Lecture 3:Metabolism

Extra Credit: Oliver

9/12/2011 6:29:00 AM

Metabolism Anabolismsynthesis and requires energy Catabolismfocus, break down of substances to yield energy Amphibolicpathways that are involved in both in anabolism and catabolism anabolismgrow and divide catabolismhousekeeping so it can make things it needs to grow and divide Enzymes ABCD (each step requiring an enzyme or each arrow ) are able to measure the cells needs for different things, regulated by how much energy it needs at a particular time activation energy is less when enzyme is present some products and reactants that are oxidized and reduced looking for the production of NADH and FADH2 (reduced forms) o oxidized forms being: NAD+, and FADH o occur in oxidized forms, and are reduced and utilmately responsible for the production of ATP o oxidized form interacts with the substrate thus reducing NAD+ and oxidizing the substrate can also occur in a reversed process Energy phosphoenolpyruvate (phosphorylates sugar across the membrane in prokaryotes) energy equivalent to ATP ADP and ATP have equal amounts of energy Low energyAMP ATP (contains an a ribose sugar, beucase of the OH at the 2 prime carbon) o adenine is attached to the first carbon o 3 phosphates are attached to the 5

o squigel bonds are high energy bondsmost of the time it is the last phosphate is removed (high energy bond) (the second bond is also a high energy bond) o ATP, GTP, or PEP are all considered to be equilavent in energy when the first bond (last phosphate) is removed o actual vs. ATP equalivalents (after one high energy bond broken) ATP ATP GTP ATP PEPATP o the first phosphate bond to the 5 carbon is a low energy bond o the cell is constantly able to measure the amt of energyit needs and constantly able to make it Enzyme Mediates Reaction enzyme looks like a pacman, and the substrate = missing piece how the enzyme measure how much energy is needed that allows the enzyme to either progress or stop the reaction o inhibitor o competitive inhibitorlooks like the substrate so it can bind to the active site, but as the substrate concentration increases there is a beter likely hood that the substrate will bind to the enzyme than the competitive inhibitor o allosteric activator/inhibitor o allosteric effectoran allosteric site is on the enzyme other than the active site where the allosteric effector binds causes a change in the active site an allosteric effector is an activator so that the active site changes so that the substrate can bind thus an allosteric inhibitor will change the active site so that the substrate cannot bind to the active site allosteric effectors are energy (ATP, ADP, AMP) or other things that will become energy (to tell the cell it has or doesnt have enough energy) the way the cell measures energy needs***

activator tells the cell that the energy levels are low so that the reaction will create energy ATP will shut off the enzyme (allosteric inhibitor)

Question from last time: sensing concentration along the microbe or is it sensing the concentration over time deceptive because receptor is on one side of the organism and cascade goes to the other side to the flagella over timebecause the organism is so small that they cannot tell that there is a concentration gradient different from one side of the organism to the other side the runs were longer if the organism was at all near the higher concentration

Aerobic Respiration Glycolysis each arrow correlates to an enzyme but most of the focus will be on, key point of control* --committed step (irreversible and specific to that particular pathway) starts at glucose and goes to pyruvate yellow = requires energy (PEP and ATP) glucose fructose-1,6 P ***1st step is phosphotransferase (ATP cross out and put PEP)!!!! different colors (stage 1 and stage 2) pink stage goes to pyruvate, pink because it is making ATP and NADH 6 carbon sugarsboth have 6 carbons just the ring differs in being 5 or 6 carbons long <3 orgo PEP vs ATP, PEP in the first step regulates other mechanisms in the cell which is why its needed in the first step, but not in the second step which is why ATP is used in the second step o PEP is only used in prokaryotes o in eukaryotes ATP is used for both steps yellow o 1. glucose (PEP used/phosphotransferase system) glucose-6P

group tanslocation, requires energy to bring sugar into the cell, but phosphorylates (using PEP) therefore not active transport, not the same molecule hexokinase, the enzyme used in the reaction, is inhibited by the product of the reaction (G6P), for example if G6P builds up in the cellproduct inhibition through allosteric inhibition meaning that G6P binds to some region of hexokinase and changes the enzymes active site feedback inhibitionalso allosteric inhibition

