Glycolysis: oxidizing glucose to get energy

- Glucose is the main fuel for our bodies.

- Glucose has a lot of reduced carbon (ie. energy).
- Glycolysis is the first step in recovering this energy.
Glycolysis has two phases:
1. Break glucose into two molecules of glyceraldehyde-3-phosphate.
requires spending two ATP: investment phase

2 Pyruvate

Glucose

2. Convert the two glyceraldehyde-3-phosphate into two pyruvate.

generates four ATP: payoff phase
pyruvate has several different fates
 Glycolysis I Glycolysis II

DHAP
The overall hexokinase reaction is highly exergonic and, thus, this reaction is essentially irreversible in the cell

Phosphofructokinase
Adds a phosphate to fructose-6-P
The key control point in controlling the rate of glycolysis.
Enzyme is elaborately regulated:
inhibited by high levels of ATP (why?)
this inhibition is reversed by high levels of AMP (why?)
inhibited by high levels of citrate (why?)
stimulated by fructose-2,6-bisphosphate (why?)
 Glucose enters the cell by:
1. Diffusion
2. Facilitated Diffusion using a transport
protein. These are both types of
passive transport, which requires no
energy. Both are reversible: is the
concentration inside the cell is
higher than the concentration
outside, glucose will flow back out.
3. Active transport, which requires ATP.
Glucose transport by Glut-1.
How to prevent reexport?
This is a case of facilitated
Change glucose into something else.
diffusion. It can move glucose
It will disappear from the glucose
down a concentration gradient
balance sheet, and will not be
(but not against a gradient).
recognized by glucose transport Glut-1 would export glucose if
proteins. the intercellular concentration
was higher then the extracellular
concentration
 Pyruvate - a major branch point in catabolism
PROBLEM: In the absence of oxygen, pyruvate must
be sacrificed to regenerate NAD+, which is required
for the next round of glycolysis.

More energy through the TCA Cycle and oxidative phosphorylation

Net Yield - Lactate Fermentation
One glucose -----> Two lactate

Net Yield - Ethanol Fermentation

1 glucose -----> 2 ethanol + 2 CO2
Pyruvate Still Possesses a Lot of Potential Energy that Cells
Can Use through Aerobic Metabolism

Glycolysis

Pyruvate
Dehydrogenase

Citric Acid
Cycle
 Follow the carbons
from acetyl CoA
through one round.
The two carbons lost
as CO2 each round are
NOT the two that
entered the cycle that
round. They came from
oxaloacetate.
Pratt figure 11-10.
 The TCA cycle
A very high energy yield
The glucose is oxidized as far as it can go, to CO2 and water
Occurs in the mitochondrion (in eukaryotic cells) where
the NADH can be regenerated to NAD+ by transferring
electrons
In prokaryotic cells, it occurs in the cytoplasm, but the
NADH can be regenerated in the same way
Dour phases:
0. Oxidation of pyruvate
1. The production of isocitrate
2. Two decarboxylations
3. The regeneration of oxaloacetate
 Phase 0: Entering the TCA cycle: pyruvate oxidation

The step between glycolysis and the TCA cycle Pyruvate is decarboxylated
with the production of 1 NADH, 1 acetyl CoA, and 1 CO2
Carried out by pyruvate dehydrogenase, a huge multi-enzyme complex that
contains 3 activities and 5 coenzymes (TPP, Coenzyme A, lipoic acid,
NAD+, and FAD)

Coenzymes of pyruvate dehydrogenase:

TPP: decarboxylates pyruvate, yields a hydroxyethyl-TPP anion
Lipoic Acid accepts the acetyl group (the long arm of lipoamide swings the acetyl
group between the active sites of the enzyme complex)
CoA accepts the acetyl group from lipoic acid arms
FAD reduced by lipoic acid arms
NAD+ reduced by FADH2
Multi-enzyme complexes have high catalytic efficiency!
Four electron pairs (in The thioester
blue) of acetyl-CoA that bond of acetyl
are ultimately used to
High-energy thioester
CoA is a high-
reduce NAD+ (3) and
FAD (1) in the Citric bond: energy bond
Acid Cycle DG = -7.5 kcal/mol with a DG =
-7.5 kcal/mol
 Phase 1 The TCA cycle: production of isocitrate

