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PROFESSOR: Today we're going to discuss bioenergetics, continue
that discussion and use that as a transition
into also beginning to see how that applies to understanding
glycolysis, the pathway that allows glucose breakdown.
So just as a reminder of a couple of the points
that we discussed last time, so last time
we discussed what makes reactions favorable.
And so remember, you could have any reaction, A to B--
A to B could be a single step.
A to B could be an entire pathway, all steps
in the pathway, that whether or not that reaction
or pathway happens is determined by thermodynamics.
It's not determined by enzymes.
Enzymes are important, of course,
to control the rate of reactions so they
may allow things to happen that otherwise wouldn't happen.
But whether the reaction happens or not
is determined by free energy.
And so free energy, delta G, remember
we discussed is the following relationship.
So it's related to this constant delta G0
prime, which is related to the equilibrium
constant for any pair of products and reactants
as well as this formula rt times the log of the products
over the reactants.
And so remember this means that whether a reaction happens
depends on the equilibrium constant,
but it also depends on the actual conditions present.
That is how much substrate and how much product is there.
And so what this means is that biology can come up with ways
by keeping the product concentrations low to do things
that may move in the opposite direction of what you would
predict from equilibrium.
And also by coupling these less favorable reactions
to more favorable reactions, you can also allow biology
to do unfavorable stuff.
And this is, of course, where the role of ATP comes in.
And we discussed this at the end of last time as well.
Of course, what's important then becomes the ratio of ATP
to ADP.
And to illustrate that, we use this example.
We use the-- remember ATP hydrolysis has a delta G0 prime
of minus 7.5 kcals per mole.
That, of course, tells you this is
about the equilibrium constant.
That tells us the equilibrium lies to the right.
We can couple it to this first reaction in glycolysis,
which traps glucose in the cell by adding a phosphate group.
Delta G0 prime for this is positive 3.3 kcals per mole.
This positive number tells you that the equilibrium
lies towards the left.
But when we couple these two reactions together,
we add these two numbers together,
and now we get a delta G0 prime of minus 4.2 kcals per mole,
which are the coupled reaction.
The equilibrium lies now again to the right.
And that's how we can use ATP hydrolysis in order
to carry out this unfavorable process, trapping
the glucose in the cell.
Now how much glucose will be trapped?
Well, of course, that will come from this reaction up here.
And that's because whether or not
a reaction occurs or not of course
depends not on the equilibrium constant
but on the actual conditions and the delta G of that reaction.
And so we can just plug that in for this system.
And so we see delta G0 prime minus 4.2 kcals per mole.
That says equilibrium lies to the right.
But how far really depends on the conditions.
And that's defined by this ratio of products over reactants.
And you can imagine that this term here, as long
as it's less than 4.2, you will favor glucose trapping
inside the cell, whereas, of course,
if it's greater than 4.2, then you'd favor--
the opposite direction would be favorable.
Now this is really critical because this really
explains how it is that ATP actually
works to help cells do various reactions.
And so if an ATP-ADP ratio exists such
that it can make delta G less than 0
for the reaction that it's coupled to,
that is how it provides the energy that then allows
unfavorable reactions to occur.
And so please do not make the common mistake
that many, many biologists make when they state,
oh, ATP equals energy.
There's a lot of ATP.
The cell must have a lot of energy.
Remember it's the ratio of ATP to ADP
that provides the energy.
And so that means ATP can be sky high,
but if ADP is equally high, there's no energy there.
Consequently, ATP can be very low.
But if ADP is extremely low, now you still
have a high ATP to ADP ratio.
And that is what provides the energy.
And that's because this occurs regardless of concentration.
It's because it's this ratio of ATP to ADP
that ultimately says how much energy is there for the ATP
to drive a reaction.
In other words, delta G, or whatever the reaction
is coupled to, is going to be proportional
to the ratio of ADP to ATP.
Great.
Now how much ATP do you need then?
What ratio do you need to drive a reaction?
Well, it's hard to say that in absolute terms
because it's going to be defined by how that ratio is coupled
to the ratio of whatever other reactants and products.
It also tells you, remember, as we talked about before, we
can switch the directions of these reactions
and we just flip the signs.
The directions here are arbitrary.
And so that means it also tells us
how much energy we need to put in if we're going
to charge up an ATP-ADP ratio.
In other words, if we want to synthesize
ATP, that is take ADP plus phosphate
and make an ATP, well, what's this?
What we just-- the exact opposite sign
of that delta G0 prime is going to now equal
plus 7.5 kcals per mole.
And so that means that the equilibrium of this reaction
is going to lie to the left just as that one lied to the right.
And it's going to lie to the left by that much.
And if we want to know how much energy it then
takes to make ATP, well, obviously, it's
not going to happen because equilibrium
favors this direction.
It's not spontaneous in that direction.
But at various conditions, we can
know by plugging in our formula delta G equals delta
G0 prime plus 7.5 kcals per mole plus RT times the log of ATP.
So if I have my ADP--
I'm sorry-- my ATP concentration sufficiently low relative
to my ADP concentration, I can still
make this reaction happen.
Now that's not going to give you an ATP-ADP ratio that
is actually consistent with being able to do anything
else into the cell.
And so but what it says is that if I am able to couple energy
from nutrient metabolism to this reaction
and overcome this positive 7.5 kcals per mole,
that basically will allow me to now synthesize ATP and maintain
an ATP-ADP ratio in the cell that allows cells to carry out
other functions by the same rules
that we talked about that would be unfavorable.
Now there's lots of implications here for metabolism,
because it says how much input you need to synthesize ATP.
How much energy you need to synthesize ATP
is going to depend on how charged this ratio is.
And how useful that ratio is to catalyze other reactions
is also going to depend on how charged that ratio is.
