
| Glycolysis: | 1 glucose (6C) ->->-> 2 pyruvate (3C) |
| • Steps 4, 5, 6 and 8 are | unfavorable under standard conditions. (without the enzyme) |
| • BUT “actual DG” | near 0 (with the enzyme) |
| - Actual DG is because metabolic flux in a cell is influenced by | the concentration of substrates and products. |
| - High levels of substrates pushes reaction to the | RIGHT to make products. Competition for enzyme |
| To carry out thermodynamically unfavorable, or energy-requiring (endergonic) reactions, cells couple them to other reactions that liberate free energy (exergonic reactions), so that the overall process is | exergonic: the sum of the free-energy changes is negative. |
| • Cells couple energetically unfavorable reactions to energetically favorable reactions to | drive the unfavorable reaction forward. |
| - UTP is used for | adding sugars |
| • CTP for | lipid synthesis |
| • GTP for | protein synthesis |
| ATP can be: | • made from higher energy phosphates • used as a phosphoryl group donor • used to drive unfavorable reactions |
| • ATP is the principal donor of | free energy in biological systems. It is used to fuel enzymatic reactions, transport of molecules, and to do mechanical work. |
| • ATP molecules are consumed within | 60 seconds of their generation. |
| • ATP is continually regenerated via | oxidation of carbon in fuel molecules like glucose, fats and proteins. |
| Three factors in ATP play a key role: | 1. Electrostatic repulsion of negatively charged phosphate groups 2. Resonance stabilization of ADP and Pi 3. Stabilization of ADP and Pi due to hydration |
| Mg+2 coordination makes phosphorus more | electrophilic (tendency to attract or acquire electrons) |
| 1. Electrostatic Repulsion | - At pH 7 ATP carries four negative charges. - ADP has three. - Repulsion is reduced upon hydrolysis. |
| 2. Resonance stabilization | • Orthophosphate (Pi) has 4 resonance forms • ADP also has better resonance than ATP |
| 3. Stabilization due to hydration | • Water can bind more effectively to ADP and Pi than it can to the phosphoanhydride portion of ATP phosphoanhydride portion of ATP |
| However, ATP is kinetically stable because | the hydrolysis reaction has a high Ea. |
| • Lots of energy is released when | ATP is hydrolyzed. |
| An ATPase enzyme: | grabs ATP and helps it get over the Ea barrier |
| Energy from the ATP hydrolysis is used for | the chemical reaction that needs it. |
| • H2O is organized & deprotonated by Glu (or another H2O) -> | OH- becomes the nucleophile. |
| • Arginine Finger polarizes the g phosphate, Can come from | neighboring protein. |
| • Mg2+ :organizes the b & g phosphates of ATP (which shields charge); pulls on electrons to make | gamma P more electrophilic |
| The association of a ligand (ATP) to enzyme binding pocket (Walker box) induces | conformational change to close around ligand. |
| • Amino acid side chains come close enough to interact with | ligand (e.g., Arg finger = R162). -> Proximity and Orientation -> Protein activity can start (e.g., DNA binding) |
| • Mg2+ interacts with | phosphates -> Neutralizes negative charge. |
| • After hydrolysis, the enzyme pocket | opens and releases product (ADP) |
| • In some cases, the gamma phosphate is transferred to | another molecule. -> Kinase activity |
| Many reactions are allosterically activated or inhibited by ATP levels, especially those that generate energy, making ATP | not a good choice as a molecule to store in large quantity for energy reserves. |
| • Muscle cells solve this problem by | storing high-energy phosphate bonds in the form of Creatine phosphate. |
| P-Creatine | • Replenishes ATP levels when standing ATP stores are used • Bridges the gap between ATP hydrolysis and new ATP made from metabolism |
| Larger atomic size of S (as compared to O) reduces the electron overlap btw C and S, so that | the partial C=S structure does not contribute significantly to resonance stabilization. |
| Thioester is “unstable” relative to an ester, thus | releases more energy on hydrolysis. |
| • Pantothenate (Vitamin B5) is a component of | CoA & cofactor for fatty acid synthesis. |
| • Pantothenate deficiency has | not been documented. |
| • BUT Deficiency of Pantothenate kinase, the enzyme needed to charge and activate Vitamin B5 to be incorporated in CoA, results in | neurological problems. |
| Most ATP in the cell is made through Oxidation of fuels that involves | ① the transfer of electrons from substrates to NAD+ [NADH] and FAD [FAD(2H)] and ② then to the mitochondrial electron transport chain and finally, ③ the transfer of electrons to Oxygen. |
| • Another key feature of metabolism is | electron transfer in oxidation-reduction reactions |
| • Flow of e- in oxidation-reduction reactions is responsible (directly or indirectly) for | all work done in living organisms: – e.g., Oxidative phosphorylation through oxidative metabolism |
| • Oxidation-reduction reactions involve: | - Loss of e- by one chem. species à becomes oxidized - Gain of e- by another species à becomes reduced |
| Redox reaction uses NAD+/NADH as a | redox cofactor (accepts/donates electron pairs) |
| Oxidation: | Adding an oxygen (taking away e- and H+) |
| Reduction: | Adding a H+ (proton) and e |
| NAD+ generally involved in oxidation of | alcohols and aldehydes in which 2 electrons are removed FAD generally involved in oxidations in which |
| Transfer of e- from e- donor to e- acceptor in one of four ways: | 1. Direct transfer of e- 2. As H atoms (= H+ + e- ) General equation: AH2 <--> A + 2e- + 2H+ 3. e- transferred as hydride ion (:H- , which has 2 e-) 4. Direct combination with O2 — e.g., oxidation of hydrocarbon to alcohol: R-CH3 (e donor) + ½O2 ( |
