10/2/19

Lecture 5
Glycolysis
Fermentation
Electron carriers
You should be able to explain to a friend…

ATP’s role as a short-term phosphate/energy carrier

NADH’s role as a short term electron (and later, energy) carrier
What is meant by “coupled reactions”
How the cell can harvest energy from oxidation of glucose
What’s the relationship between glycolysis and fermentation?
What problem does fermentation solve?
When the cell uses glucose as an energy source, what is harvested
and what is thrown away?

Unlike a match or spark..

• Life reduces barriers for some reactions without affecting
others… using proteinaceous catalysts called enzymes that
have very specific substrates (= reactants) and products
• Enzymes reduce activation energy barriers- this makes
both the forward and reverse reactions faster- or in most
cases, possible on a biological time scale
• Enzymes can be switched on and off, and act as stopcocks
or lockable doors to regulate the flow of energy and
materials
• Like all catalysts, enzymes regulate the rate, but not the
direction or energetics, of a reaction.

Moving on, here’s a MAJOR issue:

If ∆G determines the direction of a reaction (bulk flow is in the

direction of lower G), how can we build big complex things out of
little, simple ones?

For example: we eat plant protein, and digest it down to its

constituent amino acids, absorb these, and reassemble them into
human proteins.

The digestion (“hydrolysis”, splitting by water) makes sense:

∆G0’ = about -12 kJ/mol

(both R and R’ are the rest of the protein- really big molecules)

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How can we get protein synthesis to be energetically favorable?

∆G0’ = about +12 kJ/mol

(condensation reaction- spits out water as a product)

One possible solution?

∆G depends on concentration, right? So…. Maybe we just have tons

of amino acids (aa) and very little protein?

High concentration? Low concentration?

∆G = is negative if [amino acids] is super high??

that’s not actually the answer- the real answer’s trickier

It also wouldn’t explain how we get the aa in the right order! Think of the
reverse entropy costs there! We’ll get into this in lecture 21

Glycolysis is a pathway all living things use to get

energy (for building stuff) by breaking down/oxidizing
glucose

The overall pathway is exergonic

But some of the steps, under standard conditions, are

“endergonic”

So how can the reaction “go”????

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Clickers

The answer lies in the fact that Life does not occur generally
under standard conditions

∆G determines rx direction
∆G˚’ is just one factor in the calculation

What conditions does life change to control the reaction direction of hundreds of
metabolic reactions simultaneously?
A) Temperature
B) pH
C) Pressure
D) Concentration of reactants and products

Really?
An uphill climb in G?
No! this diagram both:
fails to take
concentration into
account (glucose is about
50x the conc. of the
Change in free energy vs. glucose

other metabolites).
-And-
Doesn’t factor in the big
negative ∆G of ATP
hydrolysis coupled to
these first few reactions.
It just follows the G˚’ of
each glucose-derived
compound, I guess.

Reactions “go” in the direction determined by ∆G.

Life can manipulate ∆G by manipulating concentrations

and also by physically coupling unfavorable (positive ∆G)
reactions to very favorable ones (larger negative ∆G).

Essentially “paying” for one reaction with an “energy

currency”- and often that’s ATP.

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ATP
The business end

You need to learn this vocabulary, but you don’t need to memorize the structure.

Both of these are “high-energy”* bonds

ATP

O- O- O-
O= P - O - P - O - P - O-A
O O O
“A” means adenosine-
see previous slide

*Meaning HERE- CONFUSINGLY- that we’re going to get a lot of energy out when we
rearrange them.
The bonds themselves are actually pretty weak (= unstable).

It’s all in hydrolysis (adding water to break a bond)

O- O- O-
O= P - O - P - O - P - O-A ATP
O O O
H2O
ADP
O- O- O-
O=P-OH + HO-P - O - P - O-A
O Pi O O
Inorganic phosphate What’s the big difference?
ENERGY RELEASED

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Why is this reaction exergonic?

Like charges repel!

Two molecules from one (# water molecules, isn’t really changing)
Electrons gain a lot of freedom as free inorganic phosphate (Pi)!

ATP can also give up a DOUBLE dose of energy

O- O- O- ATP
H2O + O= P - O - P - O - P - O-Ade
O O O

O- O-
O=P-O-P-OH + O-
O O PP i HO- P - O-Ade
(pyrophosphate)
O AMP
(Adenosine monophosphate)
...but wait, there’s more !

It also can give up a DOUBLE dose of energy (more pulling)

O- O- O- ATP
H2O + O= P - O - P - O - P - O-Ade
O O O

O- O-
O=P-O-P-OH + O-
O O PP i HO- P - O-Ade
+ H2O O AMP
This second reaction “pulls” the first- it’s all
2 Pi about concentration- the cell keeps [PPi] low, in
order to enhance that -∆G of ATP->AMP.