And in essence, this is why we cannot store energy as ATP.
This is why we didn't eat ATP for breakfast
and instead ate cereal that has a bunch of glucose in it,
because we can use that glucose to get ATP but the ATP itself
is not something that we really can burn to get energy
in a sustained way.
In other words, the delta G of ATP synthesis,
making ATP is going to be proportional
to the log of the ratio of ATP over ADP for all the reasons
I described.
The delta G of using ATP, ATP hydrolysis,
using ATP to drive reactions is also
proportional to RT times the log, in this case, of the ADP
to ATP ratio.
And so because you need to consume this ratio to get
energy, and you need to put in energy to create this ratio,
you are constant-- and how much is an exponential property--
you are constantly having to generate
ATP in a way that allows you to then
have a ratio that can then be consumed
to fuel other reactions.
And that's what's shown here on the slide.
So I just graphed here what the ATP-ADP ratio
is relative to a theoretical delta
G that's shown here as a linear scale, here on a log scale,
that basically there's this logarithmic relationship
and so you need an exponential increase in energy input
to drive this ratio up higher in order to, quote unquote,
"store" more energy that can be used for later.
And so this system is not one that you can use as an energy
storage system and ultimately explains
why cells have to do constant metabolism,
have to constantly do some sort of metabolism
to keep this ATP-ADP ratio charged
for it to be in a useful range to have an ATP-ADP ratio that
can be coupled to drive otherwise
unfavorable reactions in our cells.
So to say this another way, our cells
need to constantly catabolize nutrients, burn glucose,
burn some other fuel.
They have to do this because they
have to keep that ratio charged in a range
where that can then support other cell functions,
allow them to fight entropy and maintain order in biology.
We alluded last time if you stop making ATP, well,
what's going to happen?
Well, that ATP-ADP ratio the cell
has is going to fuel whatever reactions it
can do until suddenly that ratio falls below the point where
it's useful to drive those unfavorable reactions.
In fact, if you stop ATP production,
you will consume that ATP-ADP ratio in seconds.
And this is why if you poison ATP production,
cells die very quickly.
Remember last time we mentioned you have a heart attack,
blood flow stops going to a tissue.
No more nutrients and oxygen. Cells die quickly.
Cyanide poisoning does the same thing.
Cells die quickly if you stop their ability
to maintain this ATP-ADP ratio in the right range.
And so remember the concentration,
the absolute concentration of ATP for these reasons
is not a good indicator of cell energy.
The correct indicator of cell energy
is the ratio of ATP to ADP.
This term is sometimes referred to as the energy charge.
And so that can be the ATP to ADP ratio,
or oftentimes more correctly is the ATP to AMP ratio.
Why the ATP to AMP ratio?
Well, that's because once you understand this,
you can now understand that cells have a failsafe mechanism
to protect themselves if this ratio of ATP to ADP
becomes too low.
What is that mechanism?
Well, you can carry out this reaction.
Take two ATP molecules and turn them
into transfer a phosphate to make an ATP plus an AMP.
Why is that a failsafe mechanism?
Well, if they care about this, if the ATP-ADP ratio is
what maintains your ability to drive
these thermodynamically unfavorable reactions,
if I sacrifice the denominator by removing an ATP
and get an ATP out of it, I've now at least temporarily
fixed that ratio.
Now this is something that obviously cannot go on forever.
But in the short term, it does allow
cells to protect that ratio.
And in fact, what cells actually defend is this ATP-AMP ratio.
And it's very, very difficult to change the ATP-AMP ratio
in a cell and actually have it live.
So it's really this ATP-AMP ratio
that is most formally related to what
would be called energy charge.
But this is really why this is important is
for all the reasons that I just described.
All right.
So hopefully, it's clear to you now
why cells have to keep ATP-ADP ratio in the right range, why
this is useful for them to now carry out
all of these unfavorable reactions-- fight entropy,
stave off death.
And they can even have a failsafe here and sacrifice ADP
as a way to try to maintain that as a very last resort.
It also says why cells have to always do catabolism,
because you always need the energy input to keep
that ratio in the right range.
However, you might imagine that, in real life,
the messiness of biology, that there's
lots of situations where the demand for ATP
will suddenly increase.
Well, of course, maybe you can just metabolize more sugar,
do more nutrient catabolism to try to keep that ratio high.
But that takes time.
And so you need ways for cells to quickly respond
to then allow them to mobilize all of this extra thing to--
extra fuel to do that.
For example, we're walking around our ancestors
on the plains somewhere and a lion appears.
We don't have time to mobilize a bunch of new fuel.
We have to be able to run away really quickly
or the lion's going to eat us.
And so cells have systems to suddenly respond
to I need a lot more ATP demand to run away by having ways
that one can buffer this ATP-ADP ratio,
protect it in the short term, and allow cells time
to ramp up nutrient metabolism in order to keep this high.
All right.
Well, if we wanted to create such a buffer system,
how would we actually do it?
Well, obviously, we have to follow
all the rules of thermodynamics that we discussed.
And we have to do so in a way that
actually illustrates how it is that
very well how metabolism works.
In other words, if suddenly the ATP-ADP ratio
drops, if we have a sudden drop in ATP-ADP ratio
because we have increased demand,
needing to run away from a lion, how
could we actually create a buffer system
that protects this?
Well, to do it, we would need to couple ATP-ADP
to some other product reactant pair,
let's call them x and y, such that the ratio of y to x
is coupled to the ratio of ATP to ADP.
And you want it coupled in such a way
that if that ratio goes down, this ratio can now protect ATP,
basically favor ATP to ADP synthesis.
However, when nutrients are plentiful
and this ratio is really high, it then in turn
can charge up this ratio so that it can buffer it later
when the ATP-ADP ratio starts to fall.