| Pellagra is caused by a | chronic lack of Niacin (vitamin B3 or nicotinic acid) |
| • If oxygen is NOT available, the energy from oxidation of fuels (electrons carried by NADH and FAD(2H)), cannot be | transferred to oxygen, and most ATP production will cease. |
| Only anaerobic glycolysis can proceed in the absence of Oxygen, which produces very little ATP (2 ATP) in comparison to | the oxidation of glucose through oxidative phosphorylation (30-32 ATP). |
| 1. Plasma membrane (PM) components: | • Lipids, Proteins and Sugars. |
| 2. PM is a a selective barrier | • restricts entry and exit of compounds from cells. |
| 3. PM allows cell-cell interactions through | specific ligands/receptors. |
| 4. Transporters - | allow the concentration (or exclusion) of compounds, and the establishment of electrical potentials. |
| 5. Organelle membranes (inside of PM) | • compartmentalization of biochemical reactions. |
| 6. Membranes permit the two-dimensional organization/ acceleration of | biochemical reactions compared to similar reactions in (3D) solution. |
| • Glycocalyx : | carbohydrate layer on the cell surface. |
| • Lipids and proteins linked to carbohydrate moieties -> glycolipids and glycoproteins | • Help with cell-cell communication and adhesion |
| • Glycocalyx allows for distinguishing between | “self” and “non-self” (transplanted tissues). |
| • Glycocalyx is basis for blood types | • A • B • AB (universal acceptor) • O (universal donor) |
| • Recognition of diseased cells, | • Breast cancer cells have high sialic acid -> has led to immunotherapy • or invading organisms. |
| Integral membrane proteins traverse the lipid bilayer. Hydrophobic amino acids face | the acyl chains of lipids. |
| • Peripheral membrane proteins – | Can interact with other proteins or lipids. Lipid binding proteins have specific interactions, through electrostatic interactions or an amphipathic domain |
| • Membrane protein anchored through | a covalently attached lipid molecule. |
| 1. Membrane processes depend on | the fluidity of the lipids, • Determined by the Fatty Acid (FA) length and saturation • Cholesterol content (in eukaryotes) |
| 2. Long saturated FA – | pack closely together and have stronger van der Waals interactions -> Lower fluidity (butter – solid at RT) |
| 3. Unsaturated FA (one or more cis double bonds) - | produces a bend in the hydrocarbon chain -> loose packing à increases fluidity (Veg. oil – liquid at RT) |
| 4. Cholesterol - | Flat hydrophobic steroid core with a hydroxyl group at one end and a flexible hydrocarbon tail at the other end. • Interacts with acyl chains of other lipids. Stronger interactions with saturated FA. |
| 5. Bacteria lack cholesterol - | regulate the fluidity of their membranes by varying the number of double bonds and the length of their FA chains. |
| Phospholipase (PLx) X = | type of PL (A1, A2, C, D) -> Tells you where it cuts |
| Basic Glycerophospholipid: | • Glycerol backbone (3 carbons and 3 –OH) • 2 –OH linked to FA through ester bond • 3rd –OH linked to a phosphate (polar/hydrophilic head group) • * Phosphate can be further linked to other molecules (e.g., choline = phosphatidylcholine/PC) |
| 1. Rapid lateral movement of membrane constituents can be visualized using | fluorescence recovery after photobleaching (FRAP) technique. |
| 2. Lateral diffusion of lipids is much more rapid than | transverse diffusion (flipflopping). |
| 3. Transverse movement of lipids can be | spontaneous (Slow for GPL & SL. Faster for Chol.). • Protein lipid transporters helps translocation • Can establish asymmetry or eliminate it. |
| Membrane asymmetry: | • Keeps PS on the inside. Important because PS is a signal for cell damage and will lead to its destruction by white blood cells • Keeps signaling lipids on the inside. PS and PI variants interact with cytoplasmic proteins to carry out cellular functio |
| ⇒Like enzymes, facilited transporters exhibit | saturation kinetics |
| Hydrolyze ATP as energy source to move things against concentrations gradients (swimming upstream) | 1. P-type ATPases • transport Na+, K+, Ca+2, etc… 2. V-type(vacuolar) ATPases • Transports H+ • Responsible for the acidification of lysosomes, endosomes, Golgi, and secretory vesicles 3. ABC transporters (various cargo) • Toxic metals • Drugs |
| Antiporters are similar but | move cargo in opposite directions |
| Secondary active transporters- | Energy from primary transport (cargo going downstream) is used to move 2nd cargo upstream -> Symporter |
| SGLT1 - | secondary active transport of glucose in intestinal epithelial cells |
| Specificity: | Signalling molecule: receptor |
| Combinatorial code: | A combination of signals lead to an individual cell behaviour |
| Continuous input: | Absence of any signal: Death |
| Three general categories of chemical signaling: | 1. Cytoplasmic connections (tube) between cells (Gap junction) 2. Cell-to-cell contact-mediated signaling (MHCII – T-cell Receptor) 3. Free diffusion between cells • Distant cells • Adjacent cells |
| All of latter involves the physical movement of | Ligands That is, Ligand Reception by a Protein |
| Note that Reception means | Molecule-to-Molecule Contact |
| The nervous system | 1. Neurotransmitters or biogenic amines. (recycled) Acetylcholine GABA Epinephrine 2. Neuropeptides (not recycled) neuropeptide Y (vasoconstrictor) Agouti-related peptide (appetite) β-endorphin (analgesic) |
