Lecture Outline
Bch462 – Spring 05
Arizona State University
Neal Woodbury
Glycolysis is probably the oldest of the major metabolic pathways. It likely was around before there was a significant amount of oxygen in the Earth’s atmosphere.
The
function of glycolysis is to take sugars that can be converted to glucose or to
simple 3-carbon carbohydrates such as glyceraldehydes and perform anaerobic
catabolism generating ATP.
Overall, the basic pathway is thought of as starting at glucose (a number of other sugars can be converted to glucose) and ending at pyruvate.
2
ATP molecules are consumed and 4 are produced, giving a net ATP production of
2.
2 molecules
of NADH are generated by reduction of NAD+. This is an extra bonus for an aerobic
organism (such as us) because as we will later see, this results in additional
ATP formation when the NADH is reoxidized by oxygen to NAD+.
For
an anaerobic organism, this represents a problem. The NADH has to be reoxidized by a sacrificial electron acceptor
which then builds up in the environment (like ethanol when fermenting grape
juice by yeast).
The whole pathway is given below:
Fig.
18.1
In
this pathway, the catabolic reactions are directly coupled to ATP
synthesis. In other words, a phosphate
group is transfered directly from the substrate to ADP to form ATP.
This
is called substrate level phosphorylation. It involves direct chemical coupling.
Later
we will see another way to make ATP called oxidative phosphorylation. This involves an indirect “bulk” coupling of
one type of chemical reaction (electron transfer/proton transfer) to another
(phosphorylation) via a mechanical motor.
The
first phase of glycolysis actually USES ATP rather than generates it. The result of this part of glycolysis is two
phosphorylated 3-carbon sugars. These
then are used in phase 2 to generate two molecules of the three carbon
alpha-keto carboxylic acid, pyruvate and a total of four ATP molecules.
Let’s
look at the reactions in phase I
Fig.
18.2
Think
back to organic chemistry. Look at the
end products. What do you need to do to
make them from glucose?
First
we generate a phosphate ester with the number 6 carbon of glucose. Why?
What is the advantage of sticking a charged group on glucose
(particularly in single celled organisms)?
What are the thermodynamic advantages of this? It takes energy, so it better be important.
Fig.
18.4
Next
we shift the carbonyl group down one carbon.
What does this do to the carbon below it? How reactive are carbons next to (or one removed from) carbonyl
groups? This is also required for the
next step which is to phosphorylate the other end of the molecule. That phosphorylation also “locks” the system
in place.
Finally
we cleave. This is actually the
opposite of an aldol condensation.
| | | | | |
C=O + HC-C=O -> R-C-C-C=O
| | OH|
The
aldol condensation arises from a nucleophilic attach of the alpha carbon of the
second molecule on the carbonyl of the first.
Usually, this is base catalyzed, with the base removing the H from the
alpha carbon forming a carbanion.
So
basically, we phosphylate one end, move the carbonyl out of the way on the
other end, phosphorylate that, then cleave the molecule into two three carbon
pieces.
The
final step is to interconvert between the ketose dihydroxy acetone phosphate
and the aldose glyceraldehydes 3-phosphate.
This is just another isomerization reaction.
Now
we have two molecules of G-3-P.
So,
let’s go through the mechanisms of these reactions.
The
first is just formation of a phosphodiester bond transferring a phosphate from
ATP to an alcohol. This we have seen
before.
The
interesting aspect of this reaction is the regulation. In most cells, this reaction is run by
hexokinase. Because it is an energy
utilizing step which, due to ATP consumption, is very thermodynamically
favorable, it is a good place for regulation to occur.
What
molecules might act to control this reaction?
What conditions would you want it to go fast or slow?
It
turns out it is feed-back inhibited by its immediate product, G-6-P.
Why
not inhibit at this point by further downstream products?
There
is another enzyme which carries out this reaction. It is glucokinase. It is
much more specific for glucose and primarily resides in the liver. It is not feedback inhibited and makes G-6P
for a different reason, glycogen production.
It also has a higher KM, above the normal glucose level in
the blood.
Why
would you want a high KM for an enzyme designed to prepare glucose
for glycogen production?
The
second reaction is more mechanistically interesting.
Fig.
18.6
HC=O HCOH H2COH
| || |
HC—OH à COH à C=O
| | |
This
goes through an enediol intermediate (a double bond between two alcohol groups)
and is the typical intermediate between aldehydes and ketones. We will see this again later.
The
third reaction is another ATP consuming reaction, a big thermodynamic driving
step and therefore a major regulation point in the pathway.
This
is catalyzed by phosphofructokinase and is another phosphorylation reaction on
the opposite end of the sugar.
Fig.
18.7
The
most interesting thing about this enzyme is that it is inhibited by its
SUBSTRATE ATP. This is sort of opposite
of our normal chemical sense. We might
think that the reaction should go better the more ATP we add.
How
do you think this inhibition occurs mechanistically?
Given
that the reaction needs ATP to run, why would we want ATP to inhibit it?
