Transcript Chapter 15

Chapter 15
Glucose Catabolism
Chapter 15
Overview of Glycolysis
• Glycolysis involves the breakdown of glucose to pyruvate
while using the free energy released in the process to
synthesize ATP from ADP and Pi.
• The 10-reaction sequence of glycolysis is divided into two
stages: energy investment and energy recovery.
Chapter 15
The Reactions of Glycolysis
• The 10 steps of glycolysis can be described in terms of their
substrates, products, and enzymatic mechanisms.
• Glycolytic enzymes catalyze phosphorylation reactions,
isomerizations, carbon–carbon bond cleavage, and
dehydration.
• ATP is consumed in Steps 1 and 3 but regenerated in Steps
7 and 10 for a net yield of 2 ATP per glucose.
• For each glucose, 2 NADH are produced in Step 6.
Glycolysis
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
H
2
H
1
OH
OH
glucose-6-phosphate
Glycolysis takes place in the cytosol of cells.
Glucose enters the Glycolysis pathway by conversion
to glucose-6-phosphate.
Initially there is energy input corresponding to
cleavage of two ~P bonds of ATP.
6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
Hexokinase
H
OH
3
H
H
2
H
1
OH
OH
glucose-6-phosphate
Reaction 1: Hexokinase Uses 1st ATP
Glucose + ATP  glucose-6-P + ADP
ATP binds to the enzyme as a complex with Mg++.
Mg2+-Mediated Phosphorylation
Mg++ interacts with negatively charged phosphate oxygen
atoms, providing charge compensation & promoting a
favorable conformation of ATP at the active site of the
hexokinase enzyme.
6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
Hexokinase
H
OH
3
H
H
2
H
1
OH
OH
glucose-6-phosphate
The reaction catalyzed by hexokinase is highly
spontaneous.
6 CH2OH
Induced fit:
5
H
4
Glucose binding
to hexokinase
stabilizes a
conformation
in which:
OH
O
H
OH
H
2
3
H
OH
ATP ADP
H
H
4
1
OH
Mg
2+
OH
Hexokinase
glucose
 the C6 hydroxyl of the
bound glucose is close to
the terminal phosphate of
ATP, promoting catalysis.
6 CH OPO 2
2
3
5
O
H
OH
3
H
H
1
H
2
OH
OH
glucose-6-phosphate
glucose
Hexokinase
 water is excluded from the active site.
This prevents the enzyme from catalyzing ATP
hydrolysis, rather than transfer of phosphate to glucose.
Substrate-Induced Conformational
Change
Yeast hexokinase
PDBids 1IG8 and 3B8A
glucose
Hexokinase
It is a common motif for an enzyme active site to be
located at an interface between protein domains that are
connected by a flexible hinge region.
The structural flexibility allows access to the active site,
while permitting precise positioning of active site
residues, and in some cases exclusion of water, as
substrate binding promotes a particular conformation.
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
H
2
OH
H
1
OH
6 CH OPO 2
2
3
1CH2OH
O
5
H
H
HO
4
OH
2
3 OH
H
Phosphoglucose Isomerase
glucose-6-phosphate
fructose-6-phosphate
2. Phosphoglucose Isomerase catalyzes:
glucose-6-P  fructose-6-P
The mechanism involves acid/base catalysis, with ring
opening, isomerization via an intermediate, and then
ring closure.
Reaction 3: Phosphofructokinase Uses
2nd ATP
Phosphofructokinase
6 CH OPO 2
2
3
O
5
H
H
4
OH
6 CH OPO 2
2
3
1CH2OH
ATP
HO
3
OH
O
ADP
2
5
Mg2+
H
H
fructose-6-phosphate
1 CH2OPO32
H
4
OH
HO
3
2
OH
H
fructose-1,6-bisphosphate
Reaction 3: Phosphofructokinase Uses 2nd ATP
Phosphofructokinase catalyzes:
fructose-6-P + ATP  fructose-1,6-bisP + ADP
This highly spontaneous reaction has a mechanism similar
to that of hexokinase.
The phosphofructokinase reaction is the rate-limiting step
of glycolysis.
The enzyme is highly regulated.