Now the most famous example of this
actually comes from muscle physiology.
And it's best described in terms of muscles
and how they quickly respond to increased demand,
like that running away from a lion.
However, it in fact applies to many, many cells,
including most non-muscle cells in animals.
All right.
And I should say that it turns out
that if you turn off ATP synthesis in a muscle,
people have estimated that your ATP-ADP ratio will fall
into a range where it is no longer useful to do anything
within a matter of seconds, some claim even less than a second.
And so this system ends up being very important.
Now the system that muscle uses, and many other cells
do as well, is something called the creatine/creatine phosphate
system.
Now if you guys are weightlifters or bodybuilders
out there, you probably have heard of creatinine
because it's a very popular supplement that
is taken for weightlifting, something I obviously
don't do a lot of.
But that creatine/creatine phosphate is out there.
I'll describe to you what it actually does for muscle cells.
And you can decide for yourself if this is actually
useful as a energy or weightlifting supplement.
So what is creatine/creatine phosphate?
What does it look like?
Well, it's basically looks like this.
By the way, I will use this abbreviation often
throughout this course, just a shorthand
to draw a lot of these structures.
So this here would be a carboxylic acid group
drawn like this or like that, drawn to the carboxylic acid
group just so you understand my shorthand.
OK.
So this here is the molecule creatine.
If I put a phosphate here, this now becomes creatine phosphate.
Again, just to remind you, this P with a circle over it
is a phosphate group.
I'll draw this particular phosphate group
because it's a nitrogen-phosphate bond,
a phosphoramidate bond.
This is actually a bond that is more labile than the PO bonds
that we're used to seeing in the phosphodiesters like ATP,
et cetera.
All right.
So that's creatine and creatine phosphate.
And if we draw out the reaction, creatine phosphate
goes to creatine plus inorganic phosphate, so hydrolysis
of creatine phosphate.
This has a delta G0 prime of minus 10 kcals per mole.
What does that mean?
It's negative.
Tells us about the equilibrium constant.
Equilibrium lies to the right.
Remember ATP to ADP hydrolysis shown over there,
that hydrolysis has a delta G0 prime
of minus 7.5 kcals per mole.
Equilibrium also lies to the right.
This number is more negative.
This equilibrium lies further to the right
than ATP hydrolysis does.
So now what happens if we couple this reaction
to the ATP hydrolysis reaction?
So now we can do ATP plus creatine goes
to creatine phosphate plus ADP.
So we've coupled those two reactions together.
So delta G0 prime, ATP to ADP is minus 7.5.
Creatine phosphate to creatine in
that direction is negative 10.
In this direction, it would be positive 10.
And so as I've drawn the reaction,
this delta G0 prime will be plus 2.5 kcals per mole.
10 minus 7.5 plus 2.5, which tells us then
that this equilibrium now lies to the left.
However, which direction this reaction actually goes
is not determined only by the equilibrium.
It's really determined by delta G. So we can calculate that.
So delta G equals delta G0 prime plus RT
log of, as we've drawn it, creatine phosphate
over creatine and ADP over ATP.
So you can now imagine that this term here
is the sum of those two.
So it's plus 2.5.
And so if this term is less than 2.5, what does that mean?
Well, if it's--
I'm sorry.
Less than negative 2.5, then delta G will be negative
and you will favor creatine phosphate production.
If this term is greater than negative 2.5,
then delta G will be positive and you
will favor ATP production.
Why is that important?
Well, if you think about the conditions,
how you could make this term less than 2.5
or less than negative 2.5 or greater than negative 2.5,
basically, what's going to happen
is that if the ATP-ADP ratio is high,
lots of energy around, no problems charging up,
lots of things to use, charge up this ATP/ADP to a very
high ratio, well, now you will favor this direction,
creatine phosphate synthesis.
However, if ATP/ADP begins to fall and is lower,
well, now this term is going to flip
and you'll now favor ATP production.
And so basically, by charging up this creatine/creatine
phosphate ratio, it now creates a situation
where it can buffer that ATP/ADP ratio so that when times are
good, you charge up the buffer.
When times are bad, you then consume the buffer
in a way that allows you to protect your ATP/ADP
ratio for a short time.
And this is illustrated very nicely here
in this slide that basically shows what
happens in muscle physiology.
And so this is just a theoretical curve
that if you suddenly need to contract your muscle
and you stop ATP production, well, ATP levels will fall.
That ATP/ADP ratio more correctly will fall rapidly
within the cell.
Creatine phosphate will then now kick in
to protect that ATP/ADP ratio as you can ramp up
either anaerobic or aerobic metabolism to now buffer
that ATP/ADP ratio and keep that muscle functioning.
Now I want to make one point because oftentimes biochemistry
textbooks will refer to things like ATP
or creatine phosphate as, quote unquote,
"high energy compounds."
And of course, I guess they are high energy
because they have these labile phosphate-nitrogen
or phosphate-oxygen bonds.
And because they're labile, hydrolysis is favorable.
And to a certain extent, it's that hydrolysis
that is providing energy.
But remember it's not the metabolite
or the bond in isolation that's really providing the energy.
It's these ratios of ATP to ADP, creatine phosphate to creatine,
that allow you to couple, for all the reasons
we've spent now a lecture and a half discussing,
two other reactions that really provides biological energy.
All right.
Now we're ready to discuss how it
is that we can use glucose oxidation, nutrient catabolism,
to keep ATP/ADP high, that is in a useful range, where it can
allow cells to do otherwise thermodynamically
unfavorable things.
So how could we make this happen?
Well, just like this example with creatine and creatine
phosphate, if we want to use nutrient catabolism
to do the same thing, we need to couple reactions
of glucose oxidation, nutrient catabolism, that
will allow us to synthesize ATP, ADP to ATP,
and have that synthesis be favorable
despite the high ATP/ADP ratio in cells.
