Bioenergetics and Metabolism

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Glycolysis 2Regulation 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
This is the chemical structure of which
glycolytic metabolite?
Lactose intolerance is due to insufficient
lactase enzyme
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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 glucose-6P.
  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).

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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

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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 ß 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.

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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.
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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.
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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 insulin-dependent diabetes.
Making a glucokinase knock-out mouse
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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).
Human metabolic diseases are often caused by
non-lethal recessive gene mutations in enzymes
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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
• 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.

PFK-1
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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.

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Allosteric regulators alter the activity of PFK-1
by binding to a region outside of the active site
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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?
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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 fructose-1,6-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?
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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.
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Lactose intolerance occurs in most adults as a
result of decreased lactase enzyme production

• 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.

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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?
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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 --gt
  ATP) causing liver damage.

What do you think the treatment is for people
with fructose intolerance?
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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).
Demand for Glycolytic Intermediates
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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.

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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.
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Glyceraldehyde-3-P
Anaerobic Regeneration of NAD
NAD
Pi
Glyceraldehyde-3-P dehydrogenase
NADH H
3-Pglycerate
1,3-BisPglycerate
Pglycerate kinase
Phosphoglycerol mutase
ADP
ATP
Enolase
Phosphoenolpyruvate
2-Pglycerate
ADP
Pyruvate kinase
NADH H
ATP
NAD
Pyruvate
Lactate
Lactate dehydrogenase
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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.