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The ∆G from hydrolysis of ATP could drive:

Formation of a concentration gradient
Endergonic chemical reactions
Muscle contraction

ATP + H2O
∆G˚ = -7.3 kcal/mol

G ADP + Pi

∆G˚ = -10.9 kcal/mol

(When the reaction needs a bigger kick)

AMP + Pi + Pi

You don’t eat ATP*, you use fuel to reload it

ATP is an energy carrier, like a taxi, picking up energy here,
dropping it off where its needed, picking up another passenger…

*Good thing too, as you

unload phosphate from
about 50 kg ATP/day

Glycolysis- extremely core metabolism

• Energy from oxidation of glucose- stored as ATP
• You will note that the cell needs to spend 2 ATPs first in order to build 4
ATPs later
• But… there is an inherent problem with glycolysis- glycolysis alone can’t
get the job done.

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What goes in, what comes out:

Glucose, 2ADP, 2Pi, 2 NAD+ -> 2pyruvate, 2ATP, 2NADH

All living things do this. Do memorize this!

Great! We built ATP! Plus other goodies and/or junk.

The carbon in pyruvate is more oxidized than the carbon in glucose,

even though we’re not “burning” anything, and there’s no O2
involved at all. Electrons are being allowed to hang out with O
more, which is a lower energy state than hanging around C or H.

C6(H2O)6 2 x C3H3O3
Note the C’s and O’s balance here, but
the H’s do not. Where’d they go?

Red = O (6)
Black = C (6)
White = H (12)

This diagram takes the whole glycolysis picture into account, energetically- including
concentration in the cell, and all of the compounds involved in each step. The ∆G’s are
depicted as arrows. Arrow #1 is “one way” because there is a nice -∆G in step 1. Arrow
#6 is two-headed because the overall ∆G of the reactions in step 6 is closer to zero. The
“overall arrow” (glucose -> pyruvate) is one way also.
ATP->ADP ATP->ADP

1 glucose in

2 pyruvate out

2 x 2e- redox:
2NAD+ -> 2NADH
2Pi also added

2 x (ADP -> ATP) 2 x (ADP -> ATP)

You do not need to memorize these structures!

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Arrow #1, above:

There are more ways to “push” a reaction
than just by manipulating concentration,
though that’s happening here too
[glucose]>>[G-6-P].
1The enzyme (= catalyst) hexokinase breaks
down an ATP while it builds glucose 6-P. One
reaction can’t happen
-
without the other- ATP
is the source of phosphate for G-6-P.

Although glucose-6-P has a higher G0’ than

glucose + Pi, ADP has a (much) lower G0’ than
ATP. This entire rx has a big negative ∆G. So it
“goes”.

This is also an example of both “cashing in” an

ATP (using its energy) and of using ATP as an
aggressive phosphate donor.
Glucose + ATP à G6P + ADP

Need energy? Need a phosphate group? See ATP. Does it do anything else? Oh, right, it’s the
A in the 4 letter alphabet of heredity. A is perhaps for Awesome.

What do we mean by “high energy phosphate

bond” in ATP?

A. The phosphate group is tightly bound and very stable

B. The bond between phosphates will release a lot of energy when a
phosphate is bound to water, instead of another phosphate.

We mean “B”

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How come sometimes ATP is used to drive a reaction (as in the “investment” steps),
while other times ATP is the product of a reaction?

If those 3 phosphates hate being next to each

other in ATP, there are other neighbors that they
hate even more.

Standard ∆G of hydrolysis* of some phosphorylated compounds

*losing a Pi to water 3PG= 3-phosphoglycerate

Standard ∆G of hydrolysis of some phosphorylated compounds

Could PEP act as a phosphate donor to ADP?

Informally, we could just look to see which compound has the more negative ∆G of
hydrolysis- that’s your phosphate donor. The compound with the more positive ∆G of
hydrolysis is your phosphate acceptor. These values give us a feel for how hard the Pi is
being pushed to dissociate from the molecule.

Hopefully what I mean by “more negative” and “more positive” ∆G is clear,

because you’ll see it on tests.

Standard ∆G of hydrolysis of some phosphorylated compounds

Could PEP act as a phosphate donor to ADP, forming ATP?

Formally, we could set up an equation, carefully writing each reaction in the correct
direction, to end up with an overall negative ∆G for the pair of reactions.

