Cellular Respiration Stage 1: Glycolysis 2007-2008

ATP The point is to make ATP! What’s the point? http://vcell.ndsu.nodak.edu/animations/glycolysis_overview/movie-flash.htm 2007-2008

glucose      pyruvate Glycolysis Breaking down glucose “glyco – lysis” (splitting sugar) ancient pathway which harvests energy where energy transfer first evolved transfer energy from organic molecules to ATP still is starting point for ALL cellular respiration but it’s inefficient generate only 2 ATP for every 1 glucose occurs in cytosol glucose      pyruvate 2x 6C 3C Why does it make sense that this happens in the cytosol? Who evolved first? That’s not enough ATP for me!

Evolutionary perspective Prokaryotes first cells had no organelles Anaerobic atmosphere life on Earth first evolved without free oxygen (O2) in atmosphere energy had to be captured from organic molecules in absence of O2 Prokaryotes that evolved glycolysis are ancestors of all modern life ALL cells still utilize glycolysis The enzymes of glycolysis are very similar among all organisms. The genes that code for them are highly conserved. They are a good measure for evolutionary studies. Compare eukaryotes, bacteria & archaea using glycolysis enzymes. Bacteria = 3.5 billion years ago glycolysis in cytosol = doesn’t require a membrane-bound organelle O2 = 2.7 billion years ago photosynthetic bacteria / proto-blue-green algae Eukaryotes = 1.5 billion years ago membrane-bound organelles! Processes that all life/organisms share: Protein synthesis Glycolysis DNA replication

Overview 10 reactions glucose C-C-C-C-C-C fructose-1,6bP ATP 2 enzyme 10 reactions convert glucose (6C) to 2 pyruvate (3C) produces: 4 ATP & 2 NADH consumes: 2 ATP net yield: 2 ATP & 2 NADH ADP 2 enzyme fructose-1,6bP P-C-C-C-C-C-C-P enzyme enzyme enzyme DHAP P-C-C-C G3P C-C-C-P NAD+ 2 2H 2Pi 1st ATP used is like a match to light a fire… initiation energy / activation energy. Destabilizes glucose enough to split it in two enzyme 2 enzyme ADP 4 2Pi enzyme ATP 4 pyruvate C-C-C DHAP = dihydroxyacetone phosphate G3P = glyceraldehyde-3-phosphate

Glycolysis summary -2 ATP 4 ATP endergonic invest some ATP exergonic ENERGY INVESTMENT -2 ATP G3P C-C-C-P exergonic harvest a little ATP & a little NADH ENERGY PAYOFF 4 ATP Glucose is a stable molecule it needs an activation energy to break it apart. phosphorylate it = Pi comes from ATP. make NADH & put it in the bank for later. like $$ in the bank net yield 2 ATP 2 NADH NET YIELD

1st half of glycolysis (5 reactions) Glucose “priming” CH2OH Glucose O 1 ATP hexokinase get glucose ready to split phosphorylate glucose molecular rearrangement split destabilized glucose ADP CH2 O P O Glucose 6-phosphate 2 phosphoglucose isomerase CH2 O P O CH2OH Fructose 6-phosphate 3 ATP phosphofructokinase P O CH2 CH2 O P ADP O Fructose 1,6-bisphosphate aldolase 4,5 H P O CH2 isomerase C O C O Dihydroxyacetone phosphate Glyceraldehyde 3 -phosphate (G3P) CHOH CH2OH CH2 O P NAD+ Pi 6 Pi NAD+ NADH glyceraldehyde 3-phosphate dehydrogenase NADH P O O CHOH 1,3-Bisphosphoglycerate (BPG) 1,3-Bisphosphoglycerate (BPG) CH2 O P

2nd half of glycolysis (5 reactions) DHAP P-C-C-C G3P C-C-C-P Energy Harvest NADH production G3P donates H oxidizes the sugar reduces NAD+ NAD+  NADH ATP production G3P    pyruvate PEP sugar donates P “substrate level phosphorylation” ADP  ATP NAD+ Pi Pi NAD+ 6 NADH NADH ADP 7 ADP O- phosphoglycerate kinase ATP C ATP CHOH 3-Phosphoglycerate (3PG) 3-Phosphoglycerate (3PG) CH2 O P 8 O- phosphoglycero- mutase C O H C O P 2-Phosphoglycerate (2PG) 2-Phosphoglycerate (2PG) CH2OH 9 O- H2O enolase H2O C O C O P Phosphoenolpyruvate (PEP) Phosphoenolpyruvate (PEP) CH2 O- ADP 10 ADP pyruvate kinase C O ATP ATP C O Pyruvate Pyruvate CH3

Substrate-level Phosphorylation In the last steps of glycolysis, where did the P come from to make ATP? the sugar substrate (PEP) H2O 9 10 Phosphoenolpyruvate (PEP) Pyruvate enolase pyruvate kinase ADP ATP CH3 O- O C P CH2 P is transferred from PEP to ADP kinase enzyme ADP  ATP ATP