| The endocrine system | 1. Polypeptides Insulin 2. Vitamin D3 3. Retinoids Retinol = vitamin A 4. Catecholamines Epinephrine (adrenaline) 5. Steroid Cortisol Aldosterone 6. Thyroid Triiodothyronine |
| The immune system | 1. Cytokines (small proteins <20Kd) Interleukins Interferons colony stimulating factors 2. Chemokines (chemotactic cytokines). C chemokines CC chemokines CXC chemokines CX3C chemokines |
| Hydrophilic small molecules do not cross the plasma membrane. Instead, they bind to | cell-surface receptors and generate signals inside the target cell. |
| Some small molecules diffuse across the plasma membrane and bind to receptors inside the target cell. They’re hydrophobic and are therefore transported in | the blood and other extracellular fluids after binding to carrier proteins. |
| Three stages of signal transduction | 1. Reception of extracellular signal by cell -> Conformational (shape) changes 2. Transduction of signal from outside of cell to inside of cell 3. Cellular Response— occurs entirely in the receiving cell. |
| • Signal transduction consists of a conformational change when the | ligand binds. • neurotransmitters (e.g., acetylcholine - ACh) & some neuropeptides |
| • Five subunits- 2 identical a-subunits bind ACh resulting in a conformational change that opens | the channel • K+ diffuses out • Na+ diffuse in • Muscle contraction |
| • ACh has a second kind of receptor that is a | G-coupled receptor. |
| • Protein kinases use ATP & transfer a PO4 group to the OH group on a | specific amino acid side chain (Tyr, Ser or Thr) of the target protein. |
| • Binding the ligand changes the shape of the | intracellular kinase domain - Kinase domain leads to autophosphorylation or phosphorylation of an associated protein. |
| • This activates the signal transducers- | SH2 domains, STATs or SMADs |
| • A protein that is activated by a Protein Kinase in turn is inactivated by | a Protein Phosphatase |
| • This means that the effect of protein kinase signals | can’t last forever |
| • For the cellular response to continue, | more protein kinase signals must be received |
| • Ras is a small GTPase that is “active” when bound to | GTP. Binds to “effector” proteins. |
| • GEF – guanine exchange factor – | swaps out GDP for GTP to activate Ras |
| • signal transduction turns itself off by hydrolyzing GTP to GDP with the help of | a GAP (GTPase activating protein) |
| • Dysregulation -> uncontrolled cell growth -> | cancer |
| • Insulin Receptor – | Autophos. |
| • IRS – | Insulin Response Substrate |
| • IRS activates | PI3 kinase - PI(4,5)P2 -> PIP3 |
| • PIP3 binds | PH domains - Protein kinases (PDK1 PKB…) |
| PI(4,5)P2 = | phosphatidylinositol 4,5 bisphosphate |
| PIP3 = | PI 3,4,5 trisphosphate |
| PH = | pleckstrin homology |
| • Cytokine receptors | bind to Jaks |
| • JAK (Janus Kinase) are | separate proteins that associate to receptor |
| • JAKs phosphorylate | each other and the receptor |
| • Phosphorylated receptor binds | STATs (signal transducer & activator of transcription proteins) |
| • TGF-b (transforming growth factor) receptors binds to | type II receptor |
| • Heterodimeric – | Type 2 and Type 1. |
| • Phosphorylated type 1 receptor binds to | R-SMAD -> phosphorylates R-SMAD |
| • Dysregulation associated with | cancers & immune disorders |
| G-protein coupled receptors are the largest family of integral membrane protein involved in | many biological process and pathologies. Constitute |
| 50% of all modern drugs & 25% of the best-selling drugs are estimated to target | GPCRs. |
| • GPCRs transduce the signals mediated by diverse signaling molecules, such as | ions, neurotransmitters, odorants, lipids, neuropeptides, hormones, peptides, lipids and photons, to induce different intracellular function. |
| A hormone, cytokine or neurotransmitter binds to | the receptor. |
| • The activated receptor complex activates acts as a | GEF for the hetrotrimeric G protein. • GDP -> GTP • GTP-bound alpha separates and functions in downstream events |
| • This activated receptor complex stimulates membrane bound enzymes leading to generation of | second messengers. |
| • Glycolysis occurs in | the cytoplasm in ALL cell types. • RBCs and other cells without mitochondria fully rely on |
| • The major carbohydrates in the human diet are | starch, sucrose, lactose, fructose, and glucose. |
| • Starch - | polymers of glucose • linear α(1-4) bonds • Branch α(1-6) bonds - more prevalent in glycogen than in starch |
| Starches begin to be broken down by salivary a-amylase and subsequently by pancreatic a-amylase, which both catalyze the | hydrolysis of α(1-4) glycosidic bonds, and produce α-maltose, α-isomaltose, tri/oligosaccharides and dextrins |
| • Maltose is a disaccharide of glucose linked by an α(1-4) bond and is broken down by | maltase on the surface of the brush border of the intestinal epithelial cells into glucose. |
| • Isomaltose is a disaccharide of glucose linked by an α(1-6) bond and is broken down by | isomaltase on the surface of the brush border of the intestinal epithelial cells into glucose. |
| Sucrose is a disaccharide of glucose linked to fructose in an α(1-2) linkage. It is broken down by | sucrase on the surface of the brush-border of the intestinal epithelial cells. |
| • Sucrase-isomaltase complex deficiency: | Results in an intolerance of ingested sucrose. Highly prevalent in the Inuit people. |
| • Treatment includes | dietary restriction of sucrose, and enzyme replacement therapy. |