if there is a lot of aerobic respiration, then G6P should not be building up if energy is not needed then G6P will build up o 2. glucose-6P fructose-6P (6 membered ring becomes a 5 membered ring) o 3. fructose-6P (ATP used/ phosphofructokinase) fructose-1,6 PP(di/bi-phosphate) **key point of control phosphorylation of the other side the molecule is symmetrical requires energy to do so so that the molecule can get cut in half in the next step 6 carbon sugar in a 5 carbon ring cut in half from this step on energy is PRODUCED not USED ATP inhibits the reaction ATP is binding to the active site to only when there is a low concentration of ATP, and binds to the allosteric site to inhibit the reaction when there is a high concentration of ATP present in the cell PFKasinhibited by ATP, (allosteric when ATP concentrations are high) activated by AMP (adenine monophosphate) by allosteric, binding somewhere other than the active site and thus changing the active site to confer to the subtrate ATP and AMP bind to different allosteric sites enzymes with dimers, multiple binding sites to indicate on or off

when PEP loses a phosphate pyruvate (uses the enzyme pyruvate kinase to do ) and phosphorylates the ADP to ATP enzyme = ends with ase and the name often tells the action performed kinase generally = phosphorylation and phosphotase = dephosphorylation BUT most enzymes are reversible so kinases also dephosphorylate and phosphotase can phosphorylate, ex ATPase and ATPsynthase both make ATP difference between eukaryotic and prokaryotic is that it occurs in the mitochondria vs. the cytoplasmic membrane PEP Pyruvate o 3 levels of control: o inhibition by ATP/ Alanine (amino acid), alanine is an amino acid that is produced by the pyruvate products, feedback inhibition allosteric inhibition o activation by fructose-1,6 diphosphate allosteric activation when energy levels are low, no fructose-1,6 diphosphate buildup because all being used to make pyruvate fructose-1,6 diphosphate tries to speed up/ make sure that phosphate kinase is producing pyruvate o in eukaryotes, phosphate kinase can be phosphorylated to make it less active or dephosphorylated to make the enzyme more active be able to differentiate between molecules used in the TCA cycle and glycolysis

TCA Cycle each arrow correlates to an enzyme but most of the focus will be on, key point of control* --committed step (irreversible and specific to that particular pathway )

acetyl-CoA=2 carbons; citrate, aconitate, isocirate, a-ketoglutarate = 5 carbons succinyl Co-A, succinate, fumarate, malate, oxalaacetate =6 carbons key point of control in TCApyruvate acetyl Co-A (outside of TCA cycle, but still the key point of control) both molecules are outside of the cycle itself most important in terms of energy generation NAD(P)H, GTP (or ATP in some molecules, GTP more common in prokaryotes), FADH2 are all produced substrate level phosphorylationsomething is phosphorylated directly versus going through the electron transport system,\ o ATP and GTP are considered substrate level phosphorylation o GTP can give a phosphate to ADP to make ATP oxidative phosphorylationmost energy comes via this process o ATP made through the electron transport system from NADH o typically NADH 3 ATP and FADH2 2 ATP 34 ATP typically thought per glucose but can be lower than ten depending on the conditions in which the organisms lives in Amino acids, hemes (protein component), phophorins, made from compounds shuttled out of this process ultimately have to have regeneration of some of the compounds to keep the cycle going: o pyruvate + CO2 + ATP + H2O (pyruvate carboxylaze) OAA+ ADP + Pi + 2H+ o separate reaction that keeps the cycle going o the reaction requires ATP and turns is into ADP + Pin only tends to operate under aerobic conditions, a variation than can occur under anaerobic conditions key point of control: pyruvate dehydrogenase (complex): pyruvate Acetyl Co-A o the enzyme carries an oxidative decarboxylation o the process yields NADH and carbon dioxide o 3 carbons 2 carbons (which binds with oxalacetic acid to start the cycle)

