Lecture 18

Question 1

ΔG of rxn

Answer 1

concentration independent

This is the change in ΔG between the standard state concentrations and equilibrium concentrations

Question 2

concentration dependent

Answer 2

concentration dependent

This is the influence on ΔG of the actual concentration of substrates and products in the cell

Question 3

Aldolase– 4th step in Glycolysis

Answer 3

But in a living cell:
ΔGo’ = +22.8 kJ/mol
ΔG = -5.9 kJ/mol

Question 4

TIM - 1st step

Answer 4

ΔGo’  = +7.9kJ/mol
ΔG = 0 kJ/mol

Question 5

GAPDH + PGK - 6+7 step

Answer 5

ΔGo’  = -16.7 kJ/mol
ΔG = -1.1 kJ/mol

Question 6

PK- step 10

Answer 6

ΔGo’  = +23 kJ/mol
ΔG = -13.9 kJ/mol

Question 7

Regulation of Metabolism

Answer 7

• Global regulation (at the organ level)
• Transcriptional or translational control
• Covalent post-translational modifications
• Allosteric regulation

Question 8

Transcriptional or translational control

Answer 8

Expression of glucose metabolism genes is regulated by a specific transcription factor HIF-1. (Hypoxia Inducible Factor)

Question 9

inactive to active enzyme

Answer 9

kinase= atp–>adp

Question 10

active to inactive enzyme

Answer 10

phosphatase= minu Pi

Question 11

Allosteric Regulation

Answer 11

• T state: inactive
• R state: active
• Equilibrium: T R
• Allosteric effectors shift the equilibrium
– Inhibitor shifts the equilibrium towards T
– Activator shifts the equilibrium towards R

Question 12

Regulation of Glycolysis
ΔGo’ and ΔG for the rxb in heart muscle

hexokinase
PFK
PK

Answer 12

• 27.2
• 25.9
• 13.9

Question 13

Best control is at the irreversible steps of a pathway

Answer 13

• ∆G is very negative

* Virtually all regulation occurs at the irreversible steps

Question 14

• What are the irreversible steps of glycolysis?

Answer 14

– Steps 1, 3, and 10.

1. Hexokinase: Glucose Glucose-6-P
2. Phosphofructokinase: F-6-P F-1,6-BP
3. Pyruvate kinase: PEP Pyruvate

Question 15

Regulation of Glycolysis: Hexokinase

Answer 15

–HK is inhibited by G6P
–Product negative feedback inhibition.
Example of how HK is controlled by PFK.
F6P builds up then G6P builds up then glycolysis slows and glucose remains in the blood or G6P gets converted to glycogen.

Note: In LIVER Glucokinase is NOT inhibited by G6P
(encourages glycogen storage)

Question 16

Regulation of Glycolysis: Pyruvate Kinase

Answer 16

PEP + ADP Pyruvate + ATP

Pyruvate kinase has several different forms
- Liver form – PKL
- Erythrocyte form – PKR
- Muscle form – PKM1
- Cancer cell form – PKM2
– For all forms Km is lowered by Fruc-1,6-P2.

Question 17

Regulation of Glycolysis: Pyruvate Kinase

Answer 17

Liver Pyruvate Kinase is regulated by blood glucose levels (via insulin, epinephrine or glucagon, GPCR, G-protein and cyclic AMP)

(liver uses pyruvate to make and store fat)
(liver uses PEP to make glucose via gluconeogenesis)

Other forms of PK are not regulated by Blood Glucose levels.

Question 18

Regulation of Glycolysis: Pyruvate Kinase

Answer 18

Cancer cell pyruvate kinase (PKM2) is inhibited by phosphorylation on tyrosine.
– Tyrosine kinases are activated by growth factors,
– Some tumors have highly active tyrosine kinases
– Growth requires anabolic building blocks, not energy.
–When PKM2 is inhibited then glycolytic intermediates can be used to make building blocks for amino acids and phospholipids..

Question 19

Regulation of Glycolysis: Phosphofructokinase

Answer 19

Allosteric regulation of the Km of PFK-1 by high ATP.

ATP raises the Km for F6P

Question 20

During exercise [ATP] does not change much, but AMP increases a lot!

Answer 20

ATP ADP + Pi

Question 21

These reactions restore ATP:

Answer 21

PCr + ADP Cr + ATP

ADP + ADP AMP + ATP (adenosine kinase)

Question 22

When all equilibria involving ATP, ADP and AMP are

considered:

Answer 22

[ATP] decreases: 1.0 mM 0.9 mM –> 10% decrease
[ADP] increases: 0.1 mM 0.2 mM –> 100% increase
[AMP] increases: 0.02 mM 0.1 mM –> 400% increase

Question 23

Regulation of Glycolysis: Phosphofructokinase

Answer 23

Allosteric regulation of the Km of PFK-1 by high ATP.
AMP diminishes the inhibitory effect of ATP.

Activation of PFK-1 by F-2,6BP.
The sigmoidal kinetics become hyperbolic at 1 μM F-2,6BP.

F-2,6BP is an important allosteric effector in liver that shifts the inactive state to the active state for PFK-1.

F-2,6BP is made in liver cells only when blood sugar is high

Note that Vmax increases (a little) and Km decreases for the F-6-P substrate.

Question 24

Activation of PFK-1 by F-2,6BP.

Answer 24

ATP, acting as a substrate, initially stimulates the reaction rate. As the ATP concentration increases, it acts as an allosteric inhibitor. The inhibitory effect is reversed by F-2,6BP.

Question 25

The inhibitory effect is reversed by F-2,6BP. WHY

Answer 25

• High levels of glucose in blood
• High insulin in blood.
• Insulin stimulates liver cells to make F-2,6BP.
• The extra glucose gets metabolized and ultimately stored as fat.

When there is average or low blood glucose there is less F-2,6BP and the liver does not store glucose carbons as fat.

Question 26

Glucose vs. Fructose metabolism in the liver

High Fructose Corn Syrup consumption correlates with obesity. Why?

Answer 26

• Glucokinase Km for Glucose is ~10 mM
• Fructokinase Km for Fructose is ~0.5 mM (makes F1P)
• Liver uses Glucose only when blood glucose is very high
• Liver uses Fructose whenever it is available…

• Fructose-1-Phosphate has “its own” aldolase

• (so it does not need to be made into F1,6BP)
• Regulation of PFK-1 by ATP, AMP, F2,6BP is bypassed.
• Carbons from fructose are always stored as fat.

• Fructose does not stimulate insulin production.
- Its concentration in blood is not regulated.

Question 27

Some key concepts about regulation of Glycolysis

Answer 27

• Regulation occurs at steps with the highest free energy difference.
• Hexokinase, PFK-1, Pyruvate Kinase
• Be familiar with positive and negative regulators.
• Be familiar with types of regulation.
• Regulation depends on the purpose of the tissue - example muscle vs. liver
• PFK-1 stimulation by F2,6BP and relationship to ATP and AMP regulation
• Fructose metabolism differs from glucose metabolism