The first reaction of the citric acid cycle is the condensation of acetyl CoA with oxaloacetate to
make citrate and regenerate CoA
The citrate undergoes isomerization to produce isocitrate
 Phase 2: The TCA cycle: decarboxylations
The isocitrate is decarboxylated to yield a-ketoglutarate, which is in turn decarboxylated to yield
succinyl CoA
Each step yields one molecule of NADH
Now the two-carbon acetate has yielded its equivalent in CO2
But the cell has a succinate molecule instead of oxaloacetate
The chemical logic of cycles
Pyruvate decarboxylation yields acetyl groups, which are
two carbon molecules. Each carbon will be oxidized all
the way to CO2 in the Krebs cycle.
BUT it is hard to cleave between the two carbons of
acetate so it is condensed with oxaloacetete to make
6 carbon citrate, which is easy to rearrange in order to
perform a b-cleavage reaction.

O
||
C Ca Cb

easy to cleave here

 Phase 3: The TCA cycle: regeneration of oxaloacetate
Succinyl CoA is hydrolyzed to yield succinate
The succinyl CoA bond is a high energy one, and in the course of this reaction, one ATP is
formed by substrate phosphorylation
Succinate is oxidized to fumarate yielding one FADH2
Water is added to fumarate to make malate
Malate is oxidized to oxaloacetate yielding one NADH

What happens to the NADH?

Its oxidized and oxygen is reduced to make water:
NADH + H+ + 1/2 O2--> NAD + + H2O
This reaction is highly exergonic.
its carried out in steps, and some of the energy
released is converted to ATP.
The process is called electron transport and occurs in
mitochondria in eukaryotic cells and in the cytoplasmic
membrane in prokaryotes
 b-cleavage in glycolysis
Why was glucose-6P converted to fructose-6P?
To set up the subsequent b-cleavage reaction

Could you do b-cleavage on glucose?

What products would you get?

glucose 6-phosphate fructose 6-phosphate

 Electron Transport Participants:

NADH or FADH2 (electron donors)

Oxygen (electron acceptor)

Complexes I, II, III, and IV

FMN
Iron-sulfur proteins
Ubiquinone (Co Q)
Cytochromes (protein plus heme)
b, c1, c, a, a3

Where do the high energy electrons come from?

Dehydrogenases that generate NADH and FADH2
The electron transport chain
The NADH and FADH2 produced during glycolysis, acetyl-CoA formation, and the TCA cycle enter the
electron transport chain.

The electron transport chain consists of a series of electron carriers embedded in the inner mitochondrial
membrane. Each can be reduced as it accepts electrons, and oxidized as it gives them up.

The electrons are passed along in a series of exergonic redox reactions, their energy is used in the
endergonic process of ATP formation in the process of oxidative phosphorylation.
Biochemical anatomy of a mitochondrion.

Succinate dehydrogenase is in inner membrane, all other TCA cycle

enzymes are in matrix.

The convolutions (cristae) of the inner membrane provide a very

large surface area.
The inner membrane of a single liver mitochondrion has 10,000 sets
of electron-transfer systems (respiratory chains) and ATP synthase
molecules.
Components of the Electron-Transport Chain
There are 4 respiratory complexes embedded in the inner
mitochondrial membrane in the electron-transport chain.
Each consists of several proteins that are associated with a
variety of redox-active prosthetic groups with successively
increasing reduction potential.
 Succinate Dehydrogenase
of TCA Cycle

Summary of electron and proton flow through the respiratory chain.

