BCH210H1 Lecture 27: Lecture 27

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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 irreversile
- 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 hdrol attaks 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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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.

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