Reaction 4: Aldolase
6-Carbon FBP to 3-Carbon GAP & DHAP
1CH2OPO3
2C
O
HO 3C
H 4C
H
H
2
H
Aldolase
2
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2
CH
OPO
2
3
6
dihydroxyacetone
phosphate
5
C
fructose-1,6bisphosphate
O
+
O
1C
H 2C OH
2
CH
OPO
3
2
3
glyceraldehyde-3phosphate
Triosephosphate Isomerase
Reaction 5: TIM
Triosephosphate Isomerase (TIM) catalyzes:
dihydroxyacetone-P  glyceraldehyde-3-P
Triosephosphate Isomerase
H
H
C
OH
C
O
+
H H
CH2OPO32
dihydroxyacetone
phosphate
+
H
OH
H H
C
C
+
OH
CH2OPO32
enediol
intermediate
+
H
O
C
H
C
OH
CH2OPO32
glyceraldehyde3-phosphate
The ketose/aldose conversion involves acid/base catalysis,
and is thought to proceed via an intermediate, as with
phosphoglucose isomerase.
Active site Glu and His residues are thought to extract and
donate protons during catalysis.
2-Phosphoglycolate is a transition state analog that
binds tightly at the active site of Triose Phosphate
Isomerase (TIM).
TIM is judged a "perfect enzyme." Reaction rate is limited
only by the rate that substrate collides with the enzyme.
Triosephosphate Isomerase
structure is an ab barrel, or
TIM barrel.
In an ab barrel there are
8 parallel b-strands surrounded
by 8 a-helices.
Short loops connect alternating
b-strands & a-helices.
TIM
TIM barrels serve as scaffolds
for active site residues in a
diverse array of enzymes.
Residues of the active site are
always at the same end of the
barrel, on C-terminal ends of
b-strands & loops connecting
these to a-helices.
TIM
There is debate whether the many different enzymes with
TIM barrel structures are evolutionarily related.
In spite of the structural similarities there is tremendous
diversity in catalytic functions of these enzymes and
little sequence homology.
Flexible Loop Closes
Over TIM Active Site
Yeast TIM
PDBid 2YPI
glucose
Glycolysis
ATP
Hexokinase
ADP
glucose-6-phosphate
Phosphoglucose Isomerase
fructose-6-phosphate
ATP
Phosphofructokinase
ADP
fructose-1,6-bisphosphate
Aldolase
glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
Glycolysis continued
First Stage of Glycolysis
Reaction 6: GAPDH
Forms 1st “High-Energy” Intermediate
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
1C
H
2
C
OH
OPO32
+ H+ O
NAD+ NADH
1C
+ Pi
H C OH
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
2
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
Exergonic oxidation of the aldehyde in glyceraldehyde3-phosphate, to a carboxylic acid, drives formation of an
acyl phosphate, a "high energy" bond (~P).
This is the only step in Glycolysis in which NAD+ is
reduced to NADH.
Enz-Cys
Oxidation to a
carboxylic acid
(in a ~ thioester)
occurs, as NAD+
is reduced to
NADH.
Enz-Cys
O
OH
HC
CH
SH
S
OH
OH
CH
CH
CH2OPO32
glyceraldehyde-3phosphate
CH2OPO32
thiohemiacetal
intermediate
NAD +
NADH
Enz-Cys
S
O
OH
C
CH
CH2OPO32
acyl-thioester
intermediate
Pi
Enz-Cys
SH
2
O3PO
O
OH
C
CH
CH2OPO32
1,3-bisphosphoglycerate
The “high energy” acyl thioester is attacked by Pi to
yield the acyl phosphate (~P) product.
H
O
H
H
C
C
NH2
+
N
O

NH2
+
2e + H
N
R
R
NAD+
NADH
Recall that NAD+ accepts 2 e plus one H+ (a hydride)
in going to its reduced form.
Reaction 7: Phosphoglycerate Kinase
(PGK) Generates 1st ATP
Phosphoglycerate Kinase
O
OPO32 ADP ATP O
O
1C
H 2C OH
2
3 CH2OPO3
1,3-bisphosphoglycerate
C
1
Mg
2+
H 2C OH
2
3 CH2OPO3
3-phosphoglycerate
7. Phosphoglycerate Kinase catalyzes:
1,3-bisphosphoglycerate + ADP 
3-phosphoglycerate + ATP
This phosphate transfer is reversible (low DG), since
one ~P bond is cleaved & another synthesized.
The enzyme undergoes substrate-induced conformational
change similar to that of hexokinase.