So let's think about it.
So what is this?
Nutrient catabolism.
What did we talk about last time?
Oxidizing glucose to carbon and CO2.
So here's our reaction for that.
So here's carbohydrate, glucose CH2O6 plus six O2
goes to six CO2 plus six H2O.
That's the chemical formula for burning wood.
So delta G0 prime for this is minus 686 kcals per mole.
Quite a negative number.
Equilibrium greatly favors the completely burned state,
oxidized state, of that carbohydrate.
And so you can imagine that this equilibrium is
so favorable to the right, if we couple this reaction to ATP
synthesis, you're clearly going to have plenty of energy there
in order to maintain that ATP/ADP
ratio in the right range to be compatible with cells
using it to do other things.
All right.
So how do we actually do that?
Well, if we do it, we have to follow
the same rules of thermodynamics and do the same things
that we did before.
Basically, we have to do this reaction in a way
that we can draw out a similar type of equation
as we did for creatine and creatine phosphate.
And at the highest level, that is how nutrient catabolism
will allow us to keep ATP/ADP ratio high
even as ATP hydrolysis is constantly
fueling all these other unfavorable processes
in the cell.
Now if we did this all in one step, our burning wood example,
lots of heat is generated.
That's too much energy.
Not so good for biology.
But rather what biology does, as we alluded to last time,
is that because what we talked about in the beginning,
that all of the things that we discussed
are true for individual reactions
or many, many reactions, basically, if we turn A into B
and we do it in one step, like burning wood,
or we do it in 500 steps, the thermodynamic consequences
are exactly the same.
And so what we need to do is do stepwise catabolism of glucose
and take advantage of the fact that that delta G change is
the same whether it's one step or many steps
and in that stepwise fashion build in reactions where
we create intermediate chemistries that allow us to do
things like we did with creatine and creatine phosphate
such that we can favor ATP synthesis when ATP/ADP ratio is
in the physiological range.
And in essence, this is the magic
of how biology does this and basically couples
nutrient catabolism in order to provide energy.
And so the pathway that we're going to discuss today
and in the next lecture is something
called glycolysis, which simply put is glucose lysis.
It's the stepwise chemical oxidation
of glucose, which we know from our burning wood
example is favorable.
And we're going to use this pathway that basically will
be a pathway to allow the stepwise lysis of glucose,
a favorable reaction, to individual steps that set it up
in a way such that ADP to ATP synthesis
is favored despite a physiological ATP/ADP ratio.
Now I want to point out that this really illustrates,
back to that high energy compound thing,
that ATP is not the only energy currency in the cell.
In fact, glucose is much more energy for a cell than ATP.
You get much more energy released from burning wood
than you get from hydrolyzing ATP.
And in essence, this is why we use
glucose-- why nature has used glucose,
and other molecules like it, as an energy storage
device, because here is a relatively stable molecule,
can sit around for a long time, and you
can burn it when you need it to keep your ATP-ADP ratio high.
All right.
So, at the level of reactions and pathways
now, how can we use glucose oxidation and couple
it to drive ATP synthesis?
Well, hopefully it's very clear that we
have to basically set up a situation
like this creatine-creatine-phosphate
system that we already discussed.
And so I want to start by discussing,
what are the reactions in glycolysis that ultimately
behave like this and allow this favorable ADP-to-ATP synthesis?
And then we will go ahead and consider
how we can make those reactions work
in the context of an entire pathway that
allows glucose oxidation.
All right.
Now, like creatine, phosphate, and creatine,
both of the steps in glycolysis that
favor ATP synthesis involve intermediates
where you have phosphate on some molecule,
plus ADP goes to ATP plus the phosphate
coming off that molecule.
Now, if we go to our creatine-creatine-phosphate
system, obviously this equilibrium
needs to lie to the right.
And so the first one of these is as follows.
It involves this molecule, phosphoenolpyruvate, also
referred to as PEP.
You will see throughout the course
that we love to give these little acronyms
to our metabolites because it's hard to constantly
say and write out phosphoenolpyruvate.
Much easier to say PEP.
So phosphoenolpyruvate plus ADP goes to ATP.
Plus, if I take the phosphate off of there,
I get this molecule.
This molecule is enolpyruvate.
Enolpyruvate can be rearranged as follows.
To make this molecule a ketone pyruvate.
You'll note-- hopefully remember from your organic chemistry
classes that ketones like pyruvate
can exist in two forms.
They can exist in the ketone form or the enol form,
rearranging via the chemistry that I've shown.
The keto form is greatly favored over the enol form.
However, by having a phosphate trap pyruvate the enol form--
phosphoenolpyruvate, when you remove the phosphate
and it's wanting to exist in the keto form,
greatly enhances the energetics of this--
basically makes the reaction, the equilibrium
want to lie very far to the right.
And by the way of chemical tricks like this
and others like it, you will see used over and over in biology.
In fact, this is illustrated, if we
asked what's delta G0 prime for this reaction,
it's minus 15 kcals per mole.
That tells us that the equilibrium of this reaction
lies to the right.
In fact, it lies far to the right, and in fact, this is--
you may remember ATP hydrolysis is negative 7.5 kcals per mole.
So lies twice as far to the right as ATP hydrolysis does.
And in fact, this minus 15 kcals per mole is the most favorable,
at least in terms of individual reactions
that at least I know of in metabolism.
All right.
Well, if this equilibrium for this is PEP to pyruvate,
this here is delta G0 prime for ATP-ADP.
So if we want to know what's delta G0
prime for the entire reaction, well, if PEP to pyruvate
is minus 15, ATP to ADP is minus 7.5.
Well here, we're going in the opposite direction,
so it'd be plus 7.5.
