LECTURE 12: PROTEIN-LIGAND INTERACTIONS & CATALYSIS

Where we left off:

Interactions between proteins and ligands, small molecule ligands are the proteins interacting with our
target proteins
o All these interactions can be quantitated mathematically by a dissociation constant
o Protein-ligand binding curve
Hyperbolic binding curve describing the typical behavior of a ligand binding to a target
protein
The Kd value, the dissociations constant, which is an equilibrium constant, is a typical
description of how tightly, how strongly, a ligand interacts with a target protein
Each individual protein-ligand interaction has its distinct Kd, its distinct binding affinity
If we have to distinct ligands binding to the same protein, there are two important aspects:
o 1. Where would the two lines end up if you were to use infinitely high ligand concentrations?
At 1, if you have an infinitely high ligand concentration the two curves approach full
saturation which is a theta value of 1
At an infinitely high ligand concentration you can assume that even the weakest binding
ligand will interact with the protein and saturate the protein
Sometimes people draw the curves as parallel, that is not correct, at the far right they all
end up saturating
o 2. The value of Kd
The black curve has a Kd of 1 arbitrary units whereas the green curve has a Kd of 3
arbitrary units
Which ligand is more tightly binding? Which interacts more strongly with the target
protein?
THE BLACK ONE, that ligand is more tightly binding
The concentration of that ligand thats required to bind, to saturate 50% of the
target protein is much, much lower
A tight binding ligand is a ligand with a lower Kd value
A ligand with a higher Kd value is less tight binding or more loosely binding
A couple of Kd values between protein and ligand, or protein and proteins
o Insulin receptor/ insulin: Kd = 1E-10
What does 1E-10 mean? It is the substrate concentration at Kd value, at a concentration
of 1E-10 you have 50% of the available insulin receptors already bound
o Anti-HIV immunoglobulin/ gp-41: Kd = 4E-10
Gp-41, globular protein number 41, it is a protein on the HIV viral surface
Immunoglobulin can bind to protein
o Avidion/Biotin = 1E-15
Egg white
o Haemoglobin/O2: Kd = 2E-6
o Carbonic anyhydrase/Diamox: Kd = 1E-8
o Binding constants are measured at physiological temperatures, 37 degree centigrade
o Why is there a difference in Kd between haemoglobin and ant-HIV immunoglobulin?
There are certain proteins in the whosfunction is, lets say a transporter, whose function
is to pick up a ligand and move it to a different location
And let go of the ligand once it gets to that different location
That is reflected in Kd value
Turns out that proteins, transporters or enzymes
o Enzymes are classic examples, the catalyst wants to bind a substrate,
facilitate a chemical reaction, but if it doesnt let go of the product,
whats the point?

Systems where you have a temporary binding interaction, those tend to have a
larger Kd, E-5, micromolar range
In contrast, something like immunoglobulin-gp41, the body makes sure that if it binds to
the HIV virus it doesnt let go until it is no longer
Kd values are also trenmednously helpful in drug development
o Carbon anyhydrase:
Diamox, which is the original inhibitor, is created company A has Kd of E-8
Another pharmaceutical company, B, and say darn theyre making a lot of money
making Diamox
But they cant make the same molecule
What they can do is play around with the molecule and a create a different
molecule
The Kd values are a nice reflection of how a new medication might be an
improvement of the original drug
Addition of phenol group: Kd=5E-9, 5 fold lower Kd
This compound binds around 5 times stronger to the carbonic anhydrase
Why is this good?
o Most drugs that we take have side effects
o As a general rule, the less we have to take of a drug, the less likely you
will have side effects
o Also, you can buy a smaller pill, which give you an economic
advantage
Addition of hydroxyls to phenol: Kd = 2E-11
Three orders of magnitude less, 3000 times more tightly binding
Addition of Cl and tertiary alcohol: Kd=1E-6
The Kd increases, suddenly the compound has lower affinity
The trend stops, the molecule is not a good candidate for the drug
But, we can do more than that, if you have a molecular model you know that the
side chain which part of the molecule, of the carbonic anhydrase, the side chain
interacts with
You can see how they fit, turns out that if you make it too bulky, it doesnt fit
properly into the cleft that leads into the active site

Not only can we quantify how good of a drug it is, but why a drug t is more
potent or less potent

Myoglobin/ the story of oxygen management

Obviously molecule oxygen is critical for human body for the biochemistry of our cells
o There are 2 molecules that are primary responsibility is to manage molecular oxygen supply to the
cells
1. Hemoglobin
2. Myoglobin
Myoglobin
o The secondary structure is composed of a number of alpha helices and heme group
o The heme group moiety:
Considered a cofactor, a helping molecule that the protein recruits in order to fulfill its
biological function
Heme groups, zinc metal ion in carbonic anhydrase
The heme group is a perforin ring system, a planar molecule, ability to chelate an iron
metal in center
The iron center is responsible for chelating an oxygen molecule
Oxygen chelates to the iron in the perforin ring system

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Where the action is in myoglobin