Electrons reach CoQ through Complexes I and II. The reduced Q (QH2)
serves as a mobile carrier of electrons and protons. It passes electrons to
Complex III, which passes them to another mobile carrier, cytochrome c.
Complex IV then transfers single electrons from reduced cytochrome c to
O2. Electron flow through Complexes I, III, and IV is accompanied by
proton flow from the matrix to the intermembrane space.
Complexes of the electron transport chain.
As electrons are passed through and between complexes,
they move to positions of lower free energy. The energy
released is used to pump protons, which in turn drive
ATP synthesis.
 Reduction Potential of Participants
in the Electron Transport Chain
Complex I - a measure of a
compound's potential energy,
analogous to delta G
- quantitates "how much a
compound wants electrons"
- If a compound has a
Complex II large negative Reduction
Potential, it will donate
electrons to compounds with
more positive R.P.
- If a compound has a
large positive Reduction
Complex III Potential, it will accept
electrons from compounds
with lower R.P.
- electrons flow from
negative to positive

Overall Reaction:
NADH + O2 + H+ <=> NAD+ +
H2O
Complex IV DGo = -53 kcal/mol
Complex I: harvests electrons from
NADH, produces ubiquinol (reduced
form) and regenerates NAD+ (oxidized
form). Pumps Protons!
Complex I (NADH dehydrogenase)
Accepts electrons from NADH, gives
them to CoQ.
Contains one flavin mononucleotide
(FMN) and 6-7 iron-sulfur clusters.
7 of its 43 subunits are encoded by
mitochondrial genes.
Coenzymes are FMN and ubiquinone
(CoQ).
Complex II: harvests electrons from succinate, also
produces ubiquinol (reduced form), produces fumarate
(where have we seen this reaction before?)
does not release enough energy to pump protons!
Complex II (Succinate dehydrogenase)
Contains citric acid cycle enzyme succinate
dehydrogenase and three other subunits.
Passes electrons from succinate to CoQ.
No protons transported in this step, so less ATP is
made from FADH2 than from NADH.
Does so with covalently bound FAD.
Complex III: harvests electrons from Cytochromes:
ubiquinol, produces cytochrome c (reduced
form), regenerates ubiquinone (oxidized Proteins with heme prosthetic group
form) Pumps Protons! covalently bound via thioether bonds.

Cytochromes (b, c1, c, a, a3) differ in:

protein structure
heme structure
absorption spectra
reduction potential
role in electron transport
Complex IV: harvests electrons from cytochrome c,
electrons are passed all the way to O2 to produce
water, regenerates cytochrome c (oxidized form)
Pumps Protons!
The more positive the
standard potential (measured
in volts), the greater the
affinity for electrons. To have
sequential transfer of
electrons, carriers must
transfer electrons to carriers
with higher standard
potential.
 The Chemiosmotic Model
Proposed by P. Mitchell in 1961
put bacteria in acidic media, they can synthesize ATP
even if electron transport is blocked
Electron transport establishes a proton gradient by
pumping protons out of the inner mitochondrial
membrane
This proton gradient is a form of potential energy that
is used to drive ATP synthesis by ATP synthase: this
electron diffusion is exergonic because entropy is
increased. This energy is captured in the endergonic
reaction: ADP + Pi --> ATP
ATP synthesis:

Protons flow
through ATP
synthase, causing
the F1 head to
rotate.
This rotation forms
ATP from ADP
and Pi by the
binding change
mechanism.
 The Binding Change Mechanism

The F1 region of ATP synthase has 3 a-b pairs, each of which has a
binding site. Each site has 3 possible conformations:
O (open nothing bound)
L (loose has bound ADP and Pi)
T (tight ADP and Pi combine to form ATP)

Rotation of the F0 stalk causes the 3 a-b pairs to change between

these three conformations.
Why are the phosphoanhydride bonds of ATP high energy
bonds?
The favorable DG of hydrolysis of ATP
depends on the difference in free
energy between the substrate and the
products. ATP has 4 negative charges
close together which creates strain,
hydrolysis relieves this strain in several
ways.