PEP + H2O -> pyruvate + Pi ∆G˚’ = -14.8

ADP + Pi -> ATP + H2O ∆G˚’ = 7.3 Why is the sign changed here?

PEP + ADP -> ATP + pyruvate ∆G˚’ = -7.5

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2PG

PEP + H2O -> pyruvate + Pi ∆G = -14.8 Phospho-enol pyruvate (PEP)

ADP + Pi -> ATP + H2O ∆G = 7.3
PEP + ADP -> ATP + pyruvate ∆G = -7.5
In the last step of
glycolysis, another
This is how we generated high-energy
the second batch of ATP
phosphate bond is
(step 10) traded for an ATP.

Pyruvate

The same idea applies to this: Even ignoring the fact that [glucose]
in the cell is relatively high vs
[G6P]… and using standard ∆G
values….

G6P
Change in free energy vs. glucose

Glucose + Pi à Glucose-6-P (∆G˚’ = +3.3)

ATP à ADP + Pi (∆G˚’ = -7.3)

Glucose + ATP à G-6-P + ADP

(∆G˚’ = -4.0 kcal/mol)
…our reaction (directly above) is
exergonic, even under standard
conditions.

General diagram of how cells burn fuel (so far)

ATP ADP 2x2e- 2ADP 2ATP 2ADP 2ATP

glucose A B 2C 2 2E 2F 2 pyruvate
D
ATP ADP

C6H12O6 2 x C 3H 3O 3
1 glucose -> 2 glyceraldehyde 3-phosphates -> 2 pyruvates
Invest 2 ATPs Produce 4 ATPs
note we lost track of some H’s somewhere, but we know where our C’s and O’s are

This is glycolysis

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Energetics balance sheet for glycolysis

• Taking glucose to 2 pyruvates represents a ∆G˚’ of about -140 kcal/mol
• We net 2 ATPs from 2ADP + 2Pi!
• But that represents “storage” of only 15 kcal/mol glucose metabolized!*

*at least, under the “standard conditions”.

Is this an efficient process?

C6H12O6 2 x C 3H 3O 3
1 glucose -> 2 glyceraldehyde 3-phosphates -> 2 pyruvates
Invest 2 ATPs Produce 4 ATPs

In this pathway, is glucose reduced or oxidized?

a) Reduced
b) Oxidized
c) No way to tell- I need to see the structures of glucose and pyruvate

Oxidized

What do you do with those electrons?

What is the oxidizing agent?

ATP ADP 2x2e- 2ADP 2ATP 2ADP 2ATP

Fuel A B 2C 2 2E 2F 2G
D
ATP ADP

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What happens to the electrons?

Initially, the electrons go to a temporary private internal e- carrier.
- a device that carries electrons.

They can then be used for…

Biosynthetic pathways
- some of the reducing power is used for reductive biosynthesis
-or-
Respiration
- a great trick for getting energy from excess electrons

Or they can be dumped onto some internal metabolite during

fermentation - when there’s no external electron acceptor.

(2e-, 2H +, from glycolysis for example )

OK e- acceptor Pretty good e- donor

A universal
all-purpose
internal
electron carrier,
NAD+/NADH

Like ADP/ATP, NAD/NADH is a taxi

It picks up and delivers electrons
(2 of them) and energy.

In glycolysis…

Electron source- from outside the cell

might be used up (here, glucose).

Electron carrier- from inside the cell

is recycled (here, NAD+/NADH)

Terminal electron acceptor- from outside the cell,

might be used up (haven’t gotten to this yet)

How does this differ from the role of ATP, our phosphate carrier?

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Problem (design challenge)

We can’t just keep adding electrons to NAD+ (can we?).

How are we going to unload those electrons so we can keep
oxidizing glucose during glycolysis, and keep making ATP?

DUMP them on an
OPTIONS for the internal acceptor, FEED them to a greedy
electrons on NADH: and excrete that external electron acceptor and
(fermentation) use the resulting -∆G to make
USE them for synthesis
ATP
of things that need to be (respiration- coming soon!)
reduced.

2e-, 2H+
2e-, 2H+

NAD+ NADH + H+

NAD+ NADH + H+
2e-, 2H+
2e-, 2H+
Fuel A B C D E F G

In fermentation, there is no external electron acceptor. So, you’re stuck with

options 1 and 2.

In fermentation, the NAD+ is regenerated by “dumping” the electrons on pyruvate

and excreting the resulting product (here lactate).

Spit
this
out

Yogurt, Salami, Sauerkraut, your muscles

http://www.nytimes.com/2006/05/16/health/nutrition/16run.html

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Another way- turn pyruvate into acetaldehyde (lose a CO2), then

reduce that into ethanol, then excrete the ethanol

Your muscles don’t do this, but some microbes do.

