Glycolysis learning outcomes Be able to state the 3 stages of respiration (E) Be able to describe an overview of the stages of glycolysis (C) Be able to describe in detail (e.g. name of an enzyme, why a process occurs) the process of glycolysis (A)

Respiration Things to cover: What is ATP and why is it important? What is the structure of ATP? What are co-enzymes, what is their role? What happens in: – Glycolysis

Starter Quiz Overall reaction for aerobic respiration Definition of respiration – The release of chemical potential energy from organic molecules Two uses of the energy released by respiration What does ATP stand for? – Adenosine triphosphate

Starter Quiz 1.Why does each cell require the structures associated with respiration? 1.ATP cannot cross the plasma membrane 2.2 advantages of ATP over direct energy transfer from glucose 1.Idea of packaging of energy 3.Why is all the chemical energy released during respiration not transferred to ATP 1.Lost as heat 4.How much energy is released through the condensation of ATP  ADP  AMP KJmol-1

The structure of ATP Adenosine triphosphate -ATP is a phosphorylated nucleotide (similar to the structure of DNA and RNA) (ATP cant leave the cell where it is made)

Why ATP acts as an energy store... -When 1 phosphate group is removed from each molecule in one mole of ATP, 30.5 kJ of energy’s released -This is a hydrolysis reaction (requires water), and is catalysed by enzymes called ATPases Energy released (30.5KJ mol -1 ) ADP PiPi ATPase Water

The energy released from hydrolysis of ATP adenosine monophosphate adenosine diphosphate

ATP is useful as an energy carrier (currency) because it cycles. It also “packages” the energy released from respiration into useful amounts.

Respiration provides the energy required for the condensation reaction that converts ADP  ATP i.e. For each 30.5KJ mol -1 of energy that is released by hydrolysis of ATP the same energy must also be input from respiration to reform the ATP. The energy for condensation reaction comes from the chemical energy stored in Glucose.

Glucose (high stored chemical potential energy ) Carbon Dioxide + Water (low stored chemical potential energy ) Energy Heat Chemical Energy ATP ADP ATP ADP Energy Transferred Respiration How it all fits together Condensation Hydrolysis

5 questions 1.What is respiration? 2.Give three uses of respiration? 3.What are the three main components from ATP? 4.How much energy is released from hydrolysis of ATP? 5.Why do cells require ATP?

3. Krebs Cycle 2. Link Reaction 1. Glycolysis 4. Oxidative Phosphorylation 4 main processes in aerobic respiration

Key players you need to know the structure of in respiration Glucose ADP / ATP Co-enzymes: NAD, FAD, Co-enzyme A

The primary substrate for respiration is glucose (C 6 H 12 O 6 ) Other substrates can be used. We will talk about these later!

Enzymes in Respiration Respiration releases chemical potential energy from the substrate through a series of reactions. Each stage of respiration is catalysed by a specific enzyme Reactions in respiration are examples of Oxidation and Reduction reactions. – Oxidation: loss of electrons (loss of hydrogen). – Reduction: gain of electrons (gain of hydrogen).

Co-enzymes Enzymes needed to assist other enzymes in a reduction or oxidation reaction (because they can pick up and lose hydrogen atoms) Co-enzymes used in respiration: – NAD Nicotinamide Adenine Dinucleotide – CoA Coenzyme A – FAD Flavine Adenine Dinucleotide Co-enzymes that have been reduced are used in the final stage of respiration (oxidative phosphorylation) which produces a lot of ATP.

Co-enzymes how they work – NAD as an example NAD NADH Reduced NAD NADH Co-enzyme is oxidised Substrate Oxidised Substrate When a co-enzyme takes on a Hydrogen atom it is “reduced” When it deposits a Hydrogen it is oxidised Co-enzymes are continuously cycled Hydrogen atom lost H Hydrogen atom accepted by co-enzyme NAD H Hydrogen is removed. The hydrogen atom is used to generate ATP

NAD Derived from vitamin B Dinucelotide Nicotinamide is the hydrogen acceptor NAD (oxidised); NADH = reduced Used in Glycolysis Link reaction Krebs cycle (also anaerobic respiration – lactate pathways) Do not confuse with NADP