1. Phosphoanhydride bonds have less resonance stabilization than their

hydrolysis products do. This is because the two strongly electronegative Ps
compete for the electrons of the bridging O:
2. Phosphoanhydride bonds have more
repulsion (caused by the close negative
charges) than their hydrolysis products do.
3. Phosphoanhydride bonds have less
solvation energy than their hydrolysis
products do.
 from glycolysis: 2 ATP + 2 NADH
from Ac-CoA synthesis: 2 NADH
from TCA cycle: 6 NADH, 2
FADH2, 2 ATP
in electron transport, each NADH
can yield 3 ATP so the 10 NADH
yield 30 ATP (however, the 2 NADH
from glycolysis are in the cytoplasm,
and energy is required to get them
into the mitochondrion, thereby
reducing the ATP yield)
FADH2 yields 2 ATP, so 2 FADH2
yield 4 ATP
Final yield: 38 ATP. 4 from
substrate level phosphorylation and
ATP Yield from Oxidative Phosphorylation: 34 from oxidative phosphorylation
Traditional stoichiometry:
NADH 3 ATP
FADH2 2 ATP
Revised stoichiometry:
NADH 2.5 ATP
FADH2 1.5 ATP
Gluconeogenesis
Glycolysis
Gluconeogenesis: steps 1 and 2 reverse step 10 of glycolysis Gluconeogenesis: step 9 reverses step 3 of glycolysis
(and reintroduce an old friend)

Gluconeogenesis: step 11 reverses step 1 of glycolysis

DG is different in cells because [S] and [P] differ
from standard conditions
 Reciprocal Regulation
Should cells switch back and forth between
glycolysis and gluconeogenesis?
too expensive
So how does the cell know which pathway
to perform?
It calculates its ENERGY CHARGE, which
measures how much of the adenylate pool is
in the ATP form.
Cells like their energy charge to be about
0.95. If is is lower, they will do glycolysis.
If it is higher, they will put the glucose
away for later use.

So how do cells turn on the pathway they

want and turn off the pathway they dont
want?
In energy charge, ATP counts 2
They inhibit or stimulate the enzymes at the because it has two
start of each pathway. phosphoanhydride bonds. ADP has
1 and counts 1
The inhibitors are usually end products of
the pathway or products of related pathways
(feedback inhibition).

For example, ATP inhibits glycolysis. Does

that make sense?
 high levels of this
arrows show how
compound change the
high levels of this
activity of this
compound change the
enzyme in the
activity of this
direction shown
enzyme
Glycogen Particles
Glycogen is a polymer of glucose.

Ratio of glucosidic linkages:

10-12 a-1,4
1 a-1,6

Glycogen polymers exist as spherical particles that contain 10,000

60,000 glucose residues
Glycogen particles are found in virtually every cell, but are particularly
abundant in liver and skeletal muscle
in liver, the primary function of glycogen is regulation of blood
glucose levels
in muscle, glycogen provide a rapidly available energy source

Glycogen is degraded by the enzyme phosphorylase a

Phosphorylase a:
chews one glucose at
a time from the end
of a chain.

It cant chew close to

a branch, so when a
chain gets very short,
a transferase enzyme
moves it to the end of
a longer chain.

Phosphorylase a also
cant chew a branch
(why not?)
 Glucose released as Glc-1-P is easily converted to Glc-6-P

Splitting apart the two phosphates of

PPi can pull reactions forward
It liberates energy from another
phosphoanhydride bond.
 Activation of Glucose for Glycogenesis
High-energy bonds

Glucose-1-
phosphate
UTP

Pyro-
phosph 2 Pi
ate
UDP-Glucose
 Hormone:

A substance produced by one organ, transported via the circulatory system to

another organ, where it affects some
aspect of metabolism.
 Protein Kinase A is activated by adrenaline and phosphorylates enzymes that promote
Glycogenolysis
Protein Kinase a also phosphorylates glycogen synthase to inhibit Glycogenesis

Adrenaline

Adenylate
cyclase
Receptor GTP PKA
ATP cAMP P
(inactive)
GS
(inactive)
G protein
GPK PKA
(inactive) (active)
GS
(active)
P X UDP-Glucose
GPK Glycogen
(active)
GP P
GP
(inactive)
(active)