Energy accounting of glycolysis 2 ATP 2 ADP glucose      pyruvate 6C 2x 3C 4 ADP ATP 4 All that work! And that’s all I get? 2 NAD+ 2 And that’s how life subsisted for a billion years. Until a certain bacteria ”learned” how to metabolize O2; which was previously a poison. But now pyruvate is not the end of the process Pyruvate still has a lot of energy in it that has not been captured. It still has 3 carbons bonded together! There is still energy stored in those bonds. It can still be oxidized further. But glucose has so much more to give! Net gain = 2 ATP + 2 NADH some energy investment (-2 ATP) small energy return (4 ATP + 2 NADH) 1 6C sugar  2 3C sugars

Is that all there is? Not a lot of energy… for 1 billon years+ this is how life on Earth survived no O2 = slow growth, slow reproduction only harvest 3.5% of energy stored in glucose more carbons to strip off = more energy to harvest O2 glucose     pyruvate O2 So why does glycolysis still take place? 6C 2x 3C O2 O2 O2

raw materials  products But can’t stop there! Pi NAD+ G3P 1,3-BPG NADH DHAP 7 8 H2O 9 10 ADP ATP 3-Phosphoglycerate (3PG) 2-Phosphoglycerate (2PG) Phosphoenolpyruvate (PEP) Pyruvate NAD+ NADH Pi 6 raw materials  products Glycolysis glucose + 2ADP + 2Pi + 2 NAD+  2 pyruvate + 2ATP + 2NADH Going to run out of NAD+ without regenerating NAD+, energy production would stop! another molecule must accept H from NADH so NAD+ is freed up for another round

How is NADH recycled to NAD+? with oxygen aerobic respiration without oxygen anaerobic respiration “fermentation” Another molecule must accept H from NADH pyruvate H2O NAD+ CO2 recycle NADH NADH O2 NADH acetaldehyde acetyl-CoA NADH NAD+ NAD+ lactate lactic acid fermentation which path you use depends on who you are… Krebs cycle ethanol alcohol fermentation

Fermentation (anaerobic) Bacteria, yeast 1C 3C 2C pyruvate  ethanol + CO2 NADH NAD+ back to glycolysis beer, wine, bread Animals, some fungi Count the carbons!! Lactic acid is not a dead end like ethanol. Once you have O2 again, lactate is converted back to pyruvate by the liver and fed to the Kreb’s cycle. pyruvate  lactic acid 3C NADH NAD+ back to glycolysis cheese, anaerobic exercise (no O2)

Alcohol Fermentation pyruvate  ethanol + CO2 Dead end process bacteria yeast Alcohol Fermentation recycle NADH 1C 3C 2C pyruvate  ethanol + CO2 NADH NAD+ back to glycolysis Dead end process at ~12% ethanol, kills yeast can’t reverse the reaction

Lactic Acid Fermentation animals some fungi recycle NADH Lactic Acid Fermentation O2 pyruvate  lactic acid 3C  NADH NAD+ back to glycolysis Reversible process once O2 is available, lactate is converted back to pyruvate by the liver

Pyruvate is a branching point fermentation anaerobic respiration mitochondria Krebs cycle aerobic respiration http://vcell.ndsu.nodak.edu/animations/glycolysis_reactions/movie-flash.htm

And how do we do that? ATP synthase ADP + Pi  ATP set up a H+ gradient allow H+ to flow through ATP synthase powers bonding of Pi to ADP ADP + Pi  ATP ADP P + ATP But… Have we done that yet?

Overview 10 reactions glucose C-C-C-C-C-C fructose-1,6bP ATP 2 10 reactions convert glucose (6C) to 2 pyruvate (3C) produces: 4 ATP & 2 NADH consumes: 2 ATP net: 2 ATP & 2 NADH ADP 2 fructose-1,6bP P-C-C-C-C-C-C-P DHAP P-C-C-C G3P C-C-C-P NAD+ 2 2H 2Pi 1st ATP used is like a match to light a fire… initiation energy / activation energy. Destabilizes glucose enough to split it in two 2 ADP 4 2Pi ATP 4 pyruvate C-C-C

Cellular Respiration Stage 2 & 3: Oxidation of Pyruvate Krebs Cycle 2006-2007

Glycolysis is only the start Pyruvate has more energy to yield 3 more C to strip off (to oxidize) if O2 is available, pyruvate enters mitochondria enzymes of Krebs cycle complete the full oxidation of sugar to CO2 2x 6C 3C glucose      pyruvate Can’t stop at pyruvate == not enough energy produced Pyruvate still has a lot of energy in it that has not been captured. It still has 3 carbons! There is still energy stored in those bonds. pyruvate       CO2 3C 1C

Cellular respiration

Mitochondria — Structure Double membrane energy harvesting organelle smooth outer membrane highly folded inner membrane cristae intermembrane space fluid-filled space between membranes matrix inner fluid-filled space DNA, ribosomes enzymes free in matrix & membrane-bound intermembrane space inner membrane outer matrix cristae mitochondrial DNA What cells would have a lot of mitochondria?

