### Prof. Kamakaka`s Lecture 14 Notes (PPT)

```Important to recognize that metabolic need of
individual cells is different from need of whole
organism.
Brain needs glucose even when body is starving.
Liver will synthesize glucose via gluconeogenesis
while brain uses glucose via glycolysis
2
Free energy
A-B + C --- B-C + A
If this reaction is displaced from equilibrium, some energy will be released as the reaction
returns towards equilibrium.
The energy released could be lost as heat or made to do work (ATP)
Free energy (DG) of a chemical reaction is a measure of its capacity to do work.
It is related to conc of substrate and product
DG = -RTln[eqP]/[eqS]+RTln[initP]/[initS]
If initial conc =1M, RTln[initP]/[initS]=0
You get
DGo=-RTlnK
At equilibrium free energy change is zero
Higher the substrate conc, lower the product conc, greater the DG value.
Equilibrium
At equilibrium, rate of forward and reverse reaction is identical A --- B
No net flux in either direction A----- B----
At equilibrium No free energy change
Change in free energy as a reaction proceeds towards equilibrium is the key driving force
in biological processes.
Equilibrium processes do not perform useful work (movement)
Equilibrium processes can not be easily regulated
Complex molecules (and processes) will not be made in large quantities under equilibrium
conditions
Biological systems obtain energy to prevent equilibriums from reaching.
Open and closed systems
A-B + C --- B-C + A
In a closed system, heat released will equal heat absorbed
In an open system, heat released will be lost to environment, prevents the reaction
from reaching equilibrium
Furthermore, all free energy change is not lost as heat. Some is captured as chemical
bond energy (ATP)
A-B + C ---------- B-C + A
/
\
ADP+Pi
ATP
If all free energy change were captured as ATP, the reaction would be at equilibrium
and there would be no net gain of ATP. However partial loss of energy as heat to the
environment converts this reaction into a non equilibrium reaction allowing flux through
the pathway towards ATP generation.
Dynamic steady state
Living cells are NOT AT EQUILIBRIUM
They maintain a DYNAMIC STEADY STATE
Glucose enters cells, and CO2 leaves cell, but mass and composition of cell do not change
appreciably. Cells appear to be but are not at equilibrium with surroundings.
At molecular level each metabolic pathway is unidirectional and functioning.
Rate of metabolic flow (flux) through the pathway is high, but concentration of
substrate/intermediates/products remains constant.
v1
v2
A------------S-------- P
v1=v2
If this steady state is disrupted, by external change in energy etc, temporarily the
fluxes through the pathway will change and regulatory mechanisms will be triggered and
the organism will arrive at a new steady state to achieve homeostasis.
Direction of flux in a pathway is dictated by position of equilibrium at each step of
pathway
Steady state
Steady State- all flows/fluxes are constant and unchanging.
Living systems try and maintain a steady steady.
An open system at steady state is at maximum thermodynamic efficiency.
Changes in fluxes/flow require changes, which require energy.
Changes in the environment result in perturbation of the steady state, and the organism
responds and re-obtains new steady state.
How are process maintained in non-equilibrium state?
Exchange of matter and energy between organism and environment
Substrates are derived from environment, products are returned to environment
(Metabolic processes only attain equilibrium at death)
Flux and equilibrium
10/sec
20/sec
110/sec
A<-------------B <----------------C <--------------------D
0.1/sec
10/sec
100/sec
Some reactions are close to equilibrium other are obviously far from equilibrium
All the reactions are sufficiently away from equilibrium so that the process is not at
equilibrium
While in theory all enzymes are regulated, activity of only certain enzymes regulate
flux through the pathway
Reactions are usually limited by the substrate (intermediate) conc.
Therefore: Function of enzyme- Catalysis AND Monitoring state of pathway Via conc of
substrates/products)
In this simple pathway, the intermediate B has two alternative fates. To the
extent that reaction B → E draws B away from the pathway A → D, it controls
that pathway.
10
Enzymes and Equilibrium
Almost all steps in a pathway lose heat energy and are therefore displaced slightly from
equilibrium.
Most enzyme catalyzed reactions in metabolism operate close to equilibrium.
In a few cases, they are not close to equilibrium. (Non equilibrium enzymes possess low
catalytic activity so substrates accumulate and they will limit flux through pathway)
These reactions will generate the greatest free energy change and produce the most work
River—dam—sluice gate—work turbines
While to some extents all enzymes are regulated, the bottleneck enzymes are rate
limiting and highly regulated.
Substrate energy
Energy loss as
reaction proceeds
Product energy
Product +ATP (equilibrium)
Product +ATP + heat (non-equilibrium)
Product + heat
Enzymes working farthest from equilibrium are often used in regulation of a
pathway because the reverse reaction is not easily attainable (would require a
very great increase in conc of D to reverse the reaction)
Natural bottlenecks!