Glucose-1-P Glucose
GPK = Glycogen Phosphorylase Kinase
GP = Glycogen Phosphorylase Glucose-6-P
GS = Glycogen Synthase
 Glucose Homeostasis
Concentrations of glucose in our blood
must be maintained in a narrow range
(70 105 mg/dL) for optimal health:
If blood glucose levels fall below 70
mg/dL, hypoglycemia (low blood
glucose) will result in weakness,
sweating, rapid heart beat and, if severe,
mental confusion, convulsions, coma and
death

Diabetes occurs when fasting blood

glucose levels are above 126 mg/dL
(hyperglycemia) and if it persists for long
periods of time can lead to increased risk
for cardiovascular disease, kidney
disease, blindness, difficulty of wound
healing
 Insulin
a protein hormone that
regulates blood glucose levels
secreted by the beta cells of
the pancreas
stimulates cells to take in
glucose
stimulates muscle and liver
to make glucose into
glycogen
stimulates storage of amino
acids and fat
 Insulin and Glucagon
Insulin and glucagon are polypeptide hormones
synthesized, stored and released from the pancreas
b cells in endocrine pancreas produce and store insulin
and release it in response to hyperglycemia
insulin promotes glucose uptake and utilization in important
metabolic tissues (liver, skeletal muscle, adipose)

a cells in pancreas produce glucagon and release it in

response to hypoglycemia
glucagon promotes glucose formation and export by
glycogenolysis and gluconeogenesis in liver
 Diabetes
complex, polygenic disease of glucose homeostasis
many phenotypes including kidney problems, blindness, and
poor circulation in extremities
type I (juvenile onset): the beta cells are destroyed by an
autoimmune response, patients have no insulin at all and
must inject insulin daily
type II (adult onset): patients have gradually reduced insulin
sensitivity. For examples, the muscles become resistant to
insulin and the pancreas must make more. Eventually, the
pancreas wears out and is unable to make enough insulin
Many medical problems result: blindness, poor circulation, etc.
thought to be due to problems in vascular tissues, both
macrovascular and mircovascular as well as to inappropriate
glycation of proteins
 Why is high glucose bad?
1. Inappropriate glycation of proteins.
During prolonged hyperglycemia, glucose can react
nonenzymatically with the NH3 group on the amino
terminus of hemoglobin. This form, called HbA1c, can
account for more that 12% of the total hemoglobin in a
diabetic patient.
2. Diabetic cataracts are caused by increased glycation of
lens proteins, making the lens of the eye cloudy.
3. Glycated proteins and lipoproteins can be recognized by
macrophages, which can lead to accelerated
atherosclerosis.
4. Glycated proteins have altered activities, solubilities, and
degradation properties.
 Type 2 diabetes
Patients still make insulin but are
insulin resistant
Insulin can be present at normal or
elevated levels
However, response is
impaired. b-cells dont make
enough insulin to control glucose
synthesis in the liver or to
stimulate glucose uptake by skeletal
muscle.
gluconeogenesis occurs in the liver
even though there is enough
glucose available.
fatty acid synthesis is also not shut
off causing hypertriacylglycerolemia
and high VLDL
80-90 % of all cases are type 2,
many people with type 2 diabetes
are undiagnosed
Treatment
Diet can control diabetes in some patients.
If patient is obese, losing weight will increase
the number of peripheral insulin receptors,
improving insulin sensitivity.
Medications:
sulfonylureas: stimulate insulin secretion
from b-cells
metformin: reduces liver gluconeogenesis
thiazolidinediones: sensitize peripheral
tissues to insulin
alpha glucosidase inhibitors: slow glucose
adsorption
oral insulin: insulin is a protein and is
digested by the GI tract. Lots of effort is
going into devising a way to deliver oral
insulin.
inhaled insulin: Last year, the FDA
approved the use of Exubera, the first
inhalable insulin.
 alpha glucosidase inhibitors: slow glucose adsorption

Miglitol, oral, take at start of meal

slows the digestion of starch in
the small intestine, so that glucose
from the starch of a meal enters
the bloodstream more slowly, and
can be matched more effectively
by an impaired insulin response or
sensitivity. Effective by themselves
only in the earliest stages.