Download Glycolysis and gluconeogenesis

General Biochemistry
Part II-a
Prof. Dr. E. Van Driessche
Prof. Dr. S. Beeckmans
[email protected]
Protein Chemistry Lab
Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, room E.5.23
I.
The foundations of biochemistry
1.
2.
The cell and subcellular organelles
Short overview of biomolecules:
proteins, nucleic acids, lipids, polysaccharides, and their building blocks
Fundamentals of energy and energy transfer
3.
Prof. Dr. Edilbert Van Driessche
II. Metabolism and its regulation
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Strategies to regulate metabolic pathways
Glycolysis and gluconeogenesis
Pentose-phosphate pathway and Calvin cycle
Glycogen metabolism
Fatty acid degradation (-oxidation and -oxidation)
Fatty acid biosynthesis and ketone bodies
Pyruvate dehydrogenase, citric acid cycle and anaplerotic reactions
Amino acid degradation and urea cycle
Nitrogen metabolism: biosynthesis of amino acids, nucleotides and related molecules
Oxidative phosphorylation and ATP synthesis
Photosynthesis (light reactions)
III. Intra- and intercellular organization
15.
16.
17.
18.
19.
20.
Biomembranes (including transport systems)
The cytoskeleton
Metabolons: physical organization of metabolic pathways
Protein trafficking
Protein degradation
Integration of metabolism: from molecules to cells to organs
Prof. Dr. Edilbert Van Driessche
Major pathways of glucose utilization
Glycolysis
From Greek: glycos (sugar) and lysis (dissolution)
Prof. Dr. Edilbert Van Driessche
Definition:
glycolysis is the sequence of
reactions that convert glucose
into pyruvate with the
concomitant production of ATP
and NADH.
History of glycolysis
1897 − Hans Buchner & Edward Buchner:
● sucrose added to cell-free extracts of yeast results in the formation of alcohol.
fermentation can occur outside living cells.
1905 − Arthur Harden & William Young:
● glucose added to yeast extract results in an immediate start of the
fermentation, but the rate of fermentation rapidly decreases unless inorganic
phosphate is added
● inorganic phosphate is incorporated into sugar phosphates
● yeast juice contains at least 2 kinds of substances necessary for fermentation,
i.e. “zymase” and “cozymase”.
Prof. Dr. Edilbert Van Driessche
yeast juice
50°C
dialysis
inactive
inactive
mix both: active juice
Today: zymases are enzymes, cozymases are metal ions, ATP, ADP, coenzymes
(NAD, FAD, ...), magnesium, .....
1940 − Gustav Embden, Otto Mayerhof, Otto Warburg, Carl Neuberg, Jacob
Parnas, and Gerty & Carl Cori:
elucidation of the glycolytic pathway
Prof. Dr. Edilbert Van Driessche
Glycolysis: two phases
Prof. Dr. Edilbert Van Driessche
Prof. Dr. Edilbert Van Driessche
Prof. Dr. Edilbert Van Driessche
Fate of pyruvate under anaerobic conditions:
fermentations
Prof. Dr. Edilbert Van Driessche
Aim: regeneration of NAD+
Gluconeogenesis: “formation of new sugar”
A universal pathway
Glycolysis and gluconeogenesis
both take place in the same
compartment, i.e. the cytoplasm
Prof. Dr. Edilbert Van Driessche
need for rigorous regulation
Carbohydrate synthesis from
simple precursors
Opposing pathways
glycolysis and
gluconeogenesis
Glycolysis and gluconeogenesis
share many steps
Remember:
Prof. Dr. Edilbert Van Driessche
3 steps in glycolysis are irreversible
bypases using seperate
sets of enzymes are required
Prof. Dr. Edilbert Van Driessche
Prof. Dr. Edilbert Van Driessche
Alternative paths
from pyruvate to
PEP
From pyruvate to phosphoenolpyruvate
Mitochondria:
PCX
Pyruvate + HCO3− + ATP
Oxaloacetate + NADH + H+