Reaction 8: Phosphoglycerate Mutase
(PGM) Interconverts 3PG & 2PG
Reaction 9: Enolase Forms 2nd
“High-Energy” Intermediate
Enolase
O
O

C
1
H 2 C OPO32
3 CH2OH
H

O
O

C
C
OH
O
O
1
OPO32
CH2OH
C
2C
OPO32
3 CH2
2-phosphoglycerate enolate intermediate phosphoenolpyruvate
Enolase catalyzes:
2-phosphoglycerate  phosphoenolpyruvate + H2O
This dehydration reaction is Mg++-dependent.
2 Mg++ ions interact with oxygen atoms of the substrate
carboxyl group at the active site.
The Mg++ ions help to stabilize the enolate anion
intermediate.
Phosphoenolpyruvate
Has the highest-energy phosphate bond found (-61.9
kJ/mol) in living organisms, and is involved in
glycolysis and gluconeogenesis.
In plants, it is also involved in the biosynthesis of
various aromatic compounds, and in carbon fixation
in C4 plants
2,3-BPG Affects Oxygen Carrying Ability
2,3-BPG Affects Oxygen Carrying Ability
Transport of Oxygen
Increasing levels of 2, 3-BPG mean high
metabolism and more O2 is released to
tissues.
Reaction 10: Pyruvate Kinase (PK)
Generates 2nd ATP
Pyruvate Kinase
O
O
ADP ATP
C
1
C
2
O
O
C
C
1
OPO32
3 CH2
phosphoenolpyruvate
C
2
O
O
1
OH
3 CH2
enolpyruvate
C
2
O
3 CH3
pyruvate
This phosphate transfer from PEP to ADP is spontaneous.
 PEP has a larger DG of phosphate hydrolysis than ATP.
 Removal of Pi from PEP yields an unstable enol, which
spontaneously converts to the keto form of pyruvate.
Required inorganic cations K+ and Mg++ bind to anionic
residues at the active site of pyruvate kinase.
Second Stage of Glycolysis
Glycolysis
Balance sheet for ~P bonds of ATP:
2
 How many ATP ~P bonds expended? ________
 How many ~P bonds of ATP produced? (Remember
4
there are two 3C fragments from glucose.) ________
 Net production of ~P bonds of ATP per glucose:
________
2
Glycolysis - total pathway, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2 NADH + 2 ATP
In aerobic organisms:
 pyruvate produced in glycolysis is oxidized to CO2 via
Krebs Cycle
 NADH produced in glycolysis & Krebs Cycle is
reoxidized via the respiratory chain, with production
of much additional ATP.
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
O
O
C
HC
OH
CH3
CH3
pyruvate
lactate
E.g., Lactate Dehydrogenase (LDH) catalyzes reduction
of the keto in pyruvate to a hydroxyl, yielding lactate, as
NADH is oxidized to NAD+.
Lactate, in addition to being an end-product of
fermentation, serves as a mobile form of nutrient energy,
& possibly as a signal molecule in mammalian organisms.
Cell membranes contain carrier proteins that facilitate
transport of lactate.
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
O
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Skeletal muscles ferment glucose to lactate during
exercise, when the exertion is brief and intense.
Lactate released to the blood may be taken up by other
tissues, or by skeletal muscle after exercise, and converted
via Lactate Dehydrogenase back to pyruvate, which may
be oxidized in Krebs Cycle or (in liver) converted to back
to glucose via gluconeogenesis
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
O
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Lactate serves as a fuel source for cardiac muscle as
well as brain neurons.
Astrocytes, which surround and protect neurons in the
brain, ferment glucose to lactate and release it.
Lactate taken up by adjacent neurons is converted to
pyruvate that is oxidized via Krebs Cycle.
Pyruvate
Decarboxylase
Alcohol
Dehydrogenase
CO2
NADH + H+ NAD+
O
O
C
C
O
CH3
pyruvate
H
O
C
CH3
acetaldehyde
H
H
C
OH
CH3
ethanol
Some anaerobic organisms metabolize pyruvate to
ethanol, which is excreted as a waste product.
NADH is converted to NAD+ in the reaction
catalyzed by alcohol dehydrogenase.
Glycolysis, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2 NADH + 2 ATP
Fermentation, from glucose to lactate:
glucose + 2 ADP + 2 Pi  2 lactate + 2 ATP
Anaerobic catabolism of glucose yields only 2 “high
energy” bonds of ATP.
