Biochemistry I Glycolysis (stage 1 & 2) Stage __ Stage __ Chapter 16 – part 1 Dr. Ray http://higheredbcs.wiley.com/legacy/college/boyer/0471661791/animations/1 animations.htm Glycolysis Enzyme Matchup Glycolysis: A Common Pathway Aerobic Metabolism: From Glucose to Pyruvate Glycolysis Derived from the Greek stem glyk-, "sweet," and the word lysis, "dissolution.“ Glycolysis is the sequence of biochemical reactions that metabolizes one molecule of glucose (6C) to two molecules of pyruvate (3C) with the concomitant net production of two molecules of ATP, and two molecules of NADH. Overall Transformation of Glycolysis: • is catalyzed by 10 glycolytic enzymes • cost 2 ATP, produce 4 ATP = net gain 2 ATP + Glucose + 2 NAD + 2 ADP + 2Pi + 2 2 pyruvate + 2 NADH + 2 ATP + 2H2O + 2 H Glycolysis • This is a central catabolic pathway that you need to understand and know in detail! For all 10 steps you need to know: 1. Structures (Fisher projections) of reactants & products 2. Type of chemical transformation (reaction) that occurs 3. All required cofactors 4. Specific name of enzyme catalyst & the class of the enzyme 5. The mechanism of the enzymatic reactions catalyzes by: - hexokinase (step 1) - aldolase (step 4) - pyruvate kinase (step 10) 6. Energetics and mechanism: - glyceraldehyde 3-phosphate dehydrogenase (GAPDH – step 6) 3 Voet, Voet & Pratt, Fundamentals http://www.wiley.com/college/fob/quiz/quiz14/14-1.html of Biochemistry, 2002 Fig 14.1 Text Fig 16.3 Glycolysis 1 • Glycolysis is common to virtually 2 all cells, both prokaryotic and eukaryotic. 3 • In eukaryotic cells, glycolysis takes place in the cytosol. 4 • The product pyruvate is transported into mitochondria. 5 Glycolysis is divided into 3 stages: 1) preparatory phase (stage 1) 6 2) formation of GAP (stage 2) 7 3) payoff phase (stage 3) 1) Does every intermediate metabolite in 8 glycolysis have a phosphoryl group attached? 9 2) Is every metabolite a carbohydrate? Why? 10 http://higheredbcs.wiley.com/legacy/college/voet/047121495 7/guided_ex/glycolysis_overview/glycolysis_overview.html Energy Changes in Glycolysis Reactions 1 - 5: Preparatory Stage Energy Investment • Glucose 2 GAP • 6C Glucose is phosphorylated and cleaved to TWO 3C units called DHAP and GAP. • Consume 2 molecules of ATP Reactions 6 - 10 Energy Recovery • 2 GAP 2 pyruvate • 3C GAP is oxidized to form TWO “high energy” molecules 13BPG & PEP • Produce 4 molecules of ATP per 1 glucose Campbell & Farrell, Biochemistry 4Ed 2003 Glycolysis Questions 1) Complete the following phrases: a) Glycolysis is a/an ______________ pathway as the use of O2 does not occur till a later stage of carbohydrate catabolism. b) ATP that is formed directly in glycolysis involves the transfer of phosphoryl groups from high-energy intermediates (donors) to ADP, to form ATP, and is called: ____________ c) NADH formed during glycolyisis stores “reducing power” and is used in a later pathways to form ATP by __ . As electrons flow from NADH O2 at the inner mitochondrial membrane, the free energy released is utilized (captured). 2) During glycolysis: • How many C-C bond breaking reactions occur? • How many isomeriztion reactions occur? • How many phosphoryl transfers involving kinases occur? 6 Stages of Glycolysis Stage 1 is the conversion of glucose into fructose 1,6- bisphosphate (FBP), via three steps: • a phosphorylation • an isomerization • a second phosphorylation The STRATEGY of these initial steps in glycolysis is: (1) activate glucose by phosphorylation, (2) trap it in the cell, and (3) to form a compound that can be readily cleaved into phosphorylated 3-carbon units. Stage 2 is the cleavage of the fructose 1,6-bisphosphate (FBP) into two three-carbon fragments by Retro-Aldol reaction. The resulting three-carbon units (DHAP & GAP) are readily interconvertible (via isomerization). Stage 3 is the energy recovery stage which involves harvestingATP (and NADH) as the three-carbon fragments are oxidized to pyruvate. Net gain 2 ATP (plus 2 NADH) /glucose 7 Stages 1 & 2 of Glycolysis Energy Changes (5 steps) Preparatory (Energy Investment) Stage: • Glucose 2 GAP (glyceraldehyde-3-phosphate) • 6C Glucose is phosphorylated and cleaved to 1 TWO 3C units called DHAP and GAP. • Reactions 1 to 5 CONSUME TWO molecules of ATP (costs energy) 1.What types of chemical transformations 2 (reactions) occur during 1st half of glycolysis? 