A ---- B ---- C -------- D ---- E ---- F ---- G
Factors that affect flux
Flux through pathway are regulated by
1
Availability of substrate
2
Conc of enzymes responsible for rate limiting step
3
Allosteric regulation of enzyme (Feedback regulation)
4
Covalent modification of enzyme
5
Product removal
Reduction in substrate will decrease activity of enzyme (provided enzyme is not
saturated by substrate- most biological pathways operate at suboptimal conc of
substrate)
Removal of product enables reaction to proceed in a specific direction
Substrate concentration
At substrate concentrations far below the Km, each increase in [S]
produces a correspondingly large increase in the reaction velocity, v.
For this region of the curve, the enzyme has an  of about 1.0. At [S] >>
Km, increasing [S] has little effect on v;  here is close to 0.0.
14
Dependence of glycolytic flux in a rat liver homogenate on additional
enzymes. Purified enzymes were added to an extract of liver carrying out glycolysis
in vitro. The increase in flux through the pathway is shown on the y axis.
15
Mechanism of gene regulation by
the transcription factor FOXO1.
Insulin activates a signaling cascade,
leading to activation of protein kinase
B (PKB). FOXO1 in the cytosol is
phosphorylated by PKB, and the
phosphorylated transcription factor is
degraded by proteasomes.
Unphosphorylated FOXO1 can enter
the nucleus, bind to specific gene
promoters, and trigger transcription of
the associated genes. Insulin
therefore has the effect of turning off
the expression of these genes, which
include glucose 6-phosphatase.
16
17
Regulation of glucokinase by sequestration in the nucleus. The protein
inhibitor of glucokinase is a nuclear protein that draws glucokinase into the
nucleus when the enzyme is not required but releases it to the cytosol when the
glucose concentration is high and the enzyme is required.
19
Protein phosphorylation and dephosphorylation.
Reversal of entire pathways
Reversal of entire pathway of glycolysis is difficult.
DGo’ for glycolysis (glucose to pyruvate is -73 kJ/mol
To do reverse reaction, you need to change product (pyruvate)
conc many billion fold. Not feasible!
Also reversing reaction leads to loss of ATP
F6P
Hexokinas
e
Glu
G6P
Cell uses some enzymes from glycolysis in
gluconeogenesis
Fru 1,6 bisphosphatese1
Regulation of three irreversible steps
Glucose 6-Phosphatase
Glycolysis Vs Gluconeogenesis
Reactions with small DG are used by both
pathways.
Reactions with large DG- regulated
PFK1
F1,6P2
PEP
Pyruvate
Kinase
Oxaloacetate
Pyruvate
Factors affecting enzymes
ATP requirement
Effect of ATP concentration on the initial velocity of a typical ATPdependent enzyme. These experimental data yield a Km for ATP of 5 mM. The 24
concentration of ATP in animal tissues is ~5 mM.
Glucagon and insulin
The three enzymes catalyze irreversible steps
Concentration of these enzymes is regulated by hormones
Insulin is secreted from pancreas (b-cells) when glucose levels in blood increase (Insulin
promotes storage of energy).
It induces transcription/translation of glucokinase, phosphofructokinase and pyruvate
kinase (effects occur over hours)
Glucagon is secreted from pancreas (a-cells) when blood glucose is low. It has opposite
effects (induces release of glucose into blood)
Transporters
Rate of entry of glucose into cells is regulated by transporters.
Blood glucose level is 5mM
Basal Glucose transporter in most cells has Km of 1mM. Less than blood
glucose and so glucose is taken up easily.
But in liver and pancreas glucose transporters have Km of ~15mM (close to
blood glucose levels).
This allows pancreatic cells to monitor glucose levels and thereby regulate
insulin secretion.
In liver cells, glucose is only taken up when it is very abundant. Then liver
cells acquire glucose and convert it to glycogen and fatty acids.
Regulation of glucose
Liver
Muscle
Blood glu high, (feeding)
Glut2 takes up glu,
Glucokinase induced
G6P produced and used in glycolysis or stored
Blood glu high, (feeding)
Glut4 takes up glu,
Hexokinase makes G6P
If glycogen stores are filled, high G6P
inhibits hexokinase.
Blood glu low, (starving)
Glut2 not taking up glu
Glucokinase synthesis repressed
G6P not made
Blood glu low, (starving/resting)
Glut4 taking up glu (little)
Hexokinase is constitutively active
If glycogen stores are filled, high G6P
inhibits hexokinase.
During exercise,
Blood glu low/high
Glut4 takes up glu (little/much)
Low G6P,
Hexokinase fully active
High glycolysis from glycogen stores or
blood glucose
Glucokinase and
Hexokinase
Kinetic parameter
Km
Vmax
Tissue distn
Glucokinase
Hexokinase
High (10mM)
low affinity
high
low (<0.1mM)
high affinity
low
Liver, pancreas
muscle and other tissues
Glucokinase activity increases with increased glucose but is not inhibited by increased glu6PO4. The levels of the
protein are regulated by insulin.