| Lactose - | disaccharide of galactose + glucose by (b, 1-4 linkage) |
| • Lactose is broken down by | lactase into glucose and galactose |
| • Lactose appears on the surface of | the brush border of the intestinal epithelial cells. |
| • Absence of intestinal lactase -> | lactose intolerance |
| • Treatment for lactose intolerance-> | reduce consumption of milk • Get Ca2+ from yogurts, cheeses, green vegetables (broccoli) • lactase-treated products • lactase pill prior to eating |
| 1. SGLT1 (Sodium-Glucose Linked Transporter 1), • Symporter - | transports glucose & galactose against a concentration gradient (low to HIGH). |
| • SGLT1 Energy provided by | an electrochemical gradient of sodium going (HIGH to low) |
| • SGLT1 on the apical border of | intestinal cells |
| 2. Na+/K+ antiporter uses ATP to move Na+ from | low to HIGH levels – Keeps cytoplasmic levels low -> helps continued SGLT1 operation |
| 3. GLUT2 - | facilitated glucose transporter (transports glucose down its concentration gradient – HIGH to low) |
| • GLUT2 on | basolateral side. |
| • GLUT2 has high capacity (Vmax) but | low affinity (high Km) for glucose -> Will move HIGH glucose quickly |
| 4. GLUT5 - | facilitated fructose transporter • On apical border |
| • SGLT1 deficiency causes | glucose & galactose malabsorption. |
| • GLUT5 deficiency causes | fructose malabsorption (aka dietary fructose intolerance) |
| Energy investment phase of Glycolysis | • Uses 2 molecules of ATP/glucose but makes no ATP • Traps and prepares glucose for the oxidation steps. |
| 12 Glucose is converted into | glyceraldehyde 3-phosphate (GAP) |
| GAP conversion happens in 5 steps: | 1. Phosphorylation of glucose 2. Isomerization 3. Second phosphorylation of Fructose-6P (Committed Step) 4. Cleavage into two 3-carbon molecules 5. Isomerization of DHAP to GAP |
| Why Glucose? | • Low tendency to spontaneously glycosylate proteins • Most stable of hexoses (16 conformations) - All –OH groups on equatorial plane -> less steric clashes |
| Phosphoryl group: | • Traps glucose inside the cell • Acts as “handle” for enzyme recognition and provides increased binding free energy |
| • Glucose binds when | first |
| • Cleft closing dehydrates | active site; prevents nucleophilic attack by water and nonproductive ATPase action |
| • Non-polar binding site excludes water to | favor reaction – Facilitates Aspartate to act as base and start the reaction |
| • The C6-OH must be deprotonated to act as a | nucleophile |
| • Aspartate (Base) in catalytic pocket deprotonates C6-OH -> | becomes Aspartic Acid |
| • Charged C6-O- attacks | gamma P |
| • Covalent bond (intermediate) between | C6-O and P |
| • Phosphoanhydride bond between beta and gamma Phosphates is | broken |
| • Hexokinase I - | irreversible but regulatory step. |
| - Hexokinase I has a low Km & | Lower Vmax (lower capacity) |
| - Hexokinase I permits efficient phosphorylation of | glucose even at low concentrations. |
| • Hexokinase I is feedback inhibited by | Glucose-6-P. - Prevents it from tying up all the intracellular Pi in the form of G-6-P. |
| • Hexokinase IV (Glucokinase) is expressed in | hepatocytes (liver) and pancreatic b cells. |
| - Hexokinase IV has a high Km | and high Vmax (increased capacity) |
| - Hexokinase IV is not inhibited by | G6P |
| Glycolysis Step 2: Isomerization Conversion of an aldose to a | ketose sugar |
| Isomerization's Goal is to convert the 6-Carbon starting material into | 2x 3-Carbon units |
| – Carbonyl at C-1 is in “wrong” spot for | cleavage by Aldol reaction |
| – Isomerization moves carbonyl to C-2 to | promote Aldol reaction |
| • How does this work? – First, glucose 6-P must be converted to | fructose 6-P PI mechanism step 1. Base deprotonates H2O -> |
| PI mechanism step 2. Lys organizes | :OH- |
| PI mechanism step 3. OH- deprotonates C1-OH -> | H2O |
| PI mechanism step 4. C5-O deprotonate | His |
| PI mechanism step 5. Ring | opens |
| PI mechanism step 6. Glutamate deprotonates C2-H -> | enediolate |
| PI mechanism step 7. Enediolate deprotonates Glutamic acid -> | Ketone |
| PI mechanism step 8. Lys deprotonates | C5-OH |
| PI mechanism step 9. Ring closes -> | Fructose 6-phophate |
| Enol (alkenol) – | an alkene with a hydroxyl attached to one end of the double bond |
| Glycolysis Step 3: Second Phosphorylation is a | • Committed Step - Irreversible |
| Second phosphorylation: • Cell can now split fructose 1,6-BP into | two trioses |
| Second phosphorylation: Better to have a phosphoryl group at each end of fructose, so that the resulting trioses are | both phosphorylated |
| • Second phosphorylation: It is allosterically regulated by | ATP, AMP, etc. |
| • Second phosphorylation: It is the most important | regulatory point in glycolysis |
| • PFK-1 is inhibited allosterically by ATP which acts as | an “energy rich” signal. |
| • ATP binds at 2nd site away from | catalytic site. - When in excess/Part of feedback |
| - AKA: | Allosteric inhibitor |
| • AMP reverses inhibition by ATP. PFK-1 is VERY sensitive to | AMP regulation. |
| High F6-P -> High PFK2 -> High F2,6BP -> | High PFK1 activity (increased affinity) |
| F6-P can go to G6-P -> | Inhibits HK or goes into glycogen |
| The sigmoidal dependence of velocity on substrate concentration becomes hyperbolic in the presence of | fructose 2,6-bisphosphate. |
| This indicates that more of the enzyme is active at lower | substrate concentrations in the presence of fructose 2,6-bisphosphate. |
| ATP, acting as a substrate, initially stimulates the reaction. BUT as the concentration of ATP increases, | it acts as an allosteric inhibitor. |