Coenzyme A (CoA) Derived from vitamin B Used in the link reaction to transfer products of glycolysis into the mitochondria Carries Ethanoate groups created through oxidation during the link reaction onto the krebs cycle

FAD Derived from vitamin B2 Made from Adenine Ribose and 2 phosphate groups FAD  FADH Used in the Krebs cycle

Respiration – Part 1 Glycolysis

Respiration: Glycolysis Takes place in the cytoplasm Does not require oxygen Glucose is split into two molecules of Pyruvate (a 3 carbon sugar) 2 parts: energy investment phase, energy pay off phase

Respiration: Glycolysis – outcomes Net gain of 2 ATP 2 reduced NAD (NADH) (2 molecules of pyruvate)

O CH 2 O Glucose P P P H Glucose-6-phosphate Step 1: Enzyme: Hexokinase ATP ADP Glucose enters the cell The enzyme hexokinase transfers a phosphate from ATP to the glucose. The charge on the Phosphorylation has two affects: 1.phosphate group prevents the glucose from leaving the cell because the plasma membrane is impermeable to ions 2.Makes the glucose more chemically reactive Ledger ATP : -1 (ADP: +1)

Glycolysis steps Glucose (6 Carbon) Glucose-6-phosphate (6 carbon) Fructose-6-phosphate (6 carbon) Hexose1,6-bisphosphate (6 carbon) 2 x Triose Phosphate (3 carbon) 2x intermediate compounds (3 carbon) 2x Pyruvate (3 carbon) ADP + Pi ATP 2 x Oxidased NAD 2 x Reduced NAD ATPADP ATPADP 2 x ADP + Pi ATP 2 x

Steps 1-4: Energy investment phase ATP is hydrolysed; exogonic reaction 2 ATP converted to ADP The stored energy in ATP is needed to destabilise and activate the substrate molecule by phosphorylation of the substrate. Therefore some ATP is needed in order to generate ATP

Glycolysis step 1: Phosphorylation Glucose (6 Carbon) Glucose-6-phosphate (6 carbon) ATP is hydrolysed. Energy released is used to attach P i to the Glucose at Carbon number 6 Destabilises glucose Prevents from leaving the cell ATP ADP

Glycolysis step 2: Phosphorylation Glucose-6-phosphate (6 carbon) Fructose-1-phosphate (6 carbon) Configuration of the molecule changes to form a 5 carbon ring Requires an isomerase enzyme

C C O CH 2 O H OH H HO H OH H H C C C P Glucose-6-phosphate Step 2: Enzyme: Phosphoglucoisomerase Ledger ATP : -1 (ADP: +1) The enzyme rearranges the atoms in glucose-6-phosphate to form its isomer, fructose-6-phosphate. The structure of fructose-6-phosphate can be represented like this Fructose-6-phosphate O CH 2 O P CH 2 OH

Glycolysis step 3: Phosphorylation Fructose-1-phosphate (6 carbon) Hexose1,6-bisphosphate (6 carbon) Another ATP is hydrolysed and the P i released attaches to the fructose at carbon number 6. ATP ADP

O CH 2 O Fructose-6-phosphate P CH 2 O P P P H Fructose-1, 6-bisphosphate Ledger ATP : -1 (ADP: +1) Step 3: Enzyme: Phosphofructokinase The enzyme transfers a phosphate group from ATP to the sugar With phosphate groups on each end the sugar is now ready to split in half Note: 2 molecules of ATP have been invested so far in the process Ledger ATP : -2 (ADP: +2) ATP ADP

Glycolysis step 4: Splitting hexose Hexose1,6-bisphospahe (6 carbon) Triose phosphate (3 carbon) The hexose sugar is split into two 3 carbon sugars (for the rest of the process we only follow one of the sugar molecules) Triose phosphate (3 carbon)

Step 4: Enzyme: Aldolase / Isomerase Ledger ATP : -2 (ADP: +2) The enzyme cleaves the Fructose-1,6-bisphosphate into two 3 carbon sugars: glyceraldehyde-3-phosphate dihydroacetone phosphate Both sugars are isomers of each other Isomerase catalyses the reversible conversion between the two isomers

