The first mechanism I wanna talk about is allosteric control
and I have discussed this in another presentation. So I'll move through it fairly quickly.
We remember from the discussion of enzymes, that enzyme that obey Michaelis-Menten Kinetics
display a kinetic profile like we see on the screen here. A hyperbolic plot and the plot of
V versus S. V being the velocity of the enzymatic reaction.
Now we also mentioned that enzymes that don't behave Michaelis-Menten kinetics
were oftentimes display a sigmoidal plot as shown here.
Now, the reasons that arise of this will be the subject of what I am talking about in this talk.
The substrate doesn't change the enzyme in the first scenario.
That is if the enzyme grabs the substrate
and all that happens is that the enzyme catalyzes the reaction on the substrate.
However, in the second scenario the substrate as a result of interacting with the enzyme
changes the way that the enzyme binds to substrate.
So if that enzyme is a multisubunit enzyme, as many enzymes are,
then the binding of the first substrate can affect the binding of subsequent substrates.
And that's why this group deviates from the hyperbolic curve that we saw on the first one.
Now, the classic enzyme for studying allosteric control
is aspartate transcarbamoylase which you can see on the screen.
Aspartate transcarbamoylase is also known as ATCase; because it's easier to say.
Aspartate transcarbamoylase has a structure like what you see on the screen.
It contains 12 proteins,
6 subunits that we call catalytic and 6 that we call regulatory, as we shall see.
Now ATCase catalyzes a reaction that's essential for the synthesis of pyrimidines.
We can see the reaction being shown on the screen and while the reaction is important to cells.
The actual reaction isn't that important to us except for one of the substrates.
Now aspartate is an amino acid and it is one of the substrates of the reaction.
That's the one that we are interested in here.
ATP is a high energy source and ATP, as we have discussed before,
the gasoline of the cell that provides all the energy that the cell needs.
In addition to that, ATP besides providing a high energy
is also used in making RNA and DNA and when we think about this
so too is the end product of the pathway that's initiated by ATCase;
because ATCase
initiates a pathway to synthesis CTP. The end product of this pathway is CTP
and CTP is also used to make RNA and DNA.
Now here are the substrates of the reaction and here is the enzyme catalyzing that reaction.
When aspartate binds to ATCase it changes the enzyme and it
specifically changes the enzyme affinity for additional aspartates.
That's why we see the sigmoidal plot that you can see on this graph.
So ATCase is affected by a aspartate.
It's important to remember that enzymes can exist in two different states.
One state of the enzyme is referred to as the R-state and we can think about that
as a relaxed form of the enzyme. And the relaxed form of the enzyme
favors binding of additional substrate.
The other form of the enzyme is known as the T-state or the tight state and it's a tense state of the enzyme
that, though it will bind substrate, it doesn't bind it nearly as well as it binds it in R-state.
The R-state is therefore the more active form of the enzyme and the T-state is the less active form of the enzyme.
Note that both forms are active. It's not an on off switch,
but rather affecting the amount of activity that the enzyme has.
Now this graph illustrates how that sigmoidal curve
is changed not by the binding of a aspartate but by the binding of ATP.
It turns out that ATP also affects this enzyme.
ATP is a not a substrate of the enzyme.
And in fact ATP, as I noted, is an energy source and it is also appearing.
An energy source is important; because, cells that are going to go through
replication, need to copy their DNA, and to do so they need to make more nucleotides.
If the cell is in a high energy state it's in a better position to do that
than it's if in a low energy state. If it's in a low energy state
then the cell would be taking a very big risk in making nucleotides.
Well, when the cell is in the high energy state, ATP is present in abundance
and when that happens it binds to ATCase
and causes the curve to shift, as you seen here,
from the blue line that we started at to the orange line that's shown here.
Now, the difference between these is that the orange line; because it is shifted to the left
is catalyzing a reaction faster at a lower concentration of substrate.
So the same concentration of substrate will result in increased velocity of the enzyme.
What ATP has done is it has favored the activation of the enzyme
and it's telling the enzyme "It's okay to go ahead and make
nucleotides; because we are good to go for replication."
Now ATP activates ATCase by converting it to the R-state,
just like aspartate converted the enzyme to the R-state.
Now this means, therefore, that the enzyme is able to bind more substrate more effectively
and work faster which is why we see the increase in velocity that we see here.
The activity of ATCase is affected by a third compound and that's shown on the screