BCH210H1 Lecture 27: Lecture 27
Lecture 27: Glycolysis and Enzyme Inhibition
• When phosphocreatine levels fall (bind to phosphate and act as storage to replenish ATP), additional ATP
production is required
• Glycogen breakdown generates glucose 1-phosphate (must be converted to G6P for glycolysis)
• Glucose enters muscle cells from the blood using GLUT4 transporters
- GLUT4 has 12 transmembrane helices and forms a binding site for glucose transport
• Glucose provides a substrate for the glycolysis that will lead to ATP production in muscle (and other cells)
Glycolysis
• Stage 1 – first five steps (energy input phase/ stages that need energy to
generate a lot more energy)
- Require 2 ATP for glucose
- At the end of stage 1, 2 GAP (glyceraldehyde 3-phosphate)
• Stage 2 – last five steps (generate ATP and NADH)
- Generates 4 ATPs (stage 2 runs twice) and NADHs
- At the end of stage 2 is 2 pyruvate
• All takes place in cytoplasm which will head to mitochondria if O2 is available
- In mitochondria - further oxidation and production of waste products (CO2) and
ATP through oxidative phosphorylation
• Under cellular conditions, concentration of substrates and products can change
the free energy for reaction
Free energy of glycolysis
• Negative overall pathway – favorable under standard conditions
• Negative overall pathway (greater change in free energy) for actual
free energy
- Some have VERY big negative slopes (1,3,10) - regulated
- 2.4.5.6.7.8.9 are flat - reversible
Glycolysis and beyond
• Glucose enters the glycolytic pathway (10 enzymes) found in the cytoplasm
• Glucose (6C) is converted into 2 pyruvate (3C) and energy release of 2 ATP (net)
• If O2 is available, pyruvate enters mitochondria and is converted to acetyl CoA
• Further oxidation takes place in the mitochondrial matrix and a lot more ATP is made
Glycolysis Regulation (Favorable with negative free energy changes)
1. Hexokinase can be inhibited by buildup of its product (product inhibition), glucose-6-phosphate
3. PFK-1 inhibited by the amount of ATP, and activated by the amount of AMP or β-Dfructose-2,6-bisphosphate
10. Pyruvate kinase is regulated by phosphorylation → inactivation.
• All three steps involve large ΔG°’ and are irreversile
- That is why we need regulation as above
- The availability of glucose also regulates glycolysis (no glucose = pathway off)
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1. Hexokinase (recognizes hexoses)
• Glucose is phosphorylated using ATP
• Adding phosphate to C6 from ATP
• Hydroxyl on C6 attacks y phosphate → transfer phosphate to C6
Hexokinase Binding
• Mg2+-ATP2- binds to large lobe away from glucose binding
• Substrates bind in the active site via charged and polar residues
- Negative ATP interacting with positive AAs
- Glucose with hydroxyls interact with polar AAs
• This results in a conformational change where the enzyme closes around the substrates – induced fit model of
binding (transfer of P to C6)
• C6 hdrol attaks the γ phosphate → G6P
Hexokinase Mechanism
• ATP (negative charges coordinated with Mg ion)
• 2 positively charged amino acids (Lys and Arg) coordinate
negatively charge ATP and hold in active site
• Glucose hydroxyl on C6 attacks y phosphate
• Glucose 6-phosphate + ADP
2. Phosphoglucose Isomerase
• The 6 member ring opens up to the linear form, an
isomerization reaction moves the C=O from C1 to C2
• Aldose sugar to ketose sugar
3. Phosphofructokinase (PFK-1)
• PFK-1 phosphorylates C1 hydroxyl using ATP
- 2nd ATP requiring step
- Phosphate from ATP transferred to C1 by
hydroxyl on fructose 6-phosphate attacking y
phosphate
• PFK-1 is inhibited allosterically by ATP and activated by Fructose 2,6 bisP and AMP (lower energy)
- F-2, 6-BP also activates (auxiliary enzyme)
◼ PFK-2 will transfer phosphate to C2 which can stimulate PFK-1 and
activate to speed things up
Allosteric Regulation of PFK-1
• PFK-2 (F-2,6-phosphate) and AMP allosterically activates PFK-1
• High concentration AMP, outcompete ATP binding to allosteric site
• ATP acting as allosteric regulator but also enzyme reaction using ATP
• Lots of AMP, binding allosteric site, inhibit enzyme
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BCH210H1 Full Course Notes
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Document Summary
Glut4 has 12 transmembrane helices and forms a binding site for glucose transport: glucose provides a substrate for the glycolysis that will lead to atp production in muscle (and other cells) Glycolysis: stage 1 first five steps (energy input phase/ stages that need energy to generate a lot more energy) At the end of stage 1, 2 gap (glyceraldehyde 3-phosphate: stage 2 last five steps (generate atp and nadh) Generates 4 atps (stage 2 runs twice) and nadhs. At the end of stage 2 is 2 pyruvate: all takes place in cytoplasm which will head to mitochondria if o2 is available. In mitochondria - further oxidation and production of waste products (co2) and. Atp through oxidative phosphorylation: under cellular conditions, concentration of substrates and products can change the free energy for reaction. Free energy of glycolysis: negative overall pathway favorable under standard conditions, negative overall pathway (greater change in free energy) for actual free energy.