Flux through the Glycolysis pathway is regulated by
control of 3 enzymes that catalyze spontaneous reactions:
Hexokinase, Phosphofructokinase & Pyruvate Kinase.
 Local control of metabolism involves regulatory effects
of varied concentrations of pathway substrates or
intermediates, to benefit the cell.
 Global control is for the benefit of the whole organism,
& often involves hormone-activated signal cascades.
Liver cells have major roles in metabolism, including
maintaining blood levels various of nutrients such as
glucose. Thus global control especially involves liver.
Some aspects of global control by hormone-activated
signal cascades will be discussed later.
6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
Hexokinase
H
OH
3
H
H
2
H
1
OH
OH
glucose-6-phosphate
Hexokinase is inhibited by product glucose-6-phosphate:
 by competition at the active site
 by allosteric interaction at a separate enzyme site.
Cells trap glucose by phosphorylating it, preventing exit
on glucose carriers.
Product inhibition of Hexokinase ensures that cells will
not continue to accumulate glucose from the blood, if
[glucose-6-phosphate] within the cell is ample.
6 CH2OH
Glucokinase
is a variant of
Hexokinase
found in liver.
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
4
1
OH
Mg
2+
OH
Hexokinase
H
OH
3
H
H
2
H
1
OH
OH
glucose-6-phosphate
 Glucokinase has a high KM for glucose.
It is active only at high [glucose].
 One effect of insulin, a hormone produced when blood
glucose is high, is activation in liver of transcription of
the gene that encodes the Glucokinase enzyme.
 Glucokinase is not subject to product inhibition by
glucose-6-phosphate. Liver will take up &
phosphorylated glucose even when liver [glucose-6phosphate] is high.
 Glucokinase is subject to inhibition by glucokinase
regulatory protein (GKRP).
The ratio of Glucokinase to GKRP in liver changes in
different metabolic states, providing a mechanism for
modulating glucose phosphorylation.
Glycogen
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Glucokinase,
with high KM
Glucose-1-P
for glucose,
allows liver to
store glucose
Pyruvate
as glycogen in
Glucose metabolism in liver.
the fed state
when blood [glucose] is high.
Glucose-6-phosphatase catalyzes hydrolytic release of Pi
from glucose-6-P. Thus glucose is released from the liver
to the blood as needed to maintain blood [glucose].
The enzymes glucokinase & glucose-6-phosphatase, both
found in liver but not in most other body cells, allow the
liver to control blood [glucose].
Pyruvate Kinase
O
O
Pyruvate Kinase, the
ADP
C
last step Glycolysis, is
1
2
C
OPO
controlled in liver partly
3
2
by modulation of the
3 CH2
amount of enzyme.
phosphoenolpyruvate
ATP
O
O
C
1
C
2
O
3 CH3
pyruvate
High [glucose] within liver cells causes a transcription
factor carbohydrate responsive element binding protein
(ChREBP) to be transferred into the nucleus, where it
activates transcription of the gene for Pyruvate Kinase.
This facilitates converting excess glucose to pyruvate,
which is metabolized to acetyl-CoA, the main precursor
for synthesis of fatty acids, for long term energy storage.
Phosphofructokinase
6 CH OPO 2
2
3
O
5
H
H
4
OH
6 CH OPO 2
2
3
1CH2OH
O
ATP ADP
HO
2
3 OH
H
fructose-6-phosphate
5
Mg2+
1CH2OPO32
H
H
4
OH
HO
2
3 OH
H
fructose-1,6-bisphosphate
Phosphofructokinase is usually the rate-limiting step of
the Glycolysis pathway.
Phosphofructokinase is allosterically inhibited by ATP.
 At low concentration, the substrate ATP binds only at
the active site.
 At high concentration, ATP binds also at a low-affinity
regulatory site, promoting the tense conformation.
Glycogen
Glucose-1-P
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.
Inhibition of the Glycolysis enzyme phosphofructokinase
when [ATP] is high prevents breakdown of glucose in a
pathway whose main role is to make ATP.
It is more useful to the cell to store glucose as glycogen
when ATP is plentiful.