Mitochondria – Function Oooooh! Form fits function! Mitochondria – Function Dividing mitochondria Who else divides like that? Membrane-bound proteins Enzymes & permeases bacteria! Almost all eukaryotic cells have mitochondria there may be 1 very large mitochondrion or 100s to 1000s of individual mitochondria number of mitochondria is correlated with aerobic metabolic activity more activity = more energy needed = more mitochondria What cells would have a lot of mitochondria? Active cells: • muscle cells • nerve cells What does this tell us about the evolution of eukaryotes? Endosymbiosis! Advantage of highly folded inner membrane? More surface area for membrane-bound enzymes & permeases

[ ] Oxidation of pyruvate Pyruvate enters mitochondrial matrix 3 step oxidation process releases 2 CO2 (count the carbons!) reduces 2 NAD  2 NADH (moves e-) produces 2 acetyl CoA Acetyl CoA enters Krebs cycle [ 2x ] pyruvate    acetyl CoA + CO2 3C NAD 2C 1C Where does the CO2 go? Exhale! CO2 is fully oxidized carbon == can’t get any more energy out it CH4 is a fully reduced carbon == good fuel!!!

Pyruvate oxidized to Acetyl CoA NAD+ reduction Coenzyme A Acetyl CoA Pyruvate Release CO2 because completely oxidized…already released all energy it can release … no longer valuable to cell…. Because what’s the point? The Point is to make ATP!!! CO2 C-C C-C-C oxidation 2 x [ ] Yield = 2C sugar + NADH + CO2

Krebs cycle 1937 | 1953 aka Citric Acid Cycle in mitochondrial matrix 8 step pathway each catalyzed by specific enzyme step-wise catabolism of 6C citrate molecule Evolved later than glycolysis does that make evolutionary sense? bacteria 3.5 billion years ago (glycolysis) free O2 2.7 billion years ago (photosynthesis) eukaryotes 1.5 billion years ago (aerobic respiration = organelles  mitochondria) Hans Krebs 1900-1981 The enzymes of glycolysis are very similar among all organisms. The genes that code for them are highly conserved. They are a good measure for evolutionary studies. Compare eukaryotes, bacteria & archaea using glycolysis enzymes. Bacteria = 3.5 billion years ago glycolysis in cytosol = doesn’t require a membrane-bound organelle O2 = 2.7 billion years ago photosynthetic bacteria / proto-blue-green algae Eukaryotes = 1.5 billion years ago membrane-bound organelles! Processes that all life/organisms share: Protein synthesis Glycolysis DNA replication

Count the carbons! x2 3C 2C 4C 6C 4C 6C 5C 4C 4C 4C pyruvate 3C 2C acetyl CoA citrate 4C 6C 4C 6C This happens twice for each glucose molecule oxidation of sugars CO2 A 2 carbon sugar went into the Krebs cycle and was taken apart completely. Two CO2 molecules were produced from that 2 carbon sugar. Glucose has now been fully oxidized! But where’s all the ATP??? x2 5C 4C CO2 4C 4C

Count the electron carriers! pyruvate 3C 2C acetyl CoA NADH NADH citrate 4C 6C 4C 6C reduction of electron carriers This happens twice for each glucose molecule CO2 Everytime the carbons are oxidized, an NAD+ is being reduced. But wait…where’s all the ATP?? NADH x2 5C 4C FADH2 CO2 4C 4C NADH ATP

So we fully oxidized glucose C6H12O6  CO2 & ended up with 4 ATP!

Electron Carriers = Hydrogen Carriers Krebs cycle produces large quantities of electron carriers NADH FADH2 go to Electron Transport Chain! ADP + Pi ATP

Energy accounting of Krebs cycle 2x 4 NAD + 1 FAD 4 NADH + 1 FADH2 pyruvate          CO2 1 ADP 1 ATP 3C 3x 1C ATP Net gain = 2 ATP = 8 NADH + 2 FADH2

Value of Krebs cycle? If the yield is only 2 ATP then how was the Krebs cycle an adaptation? value of NADH & FADH2 electron carriers & H carriers reduced molecules move electrons reduced molecules move H+ ions to be used in the Electron Transport Chain

What’s the point? The point is to make ATP! ATP 2006-2007

And how do we do that? ATP synthase ADP + Pi  ATP set up a H+ gradient allow H+ to flow through ATP synthase powers bonding of Pi to ADP ADP + Pi  ATP ADP P + ATP But… Have we done that yet?