Glycolysis Flashcards
Function
Breakdown glucose into 2, 3-c pyruvate molecules that can enter the TCA cycle
Reduce NAD+ to NADH (gather electrons)
Where does it occur
Cytoplasm
When does it occur
When cell needs to generate ATP
Stages of glycolysis
- Invest 2 ATP
2. Generate ATP (4 total, 2 net)
OVERALL REACTION
Glucose + 2ADP + 2 Pi + 2NAD+ –> 2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2H2O
What type of pathway?
Oxidative pathway
Glucose is oxidized & energy is released (-G)
How does glucose get into cell and why?
Transporters. Because glucose is a polar molecule
What transporters bring glucose into the cell?
Where are they located?
When do they function optimally?
What enzymes do they work with?
GLUT 2:
located in liver and pancreatic beta cells
high Km (15-20 mM) therefore will not get saturated after a meal
Works with glucokinase
GLUT 4:
Located in muscle and fat cells
Low Km (5mM)
Works with hexokinase
What will be the Km of GLUT transporters found in the brain and why?
The Km will be very low because the brain requires glucose all of the time therefore the transporters will be saturated all of the time and constantly taking up glucose
Transporters work on ______________ basis.
What is the trend?
Concentration dependent
High conc of transporters, more glucose brought into the cell (within Km range)
What are the regulated reactions?
What does that mean?
Steps 1, 3, 10
Those reactions are irreversible with that enzyme
Step 1
Phosphorylation
Glucose –> Glucose 6- Phosphate
- Adding negative charge traps glucose in cell because it can no longer be recognized by the transporter. Therefore, glucose is now committed to being metabolized
- Coupled to ATP hydrolysis (ATP donates the P).
- Reaction is favorable
- Enzymes: hexokinase and glucokinase
Step 2
Isomerization
Glucose 6-Phosphate Fructose 6- Phosphate
Important because Fructose 6- phosphate can be split into 2 similar 3-C molecules in step 5
Step 3
Phosphorylation
RATE LIMITING & COMMITTED STEP
Fructose 6-Phosphate –> Fructose 1,6 bisphosphate
Enzyme: PFK-1
- Adding an additional phosphate commits to glycolysis
Step 4
Cleavage
Fructose 1,6-bisphosphate GAP + DHAP
Enzyme: Aldolase
- DHAP is favored and more is produced
- GAP & DHAP are both 3-C molecules
Step 5
Isomerization
DHAP GAP
Enzyme: triose phosphate isomerase
- Not a favorable reaction but is made possible by using GAP as a substrate in step 6 (coupling)
Step 6
Oxidation-Reduction
GAP + NAD+ + Pi 1,3- BPG + NADH + H+
Enzyme: glyceraldehyde 3-phosphate dehydrogenase
Step 6- Part 1
Take aldehyde from GAP and oxidize it to carboxylic acid
NAD+ is reduced to NADH and picks up the electrons
- favorable
Step 6- Part 2
*Unfavorable step- coupling occurs
Add a phosphate to the carboxylic acid to form an acyl phosphate
HOW?
GAP has cysteine in its active site –> glyceraldehyde 3-phosphate dehydrogenase forms a thioester bond (high energy) with it –> thioester intermediate formed –> glyceraldehyde 3-phosphate dehydrogenase then breaks that bond –> nrg released –> Pi is added
Step 7
Transfer of a Phosphate Group
1,3-BPG + ADP + H+ 3- phosphoglycerate + ATP
Enzyme: phosphoglycerate kinase
Type of reaction: substrate level phosphorylation
Result: 2 ATP produced per glucose …. balances out 2 ATP input
Explain substrate level phosphorylation in glycolysis
Step 7
1,3- BPG has high phosphoryl transfer potential. So ATP is made by using the energy available in that molecule because it can transfer its P to a molecule of lower energy
Step 8
Isomerization
3-phosphoglycerate 2-phosphoglycerate
Enzyme: phosphoglycerate mutase
Purpose: arranging for next step
Step 9
Dehydration
2-phosphoglycerate phosphoenolpyruvate
Enzyme: enolase
- Remove an H2O molecule
Step 10
Transfer of Phosphate Group
Phosphoenolpyruvate + ADP + H+ –> pyruvate + ATP
Enzyme: Pyruvate kinase
Type of reaction: substrate level phosphorylation (phosphoenolpyruvate has high phosphoryl transfer potential)
Result: Net 2 ATP per glucose molecule
Differences in standard free energy change and overall free energy change in cell are due to ?
Concentrations are constantly changing in the cell
In aerobic metabolism, what happens to pyruvate?
Enters mitochondria where it is converted to acetyl CoA and goes into TCA cycle
Glucose –> 2 pyruvate –> 2 acetyl CoA
How are NAD+ regenerated in aerobic respiration?
The mitochondria
What is fate of pyruvate in anaerobic metabolism?
Pyruvate is reduced to lactate.
Glucose –> 2 pyruvate –> 2 lactate
Why is lactate dehydrogenase important?
It oxidizes NADH to NAD+ in absence of oxygen.
REGENERATES NAD+ for step 6. Could not carry out glycolysis without.
When is ATP input balanced out?
Step 7.