Myoglobin is a single subunit, simply a tertiary structure with a single heme group
If you look myoglobins oxygen binding properties:
Regular hyperbolic curve: y-axis is saturation of myoglobin, x-axis is partial pressure of
oxygen, which is equivalent to oxygen concentration
The partial pressure of oxygen goes from 0-10 kPa
The concentration fo oxygen in the lungs, the highest oxygen concentration our cells are
exposed to, is aroiund 13 kPa
The partial pressure, the concentration of oxygen, in our tissue, if you stuck an oxygen
probe into your little toe, it would be around 4kPa
Highest concentration in your lungs, where the oxygen is loaded, it is transported to
distant part of your organism and released to provide resources for energy production
If you look at these two partial pressures, these two oxygen concentrations, they are the two
extremes an oxygen transporter has to deal with
If you look at the binding curve of myoglobin, is myoglobin a good transport protein? Would it
make a good transport protein?
No, why not? Whats the problem?
Oxygen binds too strongly, if you look at the graph, if you go from lung partial pressure,
to the partial pressure in your cells, you can see that under those conditions, the
myoglobin would barely let go of any oxygen
It would load up very readily with oxygen in the lungs, but once it goes to your toe, it
wouldnt let go
Where do we actually have myoglobin? Our MUSCLES
Muscles are loaded with myoglobin, what purpose does it serve in our muscles? Why is it
there?
Oxygen storage, storage for what?
Myoglobin in our muscle cells is kind of like a last resort oxygen storage system
Our myoglobin is probably fully oxygenated in our muscles during resting periods, but as
we start exercising strenuously we cant breathe enough oxygen, the partial pressure of
oxygen in our big toe starts to drop
Before it reaches 0, before we go anaerobic, which is very unhealthy, the myoglobin will
start releasing the oxygen it has stored, to give us an extra boost
Evolutionary thinking is that the only time you reach this point is whne you have
run from a cheetah or tiger
Short bursts where you need the extra energy, myoglobin is a last energy
resource in place
Well if myoglobin doesnt do well for oxygen transport, what molecule does? HEMOGLOBIN!

Hemoglobin

If you look at hemoglobin, it has quarternary structure, it consists of four subunits

o Each individual subunit is very similar to myoglobin
Its almost like, at one point in evolutionary history, the cell took the myoglobin
precursor, stuck it into a tetramers, and used that for oxygen transport
o But its not just four subunits stuck together, because if the four subunits were acting
independently it would show the same behavior as myoglobin
o What happened is, upon combining these four subunits, nature tweaked the myoglobin so that the
individual subunits are talking to each other
Now, they are not chit chatting, the way they are talking is through conformational
changes
If you imagine, you have 4 proteins clustered together
One of those subunits binds oxygen, its structure changes a little bit

That structural change is sensed, its noticed by the neighboring subunits

That triggers a similar conformational change
That communication between subunits is shown in the image:
o In the original state, there are 2 subunits have come together, each with a wiggly tail
o One of those subunits binds a ligand, which stops the wiggly side, it consolidates the structure
o That consolidations results in the second wiggly unit becoming less wiggly
o Allows it to bind the second ligand
This is known as COOPERATIVITY
o All the subunits work together, they cooperate to change their behavior
o Why does a conformational change affect the binding affinity
o The proteins structure is an integral part of its function
If it changes its structure its function will also change
There are two states for hemoglobin
o 1. All four subunits are wiggly, there is no oxygen bound, this is referred to as the T STATE
o 2. If oxygen is bound tot eh first subunit, theres a conformational change in all four subunits,
referred to as the R STATE
How does this relate to oxygen binding?
o The first oxygen molecule comes in and binds to one of the subunits
o There is a conformational change, not just in the subunit that bound the oxygen, but in all 4
subunits
o What happens is the new conformation has a new Kd value, because it has a different structure
It has a differnet binding affinity for oxygen
It very quickly binds the three remaining molecules of oxygen
Which of the two states binds oxygen with greater/lesser affinity?
o The R state binds oxygen with more affinity
What happens to the graph of the hemoglobin/oxygen binding curve?
o At 0 concentration, it follows the T curve
o What happens upon oxygen binding? All of the four subunits shift to the R state
o The binding curve then changes
o What you see if that the ultimate binding behavior working together, there is a sigmoidal binding
behavior
At low oxygen concentration they behave more like the T state
After reaching a certain threshold they all switch to the R state and show very high
affinity
We like to see this behavior in hemoglobin
o When oxygen concentrations are very high, like in the lungs, all four subunits are in the R state,
they are fully loaded with molecular oxygen
o It then starts moving throughout your body, goes to your big toe, where the oxygen concentration
is lower
o What happens is over 50% of the hemoglobin starts letting go of the oxygen when it reaches your
toe
Which is exactly what you want to see
o If you exercise strenuously and the osygen concentration is even lower then hemoglobin would
even more readily unload oxygen
You can literally load up oxygen and let go very easily when it is needed
T and R state represent the structural conformation of each individual subunit
o The T state is the structural conformation of each subunit that has a low affinity for oxygen
o R state is the conformation that has high affinity for oxygen
o Because of cooperativity, even if just one subunit goes from T to R, all of the other ones must
switch as well

Protein Substrate/Enzyme substrate interactions

Why should we talk about enzyme catalysis?

o Peptide hydrolysis has 450 half life
o We need catalysts to accelerate these reactions
o Enzymes are natures answer
Can accelerate by up to 17 orders of magnitude