So it's minus 15 plus 7.5, means that for the entire reaction,
this is minus 7.5 kcals per mole.
Means that this is favorable, equilibrium lies to the right.
If we drew out--
I'm not going to do it again because I've done it a lot,
but if we redrew out this equation,
replacing pyruvate and PEP in the correct places,
there for creatine, phosphate, and creatine,
you would see that there's lots of conditions within cells
that will favor ATP synthesis even
when ATP-ADP ratio is high.
All right.
This reaction is catalyzed by an enzyme called pyruvate kinase.
And pyruvate kinase is a classic example of a, quote-unquote,
"irreversible reaction."
However, I want to point out that the enzyme pyruvate
kinase is actually named for the reverse reaction that's
supposedly irreversible.
Pyruvate kinase.
If you take pyruvate and you act on the kinase
as you learn from Professor Yaffe,
that phosphorylates something.
You phosphorylate pyruvate, you get
phosphoenolpyruvate pyruvate kinase.
And so remember that absolutely no reaction is reversible.
It's only reversed-- what they mean by irreversible when they
say pyruvate kinase step in glycolysis is irreversible,
is that it is effectively irreversible
under the conditions that are compatible with life
that exist in cells.
Of course, if you have very low ADP and very high ATP,
you can come up with a way to net synthesize PEP.
You just can't drive the reverse reaction
under the ATP-ADP and the PEP pyruvate ratios that
physiologically exist in cells.
However, this is set up in a way such
that you can favor ADP-to-ATP synthesis by converting
PEP to pyruvate in a pathway, as we
will see happens in glycolysis.
All right.
Now, the second reaction that I want to--
that allows ATP synthesis in glycolysis
is catalyzed by an enzyme called phosphoglycerate kinase.
And this carries out the following reaction.
OK.
So this molecule is 1,3-Bisphosphoglycerate,
abbreviated 1,3-BPG.
And basically the phosphate is transferred from here
to ADP, which gives you this molecule, 3-phosphoglycerate
or 3-PG.
So this reaction, the delta G0 prime
for the entire coupled reaction, both steps of the reaction,
is minus 4.5 kcals per mole.
Tells you about the equilibrium constant.
Equilibrium lies to the right, doesn't lie as far to the right
as the PEP plus ADP goes to ATP plus pyruvate reaction,
but nonetheless, is one where there's
lots of conditions that are favorable in cells to maintain
a high ATP-ADP ratio, although we will see later
that this reaction actually runs quite close to equilibrium
in cells, so there's actually conditions
where it can go in the opposite direction as well,
and we'll get to that later in the course.
I want to point out one thing about this, one thing that
makes this reaction favorable, as you notice here
that this is a phosphate on an acid group,
a so-called acid anhydride.
And this is a pretty good phosphate donor.
And so you want to lose that phosphate, which
is one of the reasons why this reaction is
favorable for ATP synthesis even at high ATP-ADP ratios.
All right.
So these are the two reactions in glycolysis
that can favor ATP synthesis at a high ATP-ADP ratio
just like creatine and creatine phosphate.
And so hopefully now it's become apparent to you that if during
our pathway of glucose catabolism we can build
a pathway that couples what we already know is favorable,
glucose oxidation, to generate these intermediates,
PEP and 1,3-Bisphosphoglycerate, lacerate we can then build
a pathway where those reactions can make synthesis of ATP
favorable even though there's a high ATP-ADP ratio in cells,
and in fact, maintain that high ATP-ADP ratio in cells.
And in essence, this is the logic or the goal,
if you will, of glycolysis and why
it's important to provide energy to cells.
OK.
All right.
Now, if we're going to make a pathway where glucose oxidation
is coupled to making PEP and 1,3-Bisphosphoglycerate,
it's obvious we need to add phosphate to the system.
There's no phosphate molecule on glucose.
Now already mentioned earlier--
and I just erased it--
that to trap glucose in the cell, the first step
in glycolysis, you add phosphate.
But that phosphate donor is ATP.
And so ATP being the phosphate donor doesn't help us here.
If we're going to use phosphate transfer here,
we need to have phosphate added into the system
if this is going to work.
We can't have ATP as our phosphate donor.
You also saw lots of reactions from Professor Yaffe
that involved phosphate transfer reactions.
But again, ATP-- or GTP is the phosphate donor.
And so for this system to work, we need a way to get inorganic
phosphate onto a carbohydrate or we can't make things like PEP
or 1,3-Bisphosphoglycerate that will allow ATP synthesis.
Now we already saw earlier that additional phosphate to glucose
is not favored.
Remember, delta G0 prime of phosphorylating glucose
was plus 3.3 kcals per mole.
And it turns out that says equilibrium
lies to the wrong side, so we can't net do that
without ATP or energy input.
And it turns out this is true for other sugars as well.
And so you need energy input to add phosphate to the sugars.
Plenty of energy input from glucose oxidation,
but we need a reaction where phosphate addition is coupled
to another reaction that makes it favorable,
and we have to do this without making ATP.
And again, it illustrates, again,
the biological energy is not just about ATP.
At a deeper level It's something different.
And so to really discuss this and understand it,
I need to introduce two concepts that
are going to come up again over and over again in this class
and across metabolism.
The first of these is a very high-level bioenergetic
concept, although it's really an energetic concept,
and it comes back to what we already talked about.
So, we know burning wood is favorable.
The CN H2O N plus oxygen going to CO2 plus water.
That's burning wood, lots of energy released.
Drew it up there for a single-glucose molecule
and what the energy release the delta G0
prime is for that reaction.
Now I want to point out chemically
what is going on in this reaction.
Chemically what is happening for this
to occur is that electrons from the carbon
are being moved to the oxygen. You
are changing the oxidation state of carbon and of oxygen
in order to carry out this reaction.