Rate of reaction is driven by substrate-glucose not by demand for product-G6P. Allows all glu available to be
converted to G6P and then if excess present, it is converted to glycogen and from there to triglycerides and fatty
acids
Hexokinase activity increases with increased glucose but activity is inhibited by increased G6P. The levels of enzyme
are constitutive. It only generates ATP when energy is required.
Glucokinase is not normally active because its Km is lower than normal blood glucose levels. Eating food increases glu
in blood, activates glucokinase which converts glu to glycogen and fatty acids.
Blood glucose is 5mM
Note the sigmoidicity for
glucokinase and the much lower Km
for hexokinase I. When blood
glucose rises above 5 mM,
glucokinase activity increases, but
hexokinase I is already operating
near Vmax and cannot respond to an
increase in glucose concentration.
Glycogen
Hexokinase
Glu
Pyruvate
Kinase
PFK1
G6P
Pentose phosphate
(nucleotides/cell div)
F6P
F1,6P2
Glycogen synthesis
PEP
Pyruvate
Control of glycogen
synthesis from blood
glucose in muscle.
Insulin affects three of the five
steps in this pathway, but it is
the effects on transport and
hexokinase activity, not the
change in glycogen synthase
activity, that increase the flux
toward glycogen.
Regulation of PFK1
Regulation of three irreversible steps
PFK1 is rate limiting enzyme and primary site of regulation
Liver
Muscle
Enzyme levels induced by insulin and reduced by
starvation
Constitutively on
Allosterically activated by
AMP (during exercise)
F2,6,BP
Allosterically inhibited by
ATP (high energy state/resting)
Citrate (high energy state- from Krebs cycle)
(fatty acid oxidation)
Allosterically activated by
AMP (during exercise)
Allosterically inhibited by
ATP (high energy state/resting)
Citrate (high energy state- from Krebs cycle)
(fatty acid oxidation)
PFK1 regulation by F2,6P2
PFK-2 catalyzes
F6P + ATP -> F2,6P2 + ADP
PFK-2 allosterically activated by F6P and insulin (insulin induced dephosphorylation)
High Glu- high F6P
Therefore PFK2 active-- high F2,6P2
F2,6P2 activates PFK1 and you get high glycolysis and fat synthesis
FBPase1
PFK2/FBPase2
F2,6P2 is made and degraded during metabolic transition
Its conc determines whether you get gylcolysis or gluconeogenesis
It is a positive allosteric effector of PFK1
It is a negative (inhibitor) of FBPase1
F2,6P2 is made and degraded by a SINGLE enzyme with two distinct domains having two
distinct activities
-kinase (PFK2) synthesizes F2,6P2
-bisphosphatase (FBPase2) degrades F2,6P2
PFK2/FBPase2 is regulated by metabolic factors/hormones
F6P activates PFK2 and inhibits FBPase2 thus regulating level of F2,6P2
In liver
Lots of glucose,
Lots of F6P made
F6P activates PFK2 (also inhibits FBPase2)
This makes more F2,6P2
G6P
F6P
PFK2/FBPase2
PFK1
F2,6 P2
F1,6 P2
PEP
F2,6P2 activates PFK1 (also inhibits FBPase1)
This now makes more F1,6P2
When glucose levels drop and decrease
F6P levels drop
Inhibition of FBPase2 is reduced, F2,6P2
levels reduce.
Lower levels of F2,6P2 reduced inhibition of
FBPase1 and the reverse reaction now
proceeds making more glucose.
PFK2/FBPase2 is also regulated by
phosphorylation by PKA and PP2A
Phosphorylation- reduces PFK2 kinase
FBPase1 activity and increases FBPase2 activity
(generating more glucose)
PKA is regulated by AMP. More AMP in cell
means less ATP energy (i.e. less glucose) so
liver makes more glucose to secrete into
blood for other organs.
Coordinated Regulation of PFK-1 and FBPase-1
Both are inducible, by opposite hormones (insulin and glucagon)
Both are affected by F2,6P2, in opposite directions
Pyruvate dehydrogenase
Pyruvate + CoA + NAD -> AcetylCoA + CO2 + NADH
Glucose
Amino acids
Lactate
PDH
Acetyl CoA
CO2+H2O
Fatty acid
Oxaloacetate
Ketone
TCA cycle
Gluconeogenesis
Regulation of PDH
Muscle
Resting (don’t need energy)
Hi energy state
Hi NADH & AcCoA & ATP
Inactivates PDH
Hi ATP & NADH & AcCoA
Inhibits PDH
Exercising (need energy)
Low NADH, ATP, AcCoA
Regulation of PDH
Liver
Just been Fed (high blood glucose)
Need to convert glucose to Fatty acids
Hi energy
Insulin activates PDH
Starved (don’t need PDH)
No insulin
PDH inactive
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