| The inhibitory effect of ATP is reduced by fructose 2,6-bisphosphate, which makes the enzyme | less sensitive to ATP inhibition. |
| Glycolysis Step 5: Isomerization of DHAP & G3-P | • A ketose-aldose isomerase (goes through enol intermediate as seen before) • Isomerization produces two molecules of G3-P from the cleavage of F-1,6,bisphosphate. |
| • Continual metabolism of G3-P in glycolysis drives | the reaction forward. |
| Glyceraldehyde 3-Phosphate (G3-P) is converted to pyruvate in 5 steps: | 6. Oxidation of G3-P to 1,3-BPG 7. Phosphorylation of ADP 8. Mutase: conversion of 3-PG to 2-PG. 9. Dehydration by Enolase 10. Phosphorylation of ADP, giving pyruvate |
| Second Stage of Glycolysis= Energy generation phase of Glycolysis 2 x 3-carbon units are oxidized to pyruvate generating | 4 molecules of ATP and 2 NADH |
| Glycolysis Step 6: Oxidation of glyceraldehyde 3-P | • DOES NOT USE ATP |
| • The oxidation of glyceraldehyde 3-phosphate powers the formation of | 1,3- bisphosphoglycerate, which has a high phosphoryl transfer potential. |
| • 1,3 bisphosphoglycerate is an acyl phosphate, which is mixed anhydride of | phosphoric acid and a carboxylic acid (carbonyl). |
| Oxidation of glyceraldehyde 3-P is achieved through | coupled “oxidation-phosphorylation” reactions |
| • Conversion of aldehyde to carboxylic acid happens before | oxidation-phosphorylation (2-step process) |
| • Transfer hydride from donor to NAD+ (energetically favorable) then couple it with dehydration to form | acyl phosphate (energetically unfavorable). |
| • GAPDH uses covalent catalysis (makes a thioester intermediate on | a Cys residue). |
| G3P Dehydrogenase Mechanism step 1. Base (His) deprotonates | Cys thiol group |
| G3P Dehydrogenase Mechanism step 2. Sulfur attacks aldehyde to make | thiohemiacetal |
| G3P Dehydrogenase Mechanism step 3. Unstable oxyanion | collapses |
| G3P Dehydrogenase Mechanism step 4. Hydride transferred to NAD+ leaving a | Thioester bond |
| G3P Dehydrogenase Mechanism step 5. Pi attacks carbonyl and makes | tetrahedral oxyanion |
| G3P Dehydrogenase Mechanism step 6. Oxyanion collapses to sever | thioester |
| G3P Dehydrogenase Mechanism step 7. Base is | deprotonated |
| • Thioester is higher energy than | carboxylic acid. - Cannot resonate - Less stable (easier cleavage) |
| Coupling of the two processes (by GAPDH) allows the conservation of | energy released by oxidation |
| Without thioester intermediate: | • Reaction of stable carboxylate with phosphate |
| • With thioester intermediate: | Energy is trapped in thioester |
| Phosphoglycerate kinase has a | • ATP generation step (=2 ATP per input glucose) |
| • Lys219 guides C1-Pi to | gamma position of ATP |
| • Phosphoglycerate kinase has “ | Substrate-level phosphorylation” |
| • Energy released in oxidation of aldehyde to | carboxylate conserved through ATP formation |
| Glycolysis Step 8: Shift of the phosphate group from carbon 3 to | carbon 2 |
| Step 8 – Phosphoglycerate Mutase Allosteric shape change – | Alleviates clash of phosphate charges |
| Phosphoglycerate Mutase step 1. His deprotonates C2-OH to attack | Phospho-His |
| Phosphoglycerate Mutase step 2. C2-O- oxyanion gets Pi from Phospho-His -> | 2,3 bisphosphoglycerate |
| Phosphoglycerate Mutase step 3. Dephosphoryalted His takes Pi from C3-Pi -> | 2 phosphoglycerate |
| • Enolase catalyzes the reversible dehydration of 2-phosphoglycerate to create | phospho-enol pyruvate, - high phosphoryl group transfer potential. |
| Pyruvate kinase has an • ATP generation step - | substrate-level phosphorylation |
| • Pyruvate kinase reaction is | irreversible and regulated |
| • Regulators: – Allosteric activator: | fructose 1,6-bisphosphate (an example of feed-forward activation) |
| – Allosteric inhibitor: | ATP – protein phosphorylation |
| Pyruvate kinase deficiency | • Not enough ATP for Red blood cell survival • RBC are removed by the spleen • Most common cause of hereditary hemolytic anemia • Build up of 2,3-bisphosphoglycerate, which helps release O2 in tissues |
| • Total [NAD+ + NADH] is very low (< 0.01 mM) relative to amount of | [glucose] metabolized in a few minutes. |
| • NADH must be rapidly oxidized to | NAD+ for glycolysis to continue. |
| • In AEROBIC glycolysis -> | Reducing power of NADH is transferred to mitochondria by the malate-aspartate and glycerol 3-phosphate shuttles, regenerating NAD+. |
| • In ANAEROBIC glycolysis -> | NADH oxidized to NAD+ by lactate dehydrogenase. |
| • In aerobic glycolysis what are produced: | 30-32 ATPs/glucose (depending on the shuttle), |
| whereas in anaerobic glycolysis what are produced: | only 2 ATPs/glucose |
| Glycolysis is very important in adipocytes to make triglycerides because they lack | glycerol kinase |
| Synthesis of 2,3-BPG is a major reaction pathway for the consumption of glucose in erythrocytes and is critical in controlling | hemoglobin affinity for oxygen. |
| Where is glycogen found? | • Found as granules in the cytosol. |
| • Hepatocytes have what glycogen concentration | Highest. Up to 8% of the fresh weight in well fed state ( |
| • Muscles have a much lower what | conc. of glycogen About 1%, but higher mass ( |
| • Glycogen storage capacity in man is approximately | 15g/kg and can accommodate a gain of approximately 0.2-0.5kg |
| • γ-particles are | protein rich subunits of 3 nm wide |
| • The β- granule includes the | carbohydrate polymer and the bound γ-particles 20-30 nm – faster energy source vs alpha |
