Download Glycolysis 2

Glycolysis 2
Lecture 26
Key Concepts
• Gluconeogenesis vs. Glycolysis
– Differences and similarities
• Regulation of the Glycolytic Pathway
– Glucokinase is a molecular sensor of high glucose levels
– Allosteric control of phosphofructokinase activity
• Supply and demand of glycolytic intermediates
• Metabolic Fate of Pyruvate
•
•
How do substrate availability and enzyme activity levels control
glycolytic flux?
Why is muscle lactate dehydrogenase activity required for short
bursts of intense exercise?
Overview of the Gluconeogenesis Pathway
Gluconeogenesis is not the reverse of glycolysis.
Many reactions are the reverse of the comparable reaction in
glycolysis.
Importantly, however, the “reverse” of the three kinase steps
of glycolysis are replaced by different reactions catalyzed
by different enzymes.
Irreversible steps have actual changes in free energies (∆G)
that are highly negative and require pathway-specific
enzymes
Differences between
Gluconeogenesis &
Glycolysis
Differing reactions have
negative ∆G in both
pathways.
Why is that the case?
Multiple Cellular Compartments are Involved
in Gluconeogenesis
• Cytosol
• Mitochondrion
• Endoplasmic Reticulum
Oxaloacetate is Produced in
the Mitochondrion
Pyruvate Carboxylase
Biotin binding domain
Malate Shuttle
Without those vitamins,
how would I ever make glucose?
Glucose is Produced on the ER Membrane
Know the difference in stoichiometry
between glycolysis and gluconeogenesis!
Interplay Between Pathways
Regulation of Glycolysis
• Glucokinase & Hexokinase
• Phosphofructokinase
• Pyruvate Kinase
Glucokinase is a molecular 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
–
–
–
–
low affinity for substrate (Km for glucose is ~10mM)
highly specific for glucose
expressed primarily in liver and pancreatic cells
not inhibited to glucose-6-P.
Multiple Roles of Glucokinase
Role in liver cells
– traps extra glucose available from the diet so that it
can be stored as glycogen for an energy source later
Role in pancreatic β cells
– glucose sensor
– triggers insulin release
Glucokinase as a Sensor of Glucose Levels
GLUT protein=
Glucose
Transporter
You should be
familiar with this
protein family
Why does glucose stinulate
Differences between
Gluconeogenesis &
Glycolysis
What do you know, the
enzymes that catalyze the
irreversible reactions are
also the regulatory
enzymes!
Allosteric control of phosphofructokinase activity
Two phosphofructokinase isozymes:
• phosophofructokinase-1 (PFK-1)
– catalyzes reaction 3 in glycolysis
– produces fructose-1,6-bisphosphate
• phosphofructokinase-2 (PFK-2)
– bifunctional enzyme that catalyzes the synthesis of fructose-2,6bisphosphate (F-2,6-BP), a potent allosteric regulator of PFK-1
activity
PFK-1 is an allosteric enzyme
• exists as a tetramer (a dimer of dimers)
• in either of two conformations
– the inactive T state or active R state
– analogous to the hemoglobin tetramer
PFK-1 is an allosteric enzyme
The equilibrium between
T and R states in a cell is
controlled by allosteric
effector molecules which
bind to a regulatory site
outside of the substrate
binding pocket.
Allosteric regulation of PFK-1 by ATP
What does this graph tell us about the role of ATP in regulating PFK-1 activity?
You should understand the relationship between AMP, ADP and ATP in
regulation of this enzyme. Why is AMP used as an allosteric regulator instead
of ADP?
Allosteric regulation of PFK-1 by F-2,6-BP
What do these graphs tell us about the role of F-2,6-BP in regulating PFK-1
activity?
Allosteric effector site is far removed from the catalytic site and in fact
maps to the interface between two PFK-1 subunits.
ATP binds with equal affinity to the catalytic site regardless of the T or R state
conformation of PFK-1
ATP binding to the allosteric effector site is highest when the protein is in the T
state which functions to decrease fructose-6-P binding to the catalytic site
AMP binding to the allosteric effector site serves to stabilize the R state, and
thereby stimulates the production of fructose-1,6-BP by ATP-mediated
phosphoryl transfer
Phosphofructokinase-2
is also regulated
The main regulatory
protein is PFK-1.
Why would this be?
The last one down here is
also involved in regulating
glycolysis
Two Isoforms of Pyruvate kinase
L form in liver
M form in muscle and brain
Only L-form is regulated by
phosphorylation
Triggered by glucagon
signaling pathway.
Both forms allosterically regulated
Why is alanine an allosteric regulator?
Glycolysis and Gluconeogenesis
are Reciprocally Regulated
Supply and Demand
of Glycolytic
Intermediates
Glucose isn’t the only sugar!
•
•
•
Maltose & β-amylase
Sucrose & Sucrase
Lactose & Lactase
(don’t confuse with lactic acid!)
All can enter glycolysis
So can glycerol
Fructose, galactose and
glycerol enter the
glycolytic pathway
through a variety of
routes, many of which
require additional
enzymatic reactions
Fructose metabolism in the liver
Fructokinase
Fructose
ATP
Fructose-1-P
Aldolase B
ADP
Glyceraldehyde + Dihydroxyacetone phosphate
Glyceraldehyde Kinase
GLYCOLYSIS
+ATP
TIM
2 Glyceraldehyde-3-P (GAP)
Net 2 ATP ← +4 ATP
Medical Implications of Alternative Sugars
•
•
•
High fructose corn syrup is the most
common added sweetener to processed
foods, however, for individuals with a
genetic disease called fructose
intolerance, fructose in the diet can be
extremely toxic.
Fructose intolerance is due to
deficiencies in the enzyme fructose-1-P
aldolase, also called aldolase B.
People with fructose intolerance cannot
eat foods containing fructose
because it leads to the build-up of
fructose-1-P which has no alternate
metabolic fates in the absence of
fructose-1-P aldolase.
Galactose metabolism in the liver
Galactose
ATP
Galactokinase
ADP
Galactose-1-P
UDP-Glucose
Uridyl transferase
Glucose-1-P
UDP-Galactose
Epimerase
UDP-Glucose
Phosphoglucomutase
Glucose-6-P
GLYCOLYSIS
Glycolytic intermediates
serve important roles in
anabolic pathways by
providing carbon
skeletons for
biosynthesis
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. The third fate of pyruvate occurs in microorganisms such as yeast
which utilize alcoholic fermentation to convert pyruvate to CO2 and
ethanol using the enzymes pyruvate decarboxylase and alcohol
dehydrogenase, respectively.
Glyceraldehyde-3-P
NAD+ +
Pi
NADH + H+
Glyceraldehyde-3-P
dehydrogenase
Anaerobic
Regeneration
of NAD+
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
Medical Implications of Lactate Dehydrogenase
Deficiency (LDHA)
• Defect in Lactate Dehydrogenase
• 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.