Oxygen is a better electron acceptor than carbon,
and so transferring those electrons
from the carbon to the oxygen is favorable.
That is, in essence, what is going on in this very favorable
reaction to burn wood.
Let me illustrate that a bit more explicitly here.
So the carbon-- most of the carbons in carbohydrates
are alcohols.
So here, you can call this our carbohydrate,
with the exception of the ketone or the aldehyde
if it's a ketose or an aldose.
All the other carbons in the sugar are alcohols.
All the other bonds there.
And so if we oxidize that carbon--
that is, if we remove electrons from the carbon,
how can we do that?
So this here generates this H- hydride ion.
This is, in essence, two electrons
that we are removing from the carbon.
That gives us a ketone.
So if we oxidize the alcohol and we remove two electrons,
we get the ketone.
We could oxidize this ketone further.
That will give us the carboxylic acid.
If we oxidize it even further, now we get CO2.
So, moving in this direction, this is oxidation.
Transferring electrons from the carbon to the oxygen
to go in this oxidation direction, that
is glucose oxidation is what's happening
when we're burning wood.
It's what happens when we burn glucose.
Energy is released.
This is largely what catabolism is about in biological systems.
Now then reasons, then, that if we're
going to move in the opposite direction, so-called reduction,
that is going in this direction.
Reduction.
That's largely energy storage or anabolism in metabolism.
We are storing energy that we can burn later.
And of course, what's the more extreme version of this?
Well, what if we add two more electrons to the carbon going
in this direction?
Well, what do we end up with?
Well, now we end up with the saturated carbon.
more reduced than the alcohol is the fully saturated carbon.
You will see later that this is what fat is.
You saw that in some of the earlier lectures.
It's also what gasoline is.
What's gasoline?
It's fully saturated hydrocarbons that we then
burn to CO2 and derive energy.
Just like burning wood, burning gasoline all in one step,
oxidizing the gasoline gives you energy.
There's more energy in gasoline.
You can release more energy per weight
from gasoline, from fully-saturated hydrocarbons
than you can from wood.
Same thing happening here.
What has more calories in your food?
Everyone knows fat has more calories per weight than sugar.
It's because the more reduced our carbon is,
the more energy it stores, and as we oxidize that carbon, that
is what releases the energy.
And so energy transduction in biological systems--
and actually, in non-biological systems,
as I just illustrated with the wood and gasoline example,
but definitely in biological systems,
is largely about oxidation and reduction reactions,
and you are going to see this coming up over and over again.
Now, based on what I just said, the real magic, if you will,
of glycolysis can be further refined to say that what
glycolysis does is it couples the favorable oxidation
of carbon with reactions that allow phosphate additions
and production of intermediates, like PEP
and 1,3-Bisphosphoglycerate, where synthesis of ATP is
favored despite the high ATP-ADP ratio in cells.
If you think about it, that process
then allows you to keep that ATP-ADP ratio
in the range where it can then be
useful for all the reasons we've been talking about now for two
lectures.
Allows the cell to couple that ATP-ADP ratio, that-- in ATP
hydrolysis to otherwise carry out
otherwise unfavorable reactions.
OK.
So, based on that, arguably the key,
or at least a very important reaction in glycolysis,
is the oxidation step which happens to add the phosphate
and also happens to make 1,3-Bisphosphoglycerate,
which we saw earlier can be used to drive ATP synthesis.
And so that reaction is the following.
So this molecule hopefully looks familiar to you
from the carbohydrate lectures.
This is D-glyceraldehyde 3-phosphate.
I will abbreviate it frequently is
G-3P for glyceraldehyde 3-phosphate,
but it's D-glyceraldehyde 3-phosphate.
Remember, it's a D, sugar, because the OH
group is pointing to the right as we drew it this way.
It's also an aldose because it's a aldehyde sugar,
and it's phosphorylated on the 3 position--
remember the way we numbered sugars was starting
at the carbonyl--
1, 2, 3.
This is carbon-3, phosphorylation on the 3
position.
Glyceraldehyde 3-phosphate.
OK.
So, this step generates 1,3-Bisphosphoglycerate.
Well, to do this, there's actually
two things that happened here.
First we had to add the phosphate to this.
It says two phosphates on it. bisphosphoglycerate,
this has a single phosphate.
So inorganic phosphate has to be added
to generate that molecule.
By the way, adding that phosphate delta G0 prime
is positive plus 1.5 kcals per mole.
That means even equilibrium, this reaction
would lie to the left, of course,
because delta G0 prime is positive.
However, remember the next step downstream of this
is the one shown over here where we do ATP synthesis.
That step is very favorable.
So this is an example where metabolism
has coupled a very favorable step to a step that's
less favorable.
That can keep the 1,3-BPG levels low and pull the entire system
forward, the metabolic trick we discussed in the last lecture.
OK.
You'll note the other thing that's happening here,
other than adding that phosphate group,
is that we've changed this aldehyde to a carboxylic acid.
That is an oxidation reaction.
Ketone to the acid is an oxidation reaction.
I will illustrate over here this so you can see
why it's an oxidation reaction.
So here's our aldehyde.
If we take electrons from the carbon
and that generates this hydride ion to electrons,
what are we left with?
Well, we're left with this intermediate
that will never exist with a positive charge on the carbon.
Water, which, of course, is abundant in biological systems.
And then come--
OK.
And it basically illustrates that as I
go from the aldehyde to the acid, of course,
I make a proton and a hydride ion,
but really, I have changed the oxidation state of the carbon
from the ketone to the acid state.
Two electrons are lost there, is shown as that hydride ion.
OK.
So, what does that mean?
Well, that means that we have to maintain that balance
in this reaction as well.
So water is going to have to come in
and a proton will be produced.