| • α-granule is composed of several β-granules bound via a | protein backbone rich in disulfide bonds. – up to 300 nm |
| • Glycogenin (GN) – | Dimer (weak binding) |
| 1. intermolecular glycosylation - | transfer of 1–2 glucose to Tyr-194 from partner GN |
| 2. intramolecular glycosylation - | elongation of a primer chain (7–16 glucose residues) |
| • Glycogen synthase (GS) - | elongation of the primer chain through α-1,4-linkage o Displaces one of the GN dimer copies |
| • Glycogen-branching enzyme (GBE) - | adds glucose residues to the granule through α1,6-linkage |
| • GN binds to actin filaments for the start of | glycogen synthesis |
| • Beta granules can have up to | 12 layers |
| o Number of glucose double with each | layer |
| o A 13th layer is impossible due to | spatial constraints |
| Why is glycogen so highly branched? | • makes if more soluble (than e.g., Starch) • allows for much faster synthesis/degradation |
| Glycogenesis step 1) | Glucokinase (Hexokinase 4) |
| Glycogenesis step 2) | Phosphoglucomutase |
| Glycogenesis step 3) | UDP-Glucose Phosphorylase |
| Glycogenesis step 4) | Glycogenin (GN) |
| Glycogenesis step 5) | Glycogen Synthase (GS) |
| Glycogenesis step 6) | Glycogen Branching Enzyme (GBE) |
| Phosphoglucomutase • Must convert glucose 1-phosphate to glucose 6-phosphate before it can | enter glycolysis |
| • Note that neither phosphoglucomutase nor glycogen phosphorylase uses | ATP |
| • Therefore in Phosphoglucomutase, we get 3 ATPs per glucose via glycolysis when starting with | glycogen |
| • In Committed step or activation of glucose, UDP-glucose is the donor of | glucose in the formation of glycogen. |
| • In Committed step or activation of glucose, Spontaneous hydrolysis of the | P bond in PPi (P |
| Cleavage of PPi is the only | energy cost for glycogen synthesis (one |
| • Glycogen synthase promotes the transfer of the glucosyl residue from UDP-glucose to | a non-reducing end of the branched glycogen molecule. |
| • Glycogenin is a | protein homodimer with a MW of 37 kDa. |
| • Glycogenin functions as a | primer. |
| • Glycogenin AUTOGLUCOSYLATES at a | specific Tyr 194. |
| • Glycogenin acts as a | Glucosyltransferase. |
| • Glycogenin forms a tight 1:1 complex with | glycogen synthase. |
| Glycogen Step 1) Phosphorylase: | Release of glucose1- phosphate |
| Glycogen Step 2) Transferase: | Remodeling of the glycogen to allow further degradation -> 3 residues from branch get moved to the end of a chain |
| Glycogen Step 3) Alpha 1,6 Glucosidase removes | branch point residue |
| Glycogen steps 2 & 3 are performed by | the same debranching enzyme |
| Next after glygenolysis: Conversion of glucose 1-phosphate to | glucose 6-phosphate by phosphoglucomutase |
| Glycogen phosphorylase: • Catalyzes the phosphorolysis (not hydrolysis) of | glucose units from glycogen - Inorganic phosphate is the nucleophile |
| • Glycogen phosphorylase releases glucose units as | glucose 1-phosphate |
| • Glycogen phosphorylase does not use | ATP |
| • Energy of the glycosidic bond is captured in the | sugar phosphate |
| Glycogen phosphorylase step 1. Pyridoxal phosphate (PLP) covalently links to Lys680 -> | Schiff-base linkage (R3 cannot be a H) |
| Glycogen phosphorylase step 2. PLP Phosphate donates H+ | to Pi |
| Glycogen phosphorylase step 3. Oxygen at a1-4 linkage abstracts H+ from Pi -> | creates leaving group |
| Glycogen phosphorylase step 4. Terminal Glucose left with | carbocation at C1 |
| Glycogen phosphorylase step 5. PLP deprotonates | Pi |
| Glycogen phosphorylase step 6. Deprotonated Pi acts as | nucleophile and forms bond with carbocation -> Glucose 1-Phosphate |
| Glucose 6 phosphatase is uniquely present in the liver which can generate | free glucose which in turn can be shunted to other tissues for use |
| Glucose 6-phosphate fate 1) It is the initial | substrate for glycolysis. |
| Glucose 6-phosphate fate 2) It can be processed by | the pentose phosphate pathway to yield NADPH and ribose derivatives. |
| Glucose 6-phosphate fate 3) It can be converted into | free glucose for release into the bloodstream. |
| Glycogenolysis in Liver: • Driven by mostly by glucagon but also requires | epinephrine signaling |
| • Glycogenolysis in Liver: Glucagon from | alpha cells in pancreas |
| • Glycogenolysis in Liver: Epinephrine from adrenal glands & neurons – | binds adrenergic receptors |
| • Epinephrin can bind | alpha- and beta-adrenergic receptors (AR) in the liver |
| • Alpha-AR triggers the | phosphoinositide pathway -> Calcium signaling |
| • Beta-AR signaling like the Glucagon signal pathway -> | PKA |
| • Both PKA and Ca2+ are needed to activate | phosphorylase kinase |
| Glycogenolysis provides glucose in the first few hours after | birth |
| • Transplacental glucose transfer stops | during birth |
| • Brain & vital organs sill need | glucose |
| • Glycogen is a major fuel for the newborns and accumulates in fetal liver to very high levels just prior to birth. This is because activity of glycogen synthase increases but | glycogen phosphorylase remains low just prior to term. |
| • During delivery - levels of catecholamines (epinephrin) and glucagon in the neonate are increased, and insulin is | decreased. |
| Glucagon also activates gluconeogenesis: Lactate to pyruvate -> | glucose) |
| • These hormonal changes result in stimulation of glycogenolysis. In a full-term infant liver glycogen lasts about | 10 hrs. |
| Glycogen storage disorders arise when glycogen metabolic enzymes are | defective, deficient, or absent. |