Cytoplasm:
oxaloacetate + ADP + Pi
mMDH
cMDH
Malate + NAD+
Prof. Dr. Edilbert Van Driessche
Oxaloacetate + GTP
malate + NAD+
oxaloacetate + NADH + H+
PEP-CK
Pyruvate + ATP + GTP + HCO3−
phosphoenolpyruvate + CO2 + GDP
PEP + ADP + GDP + Pi + CO2
ΔG’° = +0.9 kJ/mol
ΔG = −25 kJ/mol
PCX
MDH
PEP-CK
pyruvate carboxylase
malate dehydrogenase
phosphoenolpyruvate carboxykinase
Synthesis of PEP from pyruvate
Prof. Dr. Edilbert Van Driessche
Reaction in the mitochondria
Reaction in the cytoplasm
Regulation of glycolysis and gluconeogenesis
Remember:
 Glycolysis and gluconeogenesis are both occurring in the cytoplasm
 The one is not simply the reversal of the other
 7 of the glycolytic reactions are freely reversible and the enzymes
catalyzing them also operate in gluconeogenesis
 3 reactions of glycolysis are essentially irreversible (large negative G’)
Prof. Dr. Edilbert Van Driessche
in gluconeogenesis these reactions are bypassed
Glycolysis:
Gluconeogenesis:
hexokinase
phosphofructokinase-1
pyruvate kinase
glucose-6-phosphatase
fructose-1,6-bisphosphatase
pyruvate carboxylase
PEP carboxykinase
At each of these points, when glycolysis and gluconeogenesis would
simultaneously operate, ATP would be consumed without accomplishing any
chemical or biological work.
Example:
ATP + fructose-6-phosphate
PFK-1
fructose-1,6-bisphosphate + H2O
Prof. Dr. Edilbert Van Driessche
ATP + H2O
ADP + fructose-1,6-bisphosphate
FBPase-1
fructose-6-phosphate + Pi
ADP + Pi +
heat
Requirement for a coordinated regulation of both pathways
Differential regulation of hexokinase isoenzymes in liver
and muscle by glucose-6-phosphate
 Four hexokinase isoenzymes exist, encoded by 4 different genes
(isoenzymes are different proteins that catalyze the same reaction)
 In myocytes: predominantly hexokinase II (also I and III)
 In hepathocytes: predominantly hexokinase IV (also called glucokinase)
Prof. Dr. Edilbert Van Driessche
The different hexokinase isoenzymes of liver and muscle reflect
the different roles of these organs in carbohydrate metabolism.
Muscle: consumption of glucose for the production of energy
Liver: maintains blood glucose homeostasis by removing or producing
glucose depending on the prevailing glucose concentration
Comparison between hexokinase II and hexokinase IV
Prof. Dr. Edilbert Van Driessche
Hexokinase I: very high affinity for glucose (half
saturated at 0.1 mM).
At intracellular [glucose] it acts at or near its
maximal rate.
In muscle, hexokinase I & II are allosterically
inhibited by glucose-6P.
In liver, hexokinase IV is half saturated at
[glucose] of about 10 mM, i.e. higher than the
[glucose] in the blood (4-5 mM).
Hexokinase IV can respond to the level of
glucose in the blood.
Prof. Dr. Edilbert Van Driessche
Regulation of hexokinase IV by sequestration in the nucleus
After a meal, blood glucose concentration increases and excess glucose is
transported into the hepatocytes and converted by hexokinase IV into glucose-6P.
Because hexokinase IV is not saturated at 10 mM, its activity continues to
increase as the [glucose] increases to 10 mM or more.
Hexokinase IV is inhibited by the reversible binding of a regulatory protein that is
specific for the liver.
Hexokinase IV is NOT inhibited by glucose-6P.
The complex control of phosphofructokinase I (PFK-1)
Remember:
Glucose-6P
glycolysis
glycogen synthesis
pentosephosphate pathway
Is a major regulatory enzyme of glycolysis !!
Prof. Dr. Edilbert Van Driessche
The metabolically irreversible reaction catalyzed by
PFK-1 commits glucose to glycolysis.
