Glycolysis 2:
Regulation of glycolytic flux, entry and exit of
glycolytic metabolites, and pyruvate metabolism
Bioc 460 Spring 2008 - Lecture 26 (Miesfeld)
Glycolytic flux is regulated in part
by PFK-1 activity; a metabolic
“valve” in the pathway
Lactose intolerance is due to
insufficient lactase enzyme
This is the chemical structure
of which glycolytic metabolite?
Key Concepts in Glycolysis
•
Glucokinase (hexokinase IV) catalyzes reaction 1 in the glycolytic pathway in
liver and pancreas cells when blood glucose levels are high. Unlike hexokinase
I, glucokinase as a very low affinity for glucose and is not inhibited by glucose6P. Therefore after a meal, the liver accumulates glucose for glycogen
synthesis, and the insulin secretion pathway is activated in pancreatic cells.
•
Phosphofructokinase 1 (PFK-1) is one of three metabolic “valves” in the
glycolytic pathway. PFK-1 is allosterically activated by fructose-2,6-BP, AMP,
and ADP (low energy charge), whereas, it is allosterically inhibited by ATP and
citrate (high energy charge). AMP stabilizes the R form of the enzyme (active),
and ATP stabilizes the T form (inactive). The activity of pyruvate kinase is
controlled by both phosphorylation and allosteric regulation.
•
Disaccharide sugars (maltose, sucrose, lactose) are cleaved by specific
enzymes to produce glucose and other monosaccharide sugars that enter the
glycolytic pathway. Glycolytic intermediates are metabolites in amino acid
biosynthesis, pentose phosphate pathway, and triacylglyceride biosynthesis.
•
Regeneration of NAD+ in the cytosol is critical to maintaining glycolytic flux
through the glyceraldehyde-3-P dehydrogenase reaction (reaction 6).
Glucokinase is a sensor of high glucose levels
Hexokinase I
– high affinity for substrate (Km for glucose is ~0.1mM)
– expressed in all tissues
– phosphorylates a variety of hexose sugars
– inhibited by the product of the reaction, glucose-6-P
Glucokinase (Hexokinase IV)
– low affinity for substrate (Km for glucose is ~10mM)
– highly specific for glucose
– expressed primarily in liver and pancreatic cells
– not inhibited by glucose-6-P
Two Major Roles of Glucokinase
Role in liver cells
When blood glucose levels are high, both hexokinase I and
glucokinase are active in liver cells, whereas, other tissues only
have hexokinase 1 and their ability to take up glucose after a meal
is unchanged. Since phosphorylation traps glucose inside cells,
and reaction 1 of glycolysis (same reaction catalyzed by both
hexokinase 1 and glucokinase) is highly favorable, liver cells take
up a disproportionate amount of the elevated blood glucose.
Role in pancreatic β cells
Glucokinase also sequesters glucose inside the pancreatic  cells
which initiates a complex signaling pathway leading to the release
of insulin into the blood. Since insulin signaling results in lowered
blood glucose levels by activating glucose uptake in the muscle
and fat cells (adipose), glucokinase is vital to glucose control.
Homeostatic blood glucose levels are ~5mM which saturates hexokinase I activity
in all tissues. However, after a carbohydrate-rich meal (glucose is plentiful), blood
glucose levels increase dramatically and the glucokinase reaction in liver and
pancreas cells becomes a major contributor to the formation of glucose-6-P.
Since glucose-6-P
is trapped in the
cell because of the
negative charge,
the liver and
pancreas
accumulate a large
share of the blood
glucose.
Glucokinase is a Sensor of Glucose Levels
GLUT protein is a
glucose transporter.
Glucose activation
of glucokinase
activity is at the
level of protein
synthesis, i.e.,
elevated glucose
in the cell leads to
increased
synthesis of
glucokinase
enzyme.
What happens to flux
through the glycolytic
pathway when glucokinase
is activated by glucose?
Increased ATP levels stimulate
membrane depolarization and
subsequent calcuim uptake.
Making a glucokinase knock-out mouse
The importance of
glucokinase in
insulin secretion
was confirmed using
transgenic mice that
lacked the
glucokinase gene in
pancreatic  cells.
Since these mice
cannot secrete
insulin when blood
glucose levels were
high, they
developed insulindependent diabetes.
Human metabolic diseases are often caused by
non-lethal recessive gene mutations in enzymes
Over 150 mutations in the
glucokinase gene have been found
in humans with a form of diabetes
called mature-onset diabetes of
the young (MODY2).
Allsoteric regulators control the activity of
phosphofructokinase 1 and modulate glycolytic flux
Phosophofructokinase-1 (PFK-1)
• catalyzes reaction 3 in glycolysis to generate fructose-1,6- BP
PFK-1
Phosphofructokinase-2 (PFK-2)
• bifunctional enzyme that catalyzes the synthesis of
fructose-2,6-BP (F-2,6-BP), a potent
allosteric regulator of PFK-1 activity. We will
talk more about the synthesis of F-2,6-BP
later in the course.
Allosteric regulation of PFK-1
• PFK-1 exists as a homotetramer (a dimer of dimers).
• It is stable in two conformations; the inactive T state or active R state
(similar to to the T and R conformations of hemoglobin).
• The activity of PFK-1 is allosterically activated by binding of F-2,6-BP,
AMP, and ADP, and it is allosterically inhibited by citrate and ATP.