But the key thing is we also generate
two electrons, this hydride ion that have to go somewhere.
Now, finding a place for those two electrons to go
is what brings up the second high-level concept
that I now need to spend some time discussing.
And this second high-level concept
that I need to introduce is the role of cofactors,
and cofactors also is effectively
the role of vitamins and what role
that these things play in metabolism.
Most vitamins are used to support metabolic reactions,
and we're going to learn all about what
many of the vitamins that you guys are familiar with reading
the side of your cereal box actually
do throughout the course.
Now cofactors, as I said, often involve vitamins,
are molecules that provide useful chemical groups that
facilitate the reaction chemistries that
are needed in metabolism.
So they provide some useful chemical groups
to facilitate some of the chemistries
that we need to carry out metabolic reactions.
They are, in general, in small non-stoichiometric quantities
in order to carry out this function.
What do I mean by that?
Meaning that you can imagine that the cell will convert lots
of glyceraldehyde 3-phosphate to 1,3-Bisphosphoglycerate,
but the enzyme, you don't need one enzyme for every time you
convert that reaction.
The same enzyme can do it over and over and over again.
Well, it turns out, cofactors are much more
like enzymes in that respect.
They need to be recycled in a way
that they can catalyze the turnover of reaction
many, many, many times, and it'll
be much clearer what I mean by this when we
get into some of the details.
Now, the vitamin concept comes from the fact
that often these cofactors or parts of the cofactors
are not made by animals, not made by humans.
If they're not made by us, but we
need them to do our metabolism, well now we
have to get them from the diet, and this is really
the concept of vitamins.
These are small things that we have
to get in quantities from the diet,
and it turns out they're very important to help us carry out
the metabolism we do.
As an aside, the vitamin industry
is a very big business.
Lots of unfounded claims out there.
I'm not going to get into--
that can get very political.
I'm just going to say that just know that more is not
always better, and the goal for this class
is really to understand what some of these vitamins
actually do.
OK.
Now, the relevant cofactor for the reaction that we're talking
about is a cofactor called nicotinamide adenine
dinucleotide, which is always abbreviated NAD or NAD+.
This stands for nicotinamide adenine dinucleotide.
You see why we want to abbreviate that NAD.
And basically what nicotinamide adenine dinucleotide is is it's
a factor that can solve our problem of where
to put these electrons in this oxidation reaction.
That is, you can accept two electrons
from a reaction like this one here
and cycle to another form of the cofactor called NADH.
All right.
What is nicotinamide adenine dinucleotide?
Well first, let's draw what it looks like.
OK.
Sorry it's squeeze down here a little bit.
So this here is adenine.
This group up here is nicotinamide.
So if you look at this, this is a dinucleotide.
And so down here, this, of course, is, from here down,
is AMP.
That phosphate is now over here.
This is a separate nucleotide, where
rather than having one of the AGCTU bases
that you're used to from RNA and DNA, nicotinamide is the base.
And so it's this AMP plus this nicotinamide
base is a monophosphate, or a nicotinamide adenine
dinucleotide.
This, the nicotinamide group, is derived
from a vitamin called niacin.
And so from the side of your cereal box,
this is what niacin does.
It allows you to generate the nicotinamide group that's
part of this cofactor, nicotinamide adenine
dinucleotide.
Now, nicotinamide adenine dinucleotide
is useful because of what that nicotinamide group can do.
And so I'm not going to draw out the whole molecule,
but I'll show you.
So NAD+, which is the oxidized form of nicotinamide adenine
dinucleotide, is as I drew it up there.
So this here would be attached to the rest
of the dinucleotide.
So this is the oxidized form.
I can have-- here's my hydride ion, which, remember,
is two electrons.
If I carry out that chemistry to add those two electrons
to the nicotinamide group, you now end up
with the reduced form of nicotinamide adenine
dinucleotide NADH.
So the difference between NAD+ and NADH, it's the H,
but it's really the two electrons that were added
in adding that H. So it's oxidized form and reduced form.
So remember, I want to stress this again, at least in biology
we can't create or destroy matter.
You can do that in nuclear physics,
but we can't do that in biology.
And so that means that if we do an electron transfer like we
did in this reaction where we oxidized the aldehyde
in glyceraldehyde 3-phosphate to the acid
in 1,3-Bisphosphoglycerate, those electrons have to be
accounted for.
And so if one molecule is oxidized,
another one has to be reduced.
So redox reactions always-- that is,
redox, oxidation-reduction reactions,
always have to happen in pairs.
And so over there we have the aldehyde in glyceraldehyde
3-phosphate is oxidized to form the acid
in 1,3-Bisphosphoglycerate.
As part of that reaction, we can couple it such
that the nicotinamide group, which is oxidized in NAD,
can be reduced to the nicotinamide group that
is found in NADH.
So that means we can draw this in here,
is that having that cofactor coupled to this reaction
now allows us to fix the redox problem of this reaction.
Now this reaction here is catalyzed
by an enzyme called glycerol 3-phosphate dehydrogenase.
I'll draw that out in the second.
Often abbreviated GAPDH, the loading control
in all of your western blots, is the enzyme GAPDH,
glyceraldehyde phosphate dehydrogenase.
That is the enzyme that carries out that reaction.
Now notice, the name fits.
It's a dehydrogenase, it's removing a hydrogen,
removing two electrons as a hydride ion,
but it's really-- what's important here
is the electrons moving, not the hydrogen itself.
So in that way it's a little bit misleading.
And you will see that in general, dehydrogenases
are classes of enzymes that catalyze
oxidation-reduction reactions, and as a result,
often involve NAD/NADH as cofactors
because they facilitate those electron transfer reactions.
OK.
So now we're able to describe how
GAPDH works in great detail.