PFK-1:
High levels of ATP lower the affinity for fructose-6P.
ADP & AMP allosterically releave inhibition by ATP.
Citrate allosterically increases the effect of ATP.
Is allosterically activated by fructose-2,6-bisphosphate.
The central role of citrate:
Is a key intermediate in the aerobic oxidation
of pyruvate, fatty acids and amino acids.
Prof. Dr. Edilbert Van Driessche
Regulation of pyruvate kinase
All three isozymes of pyruvate kinase are allosterically inhibited by ATP, acetyl-CoA and
long chain fatty acids (all signs of an abundant energy supply).
The liver isoenzyme (L form), but not the muscle isoenzyme (M form) is further regulated
by phosphorylation.
When the glucose level in blood decreases, glucagon release causes a cyclic-AMPdependent phosphorylation of the L form, that inactivates it;
use of glucose in the
liver is slowed down to save the available glucose as fuel for the brain and other organs.
Regulation of gluconeogenesis
Remember:
pyruvate
glucose
Fate of pyruvate:
Pyruvate in the mitochondria can either be transformed
to acetyl-CoA, or to oxaloacetate.
Prof. Dr. Edilbert Van Driessche
Q: Which way will be choosen?
A: This depends mainly on the availability of acetyl-CoA.
When fatty acids are used as fuel molecules, their
breakdown yields acetyl-CoA, a signal that further
oxidation of glucose for fuel is not required.
Conclusion:
When sufficient concentrations of citrate and ATP are present, gluconeogenesis is favored.
I.
The foundations of biochemistry
1.
2.
The cell and subcellular organelles
Short overview of biomolecules:
proteins, nucleic acids, lipids, polysaccharides, and their building blocks
Fundamentals of energy and energy transfer
3.
Prof. Dr. Edilbert Van Driessche
II. Metabolism and its regulation
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Strategies to regulate metabolic pathways
Glycolysis and gluconeogenesis
Pentose-phosphate pathway and Calvin cycle
Glycogen metabolism
Fatty acid degradation (-oxidation and -oxidation)
Fatty acid biosynthesis and ketone bodies
Pyruvate dehydrogenase, citric acid cycle and anaplerotic reactions
Amino acid degradation and urea cycle
Nitrogen metabolism: biosynthesis of amino acids, nucleotides and related molecules
Oxidative phosphorylation and ATP synthesis
Photosynthesis (light reactions)
III. Intra- and intercellular organization
15.
16.
17.
18.
19.
20.
Biomembranes (including transport systems)
The cytoskeleton
Metabolons: physical organization of metabolic pathways
Protein trafficking
Protein degradation
Integration of metabolism: from molecules to cells to organs
Pentose-phosphate pathway
Prof. Dr. Edilbert Van Driessche
Aims:
– formation of pentose-phosphates through the oxidation of glucose-6P
– generation of reducing power, i.e. NADPH for reductive biosynthetic processes
Generation of
pentose-phosphates
End products:
D-ribose 5-phosphate
CO2
Prof. Dr. Edilbert Van Driessche
NADPH
Recycling of pentose-phosphates to glucose-6P
Epimerization of ribulose 5-phosphate:
Prof. Dr. Edilbert Van Driessche
Non-oxidative reactions of the
pentose-phosphate pathway
Reactions catalyzed by transketolase and transaldolase
Prof. Dr. Edilbert Van Driessche
Transketolase:
transfers a 2-carbon fragment from a ketose donor to an aldose acceptor.
Reactions catalyzed by transketolase and transaldolase
Prof. Dr. Edilbert Van Driessche
Transaldolase:
transfers a 3-carbon fragment from a ketose donor to an aldose acceptor.
Partitioning of glucose-6P between glycolysis and
pentose-phosphate pathway
Prof. Dr. Edilbert Van Driessche
When NADPH is formed faster
than being used in biosynthetic
processes, the [NADPH] rises
and inhibits the first enzyme of
the pentose-phosphate
pathway.
Glucose-6P is available for
glycolysis
Prof. Dr. Edilbert Van Driessche