| Glycogen storage disorders causes the buildup of abnormal amounts and types of | glycogen in liver and/or muscle tissues. |
| * glucose 6-phosphatase (liver only) | • Multi-subunit • phosphate hydrolysis, • transports glucose 6-phosphate, glucose, and PPi across the ER membrane |
| Type-Ia Von Gierke disease- | Glucose 6 phosphatase activity is Deficient. |
| In type 1b (of Von Gierke disease): | The translocase activity of glucose 6 phosphate is affected. |
| Pompe’s disease (type II) | • Infantile acid maltase deficiency • metabolic myopathy • motor neuron disease that causes infantile hypotonia. |
| Pompe's disease is linked to- | Defective lysosomal a(1-4) glucosidase |
| Type III Cori’s Disease- | Amylo 1-6 glucosidase debranching activity is deficient. |
| Type IV Anderson's Disease- | Long linear glycogen, Normal glycogen content but it is unbranched. |
| Type V McArdle Disease- | Moderately increased glycogen content, Normal Glycogen structure, Muscle is primarily affected |
| Type VI HERS Disease- | • Increased glycogen content |
| Lafora disease- | bodies accumulate,Too much phosphorylation,Reduces solubility: unfolding exposes hydrophobic regions,Reduces available glycogen available for energy, Targeted to lysosome for degradation |
| Mutation in Laforin: | phosphatase |
| Laforin facilitates | branching • Phosphate could help unfold branched chain |
| For glycogen storage diseases that affect the liver, or types I, III, IV, and VI, the goal is to | maintain normal blood glucose levels. |
| For glycogen storage diseases that affect the muscles, or types V and VII, the goal is to | avoid muscle fatigue and/or cramps induced by exercise. |
| Entry of fructose into cells is not insulin dependent, and fructose does not promote | insulin secretion |
| Fructose can be phosphorylated by either | - hexokinase (higher Km for fructose vs glucose) - fructokinase (lower Km for fructose) |
| Fructokinase is found in | liver, kidney & small intestine |
| Fructokinase produces | fructose 1-phosphate |
| Fructokinase bypasses | PFK1 Regulation |
| Fructose 1-phosphate - cleaved by Aldolase B into | DHAP + glyceraldehyde (NOT glyceraldehyde phosphate) |
| DHAP can enter | glycolysis or gluconeogenesis • Only 3 out of 6 carbons are available for glycolysis |
| Hereditary fructose intolerance is a deficiency in | Aldolase B - Fructose 1-P accumulates - Cannot go back to Glucose |
| With Hereditary fructose intolerance, fructokinase | continues to work - Intracellular Pi levels fall - ATP levels fall |
| Low hepatic ATP can cause | Negative effects - Reduced gluconeogenesis - Reduced liver protein production -> cell death |
| AMP accumulates and is degraded, causing | hyperuricemia - Uric acid crystals form - Gout |
| - Insulin is not required for glucose entry into certain cells, such as | lens, retina, Schwann cells, liver, kidney & RBC. |
| - Hyperglycemia (and adequate NADPH) can result in | excessive sorbitol accumulation inside these cells |
| Main dietary source is the disaccharide, | lactose (galactosyl β1→4 glucose) |
| Galactose is released from lactose in the small intestine by | the digestive enzyme, lactase |
| Galactose entry into cells is not | insulin dependent |
| Lactose intolerance (due to deficiency in | lactase) |
| - Sorbitol does not efficiently cross | plasma membrane |
| - Osmotic effect of excess sorbitol may therefore | damage cells in hyperglycemia of uncontrolled diabetes |
| Galactokinase produces | galactose 1-P |
| UDP-galactose is produced in an exchange reaction with | UDP-glucose |
| UDP-galactose used in synthesis of | glycoproteins, glycosaminoglycans, glycolipids and lactose |
| UDP-galactose can be converted to UDP-glucose by | Epimerase – Change orientation of C4-OH |
| Galactose metabolism step 1. C4-OH deprotonated by | E |
| Galactose metabolism step 2. Oxyanion collapses to form | ketone |
| Galactose metabolism step 3. NAD+ takes other H from | C4 |
| Galactose metabolism step 4. Ketone intermediate will | rotate |
| Galactose metabolism step 5. NADH gives | back H |
| Galactose metabolism step 6. Oxyanion takes back H from | E |
| Elevated galactitol can cause | cataracts |
| Accumulation of galactose 1-phosphate and galactitol in nerve, lens, liver, and kidney tissue causes | liver damage, severe mental retardation, and cataracts |
| Pentose phosphate pathway Starting point: | Glucose 6-phosphate |
| Pentose phosphate pathway has | Oxidative and non-oxidative phases |
| Pentose phosphate pathway Function: This pathway generates three principal products: | - NADPH, necessary for biosynthetic reactions. - Ribose 5-phosphate, necessary for nucleotide biosynthesis. - Erythrose 4-Phosphate, for aromatic AA |
| In Pentose phosphate pathway No | ATP is used or made |
| Non-oxidative phase of Pentose phosphate pathway can be run backwards to make | R5P without making NADPH |
| Glucose 6-P is converted to | 6-phosphoglucono-lactone -Glucose 6-phosphate dehydrogenase (G6PD) |
| G6PD is the | primary regulation step & generates NADPH |
| NADPH inhibits the | enzyme (allosteric binding site) |
| 6-phosphogluconolactone is converted to | 6-phosphogluconate -6-phosphogluconolactone hydrolase (aka lactonase) |
| Lactonase Reaction is | irreversible and NOT rate-limiting |
| G-6P Dehydrogenase Has | 2 NADP+ binding sites |
| NADP+ is an | allosteric activator |
| NADPH inhibits | enzyme activity |
| 1. Enzyme (G6PD) is usually a homodimer and can form | tetramers |
| 2. Two NADP+ binding sites on G6PD: One is for catalysis. The second is | “Structural” and stabilizes dimer |