What would happen if our anaerobic muscles created ethanol instead of lactic acid?

Energy story example (from earlier reading)

What is the “energy story” for glycolysis?

What is fermentation’s contribution to the energy story?

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Energetics balance sheet for glycolysis

followed by fermentation
• Taking glucose to 2 pyruvates represents a ∆G˚’ of about
-140 kcal/mol
• We net 2 ATPs from 2ADP + 2Pi
• But that represents “storage” of only 15 kcal/mol
glucose metabolized!
• (should we count the energy stored in NADH?)

That’s just depressing.

However, in the presence of an external electron acceptor…

We can perform Respiration

= A way to get every last bit of goodness out of pyruvate, instead of dumping it.

When you have no external oxidizing agent, you’re stuck with

fermentation

Glycolysis gives us 2 ATP, 2 NADH,

2 pyruvates

Fermentation uses up the pyruvate, so

the cell can recycle any spare NADH

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If you didn’t have to dump the pyruvate (C3H3O3)…

Maybe you could oxidize (burn)

pyruvate a little more: maybe
even all the way to CO2 and get
some more ATPs/glucose?

Complete oxidation of glucose to 6 CO2 produces a net 4 ATPs by substrate-level

phosphorylation and a LOT of ATPs by cashing in NADH. Trust me, we’re going to use those
NADH’s.

We need to recycle all

these NADs- but we’ll use
an external electron
acceptor.

Before we allow all these

electrons (10 NADH,
2FADH2) to join with our
oxidizing agent, we’re
going to make them sing
for their supper.

Why does almost everything in glycolysis, pyruvate oxidation, and

the citric acid cycle come in sets of 2?
2 ATPs at a time, 2 NADH at a time….

A: Because NADH can only carry pairs of electrons

B: Because ADP carries a pair of phosphates
C: Because the 6 carbon chain of glucose is broken up into two 3 carbon
chains in glycolysis
D: Because an ATP is always paired with 2 NADH’s

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Where do the extra NADH’s come from? Pyruvate oxidation, plus the citric acid cycle

This redox rx produces

2 NADH/glucose- and
we generate/lose our
first CO2

Look!- here’s our first CO2

what happens to acetyl coA?

You do not need to Acetyl coA

memorize all these
intermediates.

You need to know what

goes in and what comes
out.

The citric acid cycle gives us 6 more

NADH/glucose, 2 FADH, and 2ATPs

The citric acid cycle (aka Krebs cycle, TCA cycle) gives us,
per glucose:
6 NADH, 2 FADH, and 2ATPs- in addition to what we got
from….
Glycolysis: (2 NADH +plus 2 ATP) and
pyruvate oxidation: (2 NADH)

And now ALL of our original carbon has been converted to

CO2, so we’re done with that- we’ve oxidized it as much as
we can.

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It’s nice that we made those extra 2 ATPs, but

what are we going to do with all that NADH
and FADH? We don’t even have pyruvate
around anymore to dump the electrons on!

That’s not a very

impressive energy story.

When a cell performs respiration, it can use the electrons stored

on NADH (and FADH) as a source of power. We’ll discuss this
Monday.

Glycolysis allows a heterotrophic cell to make some ATP by harvesting the

energy difference between a large and a small molecule, plus some redox
(gaining NADH). Pile up enough ATP and NADH, and you can build
something.

Fermentation is a way to balance the NADH budget, if you don’t need

NADH for something else.

Respiration is an alternative to fermentation that allows the cell to harness

the power of the oxidation of glucose all the way to CO 2

Respiration includes:
A: Harvesting high-energy electrons (during glycolysis, pyruvate oxidation,
citric acid cycle) (check!)
B: Cashing in electrons for ATPs (electron transport plus chemiosmotic ATP
synthesis, aka “oxidative phosphorylation of ATP”)

Food

Glucose
glycolysis

Pyruvate
Now two options

Fermentation (of glucose) Respiration (of glucose)

(one or two steps, simple!) (amazingly complicated, but worth it)

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Glucose is universally used as fuel (an energy

source) by living things.
Overall equation for aerobic respiration of glucose:

But does this reaction

actually take place?
No, the oxidation happens in tiny
steps, some -∆G’s are big enough
to power ADP -> ATP, or NAD+ ->
. NADH. We saw all of these…
except the O2 part! Directly
oxidizing sugar to CO2 would just
product heat and light .
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Yes- potential energy!

How do cells generate usable energy from

this redox reaction (electrons moving to O
from C and H)?

i.e., how are all those stored-up e- going to

help us?

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