o allosteric inhibitors: tend to see ATP, but NADH and Acetyl Co-A are also inhibitors (fully inhibited if all three are present, a little less if only 2 of the three, and even less if only one of the inhibitors are present o irreversible reaction Acetyl Co-A citrate (citrate synthase) o allosteric o inhibited by ATP Isocitrate a-ketogularate (isocitrate dehydrogenase) o allosteric o decarboxyalation, production of NADH or FADH2 o inhibited by ATP and activated by ADP (lower levels of ATP) o double whammy, it can measure both ATP and ADP levels a-ketogularate (a-KG dehydrogenase) succ-CoA o allosteric o inhibited by succinyl coA and NADH (products of the reaction) the TCA pathway yields the most energy o 4 NADH, 1FADH2, 1GTP per pyruvate (double for glucose) o ATP Equivalents: 4 NADH (12 ATP), 1 FADH2 (2 ATP), 1 GTP (1 ATP) = 15 ATP per pryvuate GTP through substrate level phosphorylation NADH, FADH2 through oxidative phosphorylation (ETS) o per glucose: glycolysis: 2 ATP; 2 NADH intermediate: 2 NADH TCA: 2 GTP/ATP; 6 NADH; 2FADH2 total NADH to ETS = 10 and total FADH2 = 4 34 ATP through oxidative phosphorylation & 4 ATP substrate level = 38 in prokaryotes in eukaryotes 36 ATP (transport across membrane uses energy) **glycolysis: makes 4 ATP eq (1NADH & 1 ATP) but uses 2 (1 PEP & 1 ATP), net = 2 (ATP eqv) anabolically important because products are shuttled out to make amino acids

o what would have to happen to amino acid intermediates for it to replenish the intermediates o Electron Transport System oxygen is the terminal electron acceptor ** determines whether aerobic or anaerobic proton motive forceproton gradient across the membrane is used to make ATP (NADH and FAADH2 are shuttled in to create the gradient) all organisms have variations of the molecules utilized in the ETS, determining what cytochromes are in the organisms are helpful in determining what the organism is quinonenonproteinaous, soluble in lipids (sitting within the phospholipid bilayers) o responsible for carrying protons across the membrane o only complex that can do so? two things happening in the membrane: o 1) the proteins (non proteins) setting up the proton gradient o 2) ATP synthase (operates separately from process above it synthesizes ATP but can also work backwards ( then called ATPase) if a proton gradient is needed, then ATP can be degraded to allow protons to be shuttled across the membrane F1 (hydrophilic) & F0 (hydrophobic) different functions F0= channel through which F1 = responsible for ATP production *** READ EXPERIMENT, chemiosmotic theory electron transport and ATP synthesis are two different entitities E. Coli o FP Fe-SQcyt b cty o energy yirld is different dependent on organism and environment it was found in ATP synthase

o responsbilbe for controlled dispassion oxidative phosphorylation o the protons are shuttled down its electrochemical gradient phosphorylating ATP inhibition of the ETS are one mechanism o create proton motive force, making the proton gradient o so when its inhibited no proton gradient being produced o therefore no protons are shuttled into ATP synthase and ADP is not phosphorylated o cyanide, o carbon monoxideinteracts with iron in cytochromes and prevents it from being able to carry oxygen, o azidelab setting, water bath, prevents algae growth Uncouplers are o dinitricphenol and o thernogenin (eukaryotic) (UPC1), found in brown fat or brown adpipose tissue, animals that hibernate or live in cold environment, vital in young infants found in inner mitochondrial membrane where ETS is the proton motive force is made but ADP is not phosphorylated thernogenin becomes the shuttling protein to generate heat instead of getting shuttled through ATP synthase o uncouple the two systems still get production of the protons, just shuttled through the uncoupling proteins o process occurs just prior to hibernation , increase synthesis in uncoupling proteins o produces non-shivering heat

Anaerobic Respiration Glycolysis o TCA Cycle

Electron Transport System o the terminal electron is acceptor is something other than oxygen*

Anaerobic Growth (Fermentation) Gylcolysis Fermentation some organisms can use anaerobic respiration using other inorganic (NO3 and SO4) or organic electron accepts to produce ATP proton motive force S elemental chemolithotrophic--< start using an inorganic compound photoheterotrophstart with an organic compound (glucose), light and CO2

Anabolic Nature a-KG glutamate and oxalacetate asparate mostly amino acids Fermentation (green, Stage III: Reduction) absence of oxygen, end products for fermentation are also end products for the cell: lactate, ethanol, H2, CO2 if the cells live in an environment containing these waste products, they will die this process does not make energy either the purpose of fermentation is to recycle and reform NAD+ o NAD+ is necessary to be an electron acceptor for gylycolysis as well as for the TCA cycle o NAD+ is necessary for glycolysis and TCA cycle to occur, o no energy is formed acids are the byproducts, the surrounding environement thus becomes more acidic, (lactate, acetate, formate)

9/12/2011 6:29:00 AM

9/12/2011 6:29:00 AM