Allosteric regulators alter the activity of PFK-1
by binding to a region outside of the active site
ATP and AMP binding to PFK-1 shifts the
equilibrium between the T and R conformations
What are the two roles of ATP in the PFK-1 catalyzed reaction?
The activity of liver pyruvate kinase is controlled by
both phosphorylation and allosteric regulation
When blood glucose
levels are high, glycolytic
flux is stimulated in part
by dephosphorylation of
liver pyruvate kinase.
Conversely, low blood
glucose levels leads to
phosphorylation and
partial inactivation of
pyruvate kinase.
Feed forward allosteric
regulation by fructose1,6-BP, and allosteric
inhibition by ATP and
alanine, also control
pyruvate kinase activity.
Based on what you know about the structure of
pyruvate and alanine, why do you think alanine is a
negative regulator of pyruvate kinase activity?
Supply of Glycolytic
Intermediates
Disaccharide sugars are
common nutrients in our
diet and provide much of
the carbohydrate used for
energy conversion.
- Maltose is from starch
- Sucrose is table sugar
- Lactose is from milk
Glycerol is a glycolytic
intermediate derived from
the degradation of
triacylglycerides.
Lactose intolerance occurs in most adults as a
result of decreased lactase enzyme production
Lactose is present at high levels in milk
products and can cause considerable
intestinal discomfort in people with reduce
lactase gene expression. This is most
common in people of Asian decent and is
rarely found in people from a Scandinavian
background.
High levels of intestinal lactose causes
osmosis of water into the intestine leading
to diarrhea, and moreover, anaerobic
intestinal bacteria metabolize the extra
lactose to produce H2 and CH4 gas.
Two ways for adults to still enjoy dairy
products are to predigest the lactose sugar
by adding lactase to yogurt or milk before
consuming, or to take a lactase
supplement pill before eating dairy foods.
Fructose, galactose and
glycerol enter the
glycolytic pathway
through a variety of
routes, many of which
require additional
enzymatic reactions.
For example, fructose is
first converted to
fructose-1-P which is
then cleaved by fructose
-1-P aldolase to
generate DHAP and
glyceraldehyde which is
then phosphorylated to
produce GAP.
Does the number of ATP required to
convert 1 mole of fructose into 2 moles of
pyruvate differ in liver and muscle cells?
The metabolic disease fructose intolerance is due
to a deficiency in the enzyme fructose-1-P aldolase
• High fructose corn syrup is the most
common added sweetener to processed
foods, however, for individuals with fructose
intolerance, fructose in the diet can be
extremely toxic.
• Prolonged ingestion of fructose, primarily
from fruit juice, leads to the build-up of
fructose-1-P which results in loss of Pi in the
liver and decreased ATP synthesis (ADP +
Pi --> ATP) causing liver damage.
What do you think the treatment is for people with fructose intolerance?
Demand for Glycolytic
Intermediates
In addition to functioning as
intermediates in the
gluconeogenic pathway
(production of glucose from
non-carbohydrate
sources), many of the
glycolytic metabolites
provide carbon skeletons
for amino acid synthesis,
the pentose phosphate
pathway (ribose-5-P), and
triacylglyceride synthesis
(glycerol).
Metabolic Fate of Pyruvate
1. Under aerobic conditions, the majority of pyruvate is metabolized
in the mitochondria to acetyl CoA, and ultimately to CO2 and H2O
which are the products of the citrate cycle and electron transport
chain.
2. Under anaerobic conditions, such as occurs in muscle cells
during strenuous exercise, or in erythrocytes which lack
mitochondria, pyruvate is converted to lactate (the ionized form of
lactic acid) by the enzyme lactate dehydrogenase.
3. Under anaerobic conditions in microorganisms such as yeast,
pyruvate can also be utilized for alcoholic fermentation to convert
pyruvate to CO2 and ethanol using the enzymes pyruvate
decarboxylase and alcohol dehydrogenase, respectively.
NAD+ must be regenerated to maintain glycolytic flux
The glyceraldehyde-3-P
dehydrogenase reaction
requires a steady supply
of NAD+ which functions
as a coenzyme in this
oxidation reaction.
Anaerobic respiration
replenishes the NAD+
through a reduction
reaction leading to lactate
or ethanol production.
Aerobic respiration
replaces the NAD+
through a metabolite
shuttle system since
NAD/H cannot cross the
mitochondrial membrane.
Glyceraldehyde-3-P
Anaerobic
Regeneration
of NAD+
NAD+ + Pi
NADH + H+
Glyceraldehyde-3-P
dehydrogenase
3-Pglycerate
1,3-BisPglycerate
ADP
Pglycerate
ATP
kinase
Phosphoglycerol
mutase
Enolase
Phosphoenolpyruvate
2-Pglycerate
ADP
Pyruvate kinase
NAD+
Lactate
NADH + H+
ATP
Pyruvate
Lactate dehydrogenase
Lactate Dehydrogenase Deficiency (LDHA)
These patients cannot maintain moderate
levels of exercise due to an inability to utilize
glycolysis to produce ATP needed for muscle
contraction under anaerobic conditions.
When lactate dehydrogenase levels are
insufficient, the level of NAD+ becomes
limiting during exercise and flux through the
glyceraldehyde-3-P dehydrogenase reaction
is inhibited.