Metabolic Fate of Pyruvate
Entry of Other Hexoses into Glycolysis
Metabolism of Fructose
Galactose & Glucose are Epimers
Metabolism of Galactose
Galactosemia: Galactitol Formation
Leads to Cataract Formation
Galactosemia is a disorder that affects how the body
processes a simple sugar called galactose. A small amount
of galactose is present in many foods. It is primarily part of
a larger sugar called lactose, which is found in all dairy
products and many baby formulas. The signs and
symptoms of galactosemia result from an inability to use
galactose to produce energy.
Researchers have identified several types of galactosemia.
These conditions are each caused by mutations in a particular
gene, and affect different enzymes involved in breaking
down galactose. Classic galactosemia, also known as type I,
is the most common and most severe form of the condition.
Galactosemia type II (also called galactokinase deficiency)
and type III (also called galactose epimerase deficiency)
cause different patterns of signs and symptoms.
If infants with classic galactosemia are not treated
promptly with a low-galactose diet, life-threatening
complications appear within a few days after birth.
Affected infants typically develop feeding difficulties, a
lack of energy (lethargy), a failure to gain weight and grow
as expected (failure to thrive), yellowing of the skin and
whites of the eyes (jaundice), liver damage, and bleeding.
Other serious complications of this condition can include
overwhelming bacterial infections (sepsis) and shock.
Affected children are also at increased risk of delayed
development, clouding of the lens of the eye (cataract),
speech difficulties, and intellectual disability.
Metabolism of Mannose
Mannose & Glucose are Epimers
The Pentose Phosphate Pathway
The pentose phosphate pathway (also called the
phosphogluconate pathway and the hexose
monophosphate shunt) is a metabolic pathway parallel to
glycolysis that generates NADPH and pentoses. While it does
involve oxidation of glucose, its primary role is anabolic
rather than catabolic.
Pentose Phosphate Pathway
There are two distinct phases in the pathway. The first is
the oxidative phase, in which NADPH is generated, and the
second is the non-oxidative synthesis of 5-carbon sugars.
For most organisms, the pentose phosphate pathway takes
place in the cytosol; in plants, most steps take place in
plastids.
Similar to glycolysis, the pentose phosphate pathway
appears to have a very ancient evolutionary origin. The
reactions of this pathway are mostly enzyme-catalyzed in
modern cells. They also occur however non-enzymatically
under conditions that replicate those of the Archean ocean,
and are catalyzed by metal ions, ferrous ions in particular.
The origins of the pathway could thus date back to the
prebiotic world.
The primary results of the pathway are:
• The generation of reducing equivalents, in the form of
NADPH, used in reductive biosynthesis reactions within
cells (e.g. fatty acid synthesis).
• Production of ribose 5-phosphate (R5P), used in the
synthesis of nucleotides and nucleic acids.
• Production of erythrose 4-phosphate (E4P) used in the
synthesis of aromatic amino acids.
Glucose-6-Phosphate Dehydrogenase
Deficiency
What is glucose-6-phosphate dehydrogenase deficiency?
G6PD deficiency is a genetic disorder that occurs most
often in males. This condition mainly affects red blood
cells, which carry oxygen from the lungs to tissues
throughout the body.
The defect in G6PD causes red blood cells to break down
prematurely. This destruction of red blood cells is called
hemolysis.
The most common medical problem associated with G6PD
deficiency is hemolytic anemia, which occurs when red
blood cells are destroyed faster than the body can replace
them. This type of anemia leads to paleness, yellowing of
the skin and whites of the eyes (jaundice), dark urine,
fatigue, shortness of breath, and a rapid heart rate.
In people with G6PD, hemolytic anemia is most often
triggered by bacterial or viral infections or by certain
drugs (such as some antibiotics and medications used to
treat malaria). Hemolytic anemia can also occur after
eating fava beans or inhaling pollen from fava plants (a
reaction called favism).
Glucose-6-dehydrogenase deficiency is also a significant
cause of mild to severe jaundice in newborns. Many
people with this disorder, however, never experience any
signs or symptoms.
How common is glucose-6-phosphate dehydrogenase
deficiency?
An estimated 400 million people worldwide have glucose6-phosphate dehydrogenase deficiency. This condition
occurs most frequently in certain parts of Africa, Asia, and
the Mediterranean. It affects about 1 in 10 African
American males in the United States.
Heinz
bodies
Helmet
cells
Relationship Between Glycolysis &
Pentose Phosphate Pathway