AMP
reverses this inhibition. Does this
make sense?
The
enzyme is also inhibited by citrate.
Citrate is an intermediate of the TCA cycle that glycolysis feeds. Does this make sense?
Finally,
the ATP inhibition of this enzyme is relieved by the molecule 2,6-fructose
bisphosphate (the product of this reaction is 1,6-fructose bisphosphate). This molecule also enhances substrate
binding.
In
the fourth reaction we are actually going to cleave the six carbon sugar to
make two three carbon sugars using Fructose Bisphosphate Aldolase. The mechanism for this varies. The simplest is shown first in Fig. 18.11
Finally,
phase I ends with the isomerization between the 3-carbon ketose dihydroxy acetone
phosphate and the 3-carbon aldose, glyceraldehyde 3-phosphate by triose
phosphate isomerase.
Fig.
18.14
Based
on the mechanism of the second reaction, can you predict the mechanism of this
one?
The
second phase of glycolysis takes the G-3-P and generates pyruvate. In the process, NAD+ is reduced
and 2 ATP molecules are generated.
Since there are two molecules of G-3-P from phase I, everything in phase
two is done in duplicate! (2 NADH
molecules and 4 ATP molecules made).
So
again lets look at the starting point and ending point and see what we need to
do.
We
have to oxidize the aldehyde to a carboxylic acid. This is thermodynamically favorable so in the process we are
going to form a phosphodiester bond to the carboxylic acid.
Next,
we complete that process by transferring the phosphate to ADP and generating
the carboxylic acid.
Next,
we move the phosphate up one position.
This stabilizes the enol we are about to form. Next, use the energy from allowing the enol to go to the ketol (much
more stable) to drive the transfer of the phosphate group to ADP.
So,
lets look at the first of these reactions.
This is carried out by glyceraldehyde-3-P dehydrogenase.
Fig.
18.17
The
mechanism is shown in figure 18.18. It
involves attacking the aldehyde with a thiol group from Cys. This then makes a tetrahedral carbon. This is oxidized by NAD+ to form
a thiohemiacetal. Finally, the thio
group is displaced by a phosphate, again through a tetrahedral intermediate.
In
the next step, we really complete the last step with the transfer of the
phosphate to ADP:
Fig.
18.20
Next,
phosphoglycerate mutase enzyme transfers the phosphate to the middle
carbon. This stabilizes the molecule
for formation of the enol in the next step.
Fig.
18.25
The
next step is a dehydration reaction which forms the enol. Fig. 18.26
This
molecule is called phosphoenolpyruvate (PEP) and as its acronym implies, it is
a high energy molecule. Note that it
would love to give the phosphate up and tautomerize to the ketone.
This
is exactly what happens in the next step, but the tautomerization is coupled to
ATP formation. This is carried out by
pyruvate kinase: Fig. 18.27
The
mechanism can be thought of as follows: Fig. 18.28
Pyruvate
kinase is another regulatory step as it is another point of large driving force
in glycolysis:
Activator:
AMP, Fructose 1,6 bisphosphate
Inhibitors:
ATP, acetyl-CoA, alanine.
Can
you explain why each of these is an inhibitor of pyruvate kinase?
This
concept that the points of thermodynamic driving force are the major regulatory
points is best shown in Fig. 18.31
Here
we immediately see why reactions 1, 3 and 10 are the ones where major
regulation occurs (11 is actually lactase dehydrogenase which happens in the
absence of oxygen – this needs to be thermodynamically favored to drive the
reactions forward, see below).
This
general concept of using thermodynamics to make major decisions in metabolic
pathways is a general one. Be sure you
understand this.
In
the presence of oxygen, pyruvate is used to feed the TCA cycle which is the
subject of chapter 20.
In
the absence of oxygen, we have a problem.
We must recycle the NADH formed during the oxidation of glyceraldehyde
3-phosphate back to NAD+. We
can’t lose the NAD+. It is
too precious. So, we need a fall guy.
Different
organisms use ways of doing this. We
reduce pyruvate to lactate (reduce the ketone to an alcohol). The lactate thus produced builds up in our muscles
when we are moving to much or too fast for oxygen to remove the pyruvate
first. Once it builds up, most of it
actually has to be taken to the liver, made back into glucose and recycled. This is one reason that it takes so long for
the tiredness to go away once you have worked your muscles for awhile (and why
it builds up over time while you are working).
Fig.
18.30
Yeast
generally speaking decarboxylate the pyruvate instead, generating
acetaldehyde. This then is reduced to
ethanol. Thus the products of the yeast
reaction are bubbles and booze.
Fig.
18.30
I
am not going to worry about the details of how other substrates can enter
glycolysis. Suffice it to say that many
sugars can be converted to one of the intermediates in the pathway. We have not yet talked about lipids and
fatty acids, but the backbone of a triglyceride (fat) molecule turns out to be
glycerol which can be converted to glyceraldhyde 3-phosphate.