1,3- BPG has high phosphoryl transfer potential so by substrate level phosphorylation ATP is produced
(2 molecules ATP per glucose)
What two steps are substrate level phosphorylation?
Step 7 - 1,3- BPG has high phosphoryl transfer potential
Step 10- phosphoenolpyruvate has high phosphoryl transfer potential
how is hexokinase regulated?
Hexokinase is feedback inhibited by its product glucose 6-phosphate
How is glucokinase regulated?
It is inducible by enzyme. Insulin triggers the increased transcription of glucokinase in the liver. Thus, it is more of an adaptive response.
How is pyruvate kinase regulated?
2 ways….allosteric and hormonal
Allosteric regulation of pyruvate kinase (step 10)
Positive modifier = fructose 1,6-bisphosphate
Negative modifier = ATP (also feedback inhibitor because product of reaction)
What is the significance of fructose 1,6-bisphosphate as positive modifier of pyruvate kinase
Connects step 3 and 10…
Positive modification by fructose 1,6-bisphosphate ensures that pyruvate kinase is keeping up with PFK-1
If not present, steps 4-9 are all reversible and would go backwards when the products start to buildup and are not converted in the forward glycolytic direction
Significance of PFK-1
Dictates flow of all its subsequent reactions
It catalyzes the rate limiting step
Pyruvate kinase is ______active in the fasted state in the liver
LESS
Glycolysis _________ in the fasted state in the liver.
Explain.
DECREASES
Glucose that is made by breaking down glycogen goes into the blood to serve brain and red blood cells.
The alternative energy source is used during fasting (i.e. Fatty acids)
What is the alternative energy source in the liver during fasting
Fatty acids
Glucose is _____ in the fasting state so the liver _____
Limiting; the liver maintains glucose homeostasis by breaking down glycogen into glucose for the brain and red blood cells while using fatty acids as an alternative energy source.
Hormonal regulation of pyruvate kinase
Fed vs. fasted state
Fed state (high I/G): dephosphorylation of pyruvate kinase making it more active. How? Insulin activates phosphatases to reverse glucagon effects.
Fasted state (low I/G): phosphorylation of pyruvate kinase making it less active. How? Glucagon activates kinases (esp. PKA)
Summary of hormonal pyruvate kinase regulation.
Fed = _____ = activity
Fasted = _______ = activity
Fed = dephosphorylation = more active
Fasted = phosphorylated = less active
Regulation of PFK- 1 is
ONLY allosteric
Positive modifiers of PFK-1
AMP and fructose 2,6- bisphosphate
Negative modifier of PFK-1
ATP
What two enzyme’s regulation are related to energy charge and how?
PFK-1 and pyruvate kinase
PFK-1 activity _______ when energy charge is low
INCREASES
PFK-1 activity _________ when energy charge is high
Decreases
How is ATP a substrate and allosteric modifier in step 3?
Which is higher affinity?
ATP is a substrate when it binds to the active site when [ATP] is low
ATP is an allosteric modifier when it binds to the allosteric site when [ATP] is high and PFK-1 activity should be decreased.
Active site has higher affinity.
Which way do they allosteric modifiers of PFK-1 shift the reaction velocity graph?
HIGH AMP –> shift to left. PFK-1 can work at lower fructose 6-phosphate concentrations.
HIGH ATP –> shift to right.
At rest, glycolysis is _______
During exercise, glycolysis is ______
Corresponding energy charges?
Rest –> slow
high energy charge
Exercise –> Stimulated
Low energy charge (need to make ATP)
Explain the regulation of glycolysis in skeletal muscle AT REST
High energy charge…
ATP allosterically binds to PFK-1 AND pyruvate kinase and because it is a negative modifier, the activity of PFK-1 AND pyruvate kinase is decreased.
Steps 4-9 are also slowed down due to PFK-1.
PFK-1 slowing down causes fructose 6-phosphate to build up, which can then isomerize back to glucose 6-phosphate and and feedback inhibit hexokinase.
Explain the regulation of glycolysis in skeletal muscle DURING EXERCISE
Low energy charge causes positive allosteric modifier AMP to bind to PFK-1 and increase its activity.
More fructose 1,6-bisphosphate is produced which goes to pyruvate kinase and positively allosterically modifies that enzyme causing increased ATP production.
How and when is fructose 2,6-bisphosphate made?
Made during FED state by enzyme: PFK-2
Activity of PFK-2 in fed vs. fasted state
Fed state: PFK-2 is dephosphorylated and more active due to insulin signaling
Fasted state: PFK-2 is phosphorylated and less active because it is degraded by FBPase-2
FBPase-2 domain is active in the ____state
What happens?
Fasted
Degrades fructose 2,6-bisphosphate
PFK-2 domain is active in the _____ state
What happens?
Fed
Makes fructose 2,6-bisphosphate which positively modifies PFK-1.
Benefit of allosteric enzymes
Can work over range of substrate concentrations with quick responses. Thus, when conditions change they are ready to go.
What does fructose 2-6-bisphosphate do and how?
Overrides negative allosteric effects of high [ATP]
Faster velocity & decline is less
Why is fructose 2,6-bisphosphate important?
In fed state, you can metabolize glucose to make ATP and use that ATP for biosynthesis. With fructose 2,6-bisphosphate, biosynthesis is a smooth process and not sputtering along.