And so the chemistry of GAPDH works as follows.
And so in the active site of GAPDH,
there's a cystine residue.
That cystine residue has a sulfur shown here.
Also bound near the active site is in NAD molecule
to catalyze the reaction shown.
That NAD has to be in the oxidized state, so it's NAD+.
And so glyceraldehyde 3-phosphate binds
in the active site.
There's glyceraldehyde 3-phosphate.
By the way, I will show you how many reactions in metabolism
work chemically.
Obviously we don't have time in this class, an overview class
to go into all the great chemical details of what's
going on and exactly how everything works,
but I'll at least try to give you
a flavor of some of the chemistry that
happens throughout the course.
All right.
So once you have, then--
OK.
So now you have this intermediate bound
into the enzyme active site.
You can now carry out the oxidation of that carbon.
So these are two electrons being removed as a hydride ion.
They can be transferred to NAD in the active site.
That generates NADH by the exact chemistry
that I just showed you.
OK.
That NADH now needs to be exchanged out
or the enzyme will not be able to catalyze
the next series of reactions, and that NADH will then
go somewhere else, and of course,
has to be cycled back to NAD, but that doesn't happen
in this reaction, per se.
OK.
Next, this here, you'll notice, is that by the way
this chemistry worked, we formed this thioester bond
in the active site.
This is another thing that you will see over and over again
in metabolism.
This sulfur in the thioester bond is a good leaving group,
and so it's really setting up a situation that will now help
with the phosphate addition.
So here, I'll draw out inorganic phosphate.
And that, of course, will give us the identical enzyme active
site that we started with plus 1,3-Bisphosphoglycerate.
And that 1,3-Bisphosphoglycerate can now be coupled to ATP
synthesis to make 3-phosphoglycerate.
And so you can see, in essence, what this reaction has done is
that it has coupled oxidation of the aldehyde to the acid,
the aldehyde in 3-phosphate to the acid
in 1,3-Bisphosphoglycerate, which can then do
phosphorylation of ADP to make ATP.
So in essence, this is oxidative phosphorylation.
Now I realize in high school or in other classes,
oxidative phosphorylation means something different to you,
and of course, we will discuss the process that
is called oxidative phosphorylation in great detail
later in this course.
But I want to point out that the chemistry that's actually
happening here in glycolysis is also oxidative phosphorylation.
It's basically how you coupled this favorable carbon oxidation
that's occurring at this GAPDH step
in a way that allows you to synthesize ATP
at a high ATP-ADP ratio, which in the end will allow cells
to do otherwise unfavorable things.
OK.
Now we're ready to discuss these reactions, the GAPDH reaction,
the pyruvate kinase reaction, the phosphoglycerate kinase
reaction, and how they fit into glycolysis
as a metabolic pathway in a way that allows cells to use
glucose metabolism-- that is, the breakdown of glucose,
the burning of glucose as a way to get energy.
And that pathway is, of course, glycolysis or glucose lysis
that we mentioned earlier.
Glycolysis is a very ancient and ubiquitous pathway.
It's used by essentially all life on Earth
and probably has been for at least 2 to 3 billion years.
Glycolysis, I guess, as a pathway was,
quote-unquote, "discovered" mid-19th century
by Louis Pasteur, and Louis Pasteur,
of course, described life, microorganisms
as being responsible for the process of fermentation.
That is, conversion of glucose into alcohol, something
many college students are interested in, as well
as other organic acids.
And of course, fermentation to make alcohol
or to store other types of organic acids
has been used in food preservation
for centuries, which is really why Pasteur's discoveries were
so impactful at the time.
Now even though Pasteur discovered this,
he didn't really understand chemically how it worked,
and it actually took decades to figure that out.
And when it was finally figured out and described
as the pathway called glycolysis,
this was done by two chemists, two chemists in Germany, Embden
and Meyerhof.
And basically what they did is they pieced together
several enzymatic or chemical activities that
have been purified from cell lysates mostly from yeast
and used those to build a pathway that described
chemically how you could start with glucose
and chemically break down that glucose in a way that made
sense in this fermentation pathway,
and that is why glycolysis is sometimes called
the Embden-Meyerhof pathway.
Now, glycolysis does not completely
convert glucose to CO2.
Instead, what glycolysis does is it converts glucose--
so I will draw here in the pyranose form.
It converts glucose by a number of steps
into two molecules of the organic acid pyruvate, which,
of course, was the product of that pyruvate kinase reaction
that we showed earlier.
Note that the fermentation that Louis Pasteur described
was further metabolism of that pyruvate into something else.
In the case of yeast, it was ethanol or ethyl alcohol,
or something else.
The fermentative product in mammals
is this organic acid lactate.
And what we will see next time is
that this fermentation of pyruvate
into one of these other molecules
ends up being an alternative to the further oxidation
of pyruvate to CO2 which requires oxygen,
and the reason oxidizing pyruvate to CO2
requires oxygen is because ultimately we
have to regenerate that NADH we made in glycolysis to allow
that cofactor to cycle and glycolysis to keep moving.
Fermentation gives us an alternative way
to do this, which we'll discuss in great detail
in the next lecture.
But first I want to focus on glycolysis
and what we can describe next time as really how
we can build a pathway to turn glucose into pyruvate.
This glycolysis, this Embden-Meyerhof pathway,
and some of the details of that pathway
are shown here on this slide.
And this is where we'll start off the next lecture,
discussing how each of these steps in glycolysis function
in a way that allow a pathway to generate phosphoenolpyruvate,
1,3-Bisphosphoglycerate, incorporate this GAPDH
reaction, and do so in a way that's overall energetically
favorable to allow the cell to couple this oxidation
of glucose to keeping ATP-ADP ratio in the right range
to support other unfavorable functions in the cell.
OK, thanks.