Steps 5 -6: Energy payback Condensation reactions convert ADP to ATP. These reactions are endogonic. Energy is transferred from the substrate to the ATP molecule. 4 molecules of ATP are formed (net gain of 2 ATP) Activation energy for the phosphorylation of ADP comes directly from oxidation of the substrate. Hydrogen atoms are lost from the substrate – they are accepted by the co-enzyme NAD which forms NADH (x2). (The NADH is used later in oxidative phosphorylation to generate ATP)

Glycolysis step 5: Oxidation of triose phosphate Intermediate 3 carbon compound Remember this step happens to each Triose molecule; therefore: 2 hydrogen atoms accepted by NAD NAD is reduced producing 2 NADH 2 ATP are formed by the process of substrate level phosphorylation. This is where ATP is formed directly using energy released from the oxidation of the substrate. Triose phospahte (3 carbon) ADP ATP NAD NADH

O CH 2 O P C CHOH NAD Step 5: Enzyme: Triose phosphate dehydrogenase Ledger ATP : -2 (ADP: +2) NADH: +2 The enzyme catalyses two steps first, the Glyceraldehyde-3-phosphate is oxidised by NAD. Electrons and H+ are transferred to the NAD forming NADH. Results in production of 2 molecules of NADH The oxidation of the sugar is very exergonic The enzyme uses released energy to attach a phosphate group to the sugar The phosphate ions come from the pool of inorganic phosphate in the cytosol H O P

O CH 2 O P C CHOH Step 5b: Enzyme: Phosphoglycerokinase Ledger ATP : -2 (ADP: +2) NADH: +2 The phosphate added in the previous step is transfered to ADP step 7 gives a gain of 2 molecules of ATP per molecule of glucose that starts glycolysis The energy gained by the oxidation of sugar in step 6 has now been released O P Ledger ATP : 0 (ADP: 0) NADH: +2 P P ATP ADP 3 - Phosphoglycerate

Glycolysis step 6: Conversion to Pyruvate Pyruvate (3 carbon compound) 2 ATP are formed; substrate level phosphorylation 4 steps each catalysed by a specific enzyme 2 molecules of pyruvate are formed Intermediate 3 carbon compound ADP ATP

O CH 2 O C CHO Step 6: Enzyme: Phosphoglyceromutase Ledger ATP : -2 (ADP: +2) NADH: +2 The enzyme relocates the remaining phosphate group in preparation for the next step O-O- Ledger ATP : 0 (ADP: 0) NADH: Phosphoglycerate H P 2-Phosphoglycerate

O CH 2 C C Step 6b: Enzyme: Endolase Ledger ATP : -2 (ADP: +2) NADH: +2 The enzyme causes a double bond to form in the molecule by extracting a molecule of water from each molecule of 2- Phosphoglycerate. This arrangement makes the remaining phosphate bond very unstable O-O- Ledger ATP : 0 (ADP: 0) NADH: Phosphoglycerate OH P 2-Phosphoenolpyruvate OH Water

O CH 2 C C Step 6c: Enzyme: Pyruvate Kinase Ledger ATP : -2 (ADP: +2) NADH: +2 The remaining phosphate group transfers to ADP A net gain in Glycolysis of 2 ATP per molecule of glucose Each molecule of glucose has produced 2 molecules of pyruvate O-O- Ledger ATP : 0 (ADP: 0) NADH: +2 2-Phosphoenolpyruvate P Pyruvate O P P ATP ADP Ledger ATP : +2 (ADP: 0) NADH: +2

Glycolysis steps Glucose (6 Carbon) Glucose-6-phosphate (6 carbon) Fructose-6-phosphate (6 carbon) Hexose1,6-bisphosphate (6 carbon) 2 x Triose Phosphate (3 carbon) 2x intermediate compounds (3 carbon) 2x Pyruvate (3 carbon) ADP + Pi ATP 2 x Oxidased NAD 2 x Reduced NAD ATPADP ATPADP 2 x ADP + Pi ATP 2 x