| 3. The Structural NADP+ binding site affects | protein stability in part through dimerization. |
| 4. G6PD Mutations can lead to deficiency (“Favism”) -> | common cause of acute hemolytic anemia |
| • G6P-DH is the only source of NADPH in RBCs. Needed for Glutathione reduction -> | blocks damage caused by oxidative stress. |
| 2nd NADPH generation step 1. B: abstracts C3-OH -> | C3=O ---- HB |
| 2nd NADPH generation step 2. C3-H taken by NAPD+ -> | NADPH |
| 2nd NADPH generation step 3. Hydride to NADP+ -> | NADPH |
| 2nd NADPH generation step 4. C2-OH H-bonds to | BH |
| 2nd NADPH generation step 5. Electrons withdrawn away from | CO2 |
| 2nd NADPH generation step 6. CO2 is released -> | Alkene formed |
| 2nd NADPH generation step 7. C3=O opens to C3-O- and abstracts proton from BH -> | C3-OH (enol) |
| 2nd NADPH generation step 8. Tautomerism with Ribulose 5-phosphate (C2 ketone) where base is | re-protonated. |
| Tautomerism – | Equilibrium between Ketone and Enol |
| In non-oxidative reaction, catalyze interconversion of | 3-, 4-, 5-, 6- and 7-carbon sugars |
| In non-oxidative reaction, allow conversion of ribulose 5-P to | ribose 5-P or glycolysis intermediates |
| Thiamine pyrophosphate (TPP) is a cofactor for | transketolase |
| Transketolase Rxn PT.1: Part A. TPP deprotonated by | Glu418 |
| Transketolase Rxn PT.1: Part B. YLID C- (carbanion) nucleophilic attack on | Xylulose 5P carbonyl -> Makes oxyanion |
| Transketolase Rxn PT.1: Part C. TPP make C-C bond with | X5P C2 |
| Transketolase Rxn PT.1: Part D. Oxyanion deprotonates TPP NH3+ -> | C2-OH |
| Transketolase Rxn PT.1: Part E. His263 deprotonates C3-OH -> | oxyanion |
| Transketolase Rxn PT.1: Part F. Oxyanion collapses -> C2=C3 -> | the bond severs |
| Transketolase Rxn PT.1: Part G. Releases | G3P |
| Transketolase Rxn PT.1: Part H. eneamine <--> | alpha-carbanion |
| Transketolase Rxn PT.2:Part A. Eneamine attacks Ribose to make oxyanion -> | 7 carbon sugar |
| Transketolase Rxn PT.2:Part B. Oxyanion deprot. | His263 |
| Transketolase Rxn PT.2:Part C. TPP deprot. | C2-OH |
| Transketolase Rxn PT.2:Part D. C2 carbanion collapses -> | severs covalent bond with TPP |
| Transketolase Rxn PT.2:Part E. Sedoheptulose 7-P is | released |
| Transaldolase Rxn PT.1:Part A. Base (Glu-106) deprotonates water -> OH- deprot. | Lys |
| Transaldolase Rxn PT.1:Part B. Lys-142 attacks C2 Carbonyl -> | Oxyanion |
| Transaldolase Rxn PT.1:Part C. Oxyanion gains proton -> | C2-OH |
| Transaldolase Rxn PT.1:Part D. Pi bond between | N=C (Schiff Base) |
| Transaldolase Rxn PT.1:Part E. C4-OH deprotonated (by | Asp-27) |
| Transaldolase Rxn PT.1:Part F. Collapse of C4 oxyanion -> To carbonyl -> | Severing of C3-C4 bond |
| Transaldolase Rxn PT.1:Part G. Erythrose 4-P is | released |
| Schiff Base – | A type of Imine where amine (Lys) reacts with an aldehyde or ketone -> aldimine/ketimine |
| Transaldolase Rxn PT.2:Step 1. Erythrose 4-P | released |
| Transaldolase Rxn PT.2:Step 2. G3P comes into | reaction |
| Transaldolase Rxn PT.2:Step 3. Attack of G3P carbonyl -> | oxyanion and making of 6C sugar |
| Transaldolase Rxn PT.2:Step 4. OH- nucleophile attacks imine -> | C2-OH |
| Transaldolase Rxn PT.2:Step 5. C2-OH deprot. by | Asp27 |
| Transaldolase Rxn PT.2:Step 6. Oxyanion collapses to | carbonyl |
| Transaldolase Rxn PT.2:Step 7. Severs | imine |
| Transaldolase Rxn PT.2:Step 8. Released | F6P |
| Transaldolase Rxn PT.2:Step 9. Lys-NH2 deprot. Aspartic acid -> | Lys-NH3+ |
| • CYP450 superfamily of enzymes with | Heme as cofactor |
| • CYP450 functions as | monooxygenase - Deliver electrons to Iron (Fe3+ to Fe2+) and eventually to O2 - O2 is reduced to one –OH and one H2O |
| NADPH oxidase converts O2 into | superoxide radical,·O2 |
| (Superoxide radical is also known as | respiratory burst or oxidative burst) |
| HOCl = | hypochlorous acid |
| OH· = | hydroxyl radical |
| ·O2- can react with water to form | hydrogen peroxide (H2O2), which is also toxic to microorganisms |
| Myeloperoxidase (MPO) in lysosomes catalyzes formation of hypochlorous acid (HOCl) from | H2O2 and Cl- |
| HOCl (bleach) is highly toxic to | microogranisms |
| Collectively (HOCL& H2O2), these are called | reactive oxygen species (ROS) |
| Nitric oxide (NO) is | a potent vasodilator |
| It also acts as a neurotransmitter, decreases platelet aggregation, and has a role in | macrophage function |
| NO is synthesized by NO synthase (NOS) from | arginine, O2 and NADPH |
| • There are three known NO synthases: | – eNOS (endothelium – constitutive) – nNOS (neural tissue – constitutive) – iNOS (inducible in hepatocytes, monocytes/macrophages & neutrophils) |
| Hereditary deficiency in G6PD causes hemolytic anemia because of inability to | detoxify oxidizing agents (due to insufficient reduced glutathione) |
| Other tissues have alternate pathways for producing | NADPH (e.g., malic enzyme) that is lacking in RBC |
| RBCs can make NADPH only via the | PPP |
| Therefore, the G6PD mutation has a much more severe effect on | RBC |
| G6PD deficiency protects against | malaria. |
| The parasites causing this disease require | NADPH for optimal growth. |
| Because the PPP is defective, the cell and parasite | die from oxidative damage. |
| Primaquine – | common antimalarial drug. However, indiscriminate use of primaquine will cause hemolysis in individuals deficient in G6PD. |
| To create ribose, the non-oxidative reactions can synthesize ribose 5-P from | glyceraldehyde 3-P and fructose 6-P (bypasses oxidative reactions) |
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