1. phosphorylation 4. Retro-aldol 2. isomerization cleavage 3 3. phosphorylation 5. isomerization 4 5 5 DHAP isomerization GAP 8 Voet, Voet & Pratt, Fundamentals of Biochemistry, 2002 Fig 14.7 Glycolysis For all 10 steps you need to know: 1. structures 1 2. chemical transformation 3. cofactors 2 4. enzyme catalyst 5. mechanism 3 - hexokinase (step 1) - aldolase (step 4) - GAPDH (step 6) <energetics> 4 - pyruvate kinase (step 10) 5 Stages 1 & 2: 1 glucose 2 GAP step 5: 1 DHAP 1 GAP DHAP O O O H O H CH2 O P O- C C CH2OH CH2 O P O- ATP ATP O O- H OH 1 H OH 2 O 3 O O- 5 + CH2OH HO H HO H HO H HO H 4 O H H OH H OH H OH H OH C H OH H OH O H OH O H OH O ADP ADP H OH O CH OH CH O P O- 2 CH2 O P O- 2 CH2 O P O- CH2 O P O- O- O- O- O- Glucose G6P F6P FBP GAP Glycolysis For all 10 steps you need to know: 1. structures 1 2. chemical transformation 3. cofactors 2 4. enzyme catalyst 5. mechanism 3 - hexokinase (step 1) - aldolase (step 4) - GAPDH (step 6) <energetics> 4 - pyruvate kinase (step 10) 5 Stages 1 & 2: 1 glucose 2 GAP step 5: 1 DHAP 1 GAP Retro-Aldol Isomeri- Cleavage DHAP zation Phospho - Isomeri- Phospho- O O O H rylation O zation rylation H CH2 O P O- C C CH2OH CH2 O P O- ATP ATP O O- H OH 1 H OH 2 O 3 O O- 5 + CH2OH HO H HO H HO H HO H 4 O H H OH H OH H OH H OH C H OH H OH O H OH O H OH O ADP ADP H OH O CH OH CH O P O- 2 CH2 O P O- 2 CH2 O P O- CH2 O P O- O- O- O- O- Glucose G6P between at C6 C1 C2 F6P at C1 FBP C3 / C4 GAPC1 C2 Glycolysis Stages 1 & 2: Energy Investment & Cleavage 1 2 3 5 4 Stage 3: Energy Recovery DHAP O O O H O H CH2 O P O- C C CH2OH CH2 O P O- O O O- H OH H OH O O O- P O O- O O- CH OH O O- HOO H H O O O- O HOO- H 2 HO O-H C HO H C C C 6 C 7 C 8 O 9 O O H 10 H OH H OH H OH H OH C H OH O H OH O H OH O CH O P O- C O P O- C O H OH H OH O H OH O H OH O CH2 O P O- CH2 O P O- CH2 O P O- O- H O- OH O CH2OH C CH2OH CH O P O- CHO2- O P O- CH O P O- CH3 O- O2- 2 H H CH2 O P O- GAP 13BPG O- 3PG O- 2PG O- OP- yruvate Glucose G6P PEP F6P FBP GAP Logic of Glycolysis C-C bond cleavage in Key Step 4: FBP DHAP + GAP • Reaction catalyzed by aldolase cleaves Aldol 6C bis-phosphorylated fructose into two condensation 3C mono-phosphorylated sugars, via a retro-aldol (cleavage) reaction. a 1. What is an aldol reaction? b 2. Which functional groups in the reactant FBP are critical for the aldolase reaction? Reverse Aldol 3. Based on the C-C bond that is broken, cleavage why is it necessary to convert glucose to fructose prior to this step? 4. What types of transformations are necessary to turn glucose into fructose-1,6-bisphosphate (FBP)? 5. Why are TWO phosphorylation steps required prior to the aldolase step? 12 Voet, Voet & Pratt, Fundamentals of Biochemistry, 2002, p.388 Step 1 – Phosphorylation of Glucose: Glucose G6P Glucose + ATP G-6P + ADP DGo’ = - 16.7 kJ/mol • Glucose enters most cells through specific transporters. • Glucose is phosphorylated to Glucose-6-P by Hexokinase which catalyzes a phosphoryl group transfer from ATP to a primary alcohol of an aldohexose (broad specificity). • Hexokinase, like all other kinases, requires Mg2+(or another divalent metal ion such as Mn2+) for activity. The divalent metal ion forms a complex with ATP. • Anionic G6P will not leave cell, so phosphorylation is the first step Phosphoryl transfer (and shifts) Transferase - + ROH + ATP ROPO3 + ADP + H • Proteins that catalyze this reaction are called: ________ • Phosphate transfer almost always occurs from ATP (and not ADP) 1) What drives this reaction? Energy __________ upon phosphate transfer from an ____________________ phosphoryl donor ATP to form a ______________ phosphorylated alcohol 2) Why is the CH2OH group at Glucose + ATP G-6P + ADP C6 phosphorylated rather DGo’ = - 16.7 kJ/mol than any of the other alcohols? Glycolysis steps 1 and 3 use this reaction: C6 is a ________ while Step 1: on aldohexose (glucose) rest are __________ . A_________ is sterically Step 3: on ketohexose (fructose-6-P) more accessible. Step 1 – Phosphorylation of Glucose: Glucose G6P 1. Why does phosphorylation of an alcohol or other low energy group require the source of the phosphate to be ATP, rather than Pi (inorganic phosphate)? D Go’ of hydrolysis = -13.8 kJ/mol for Glucose-6-phosphate (G6P) • R-O-P group has some phosphoryl group transfer potential (is activated), so energy is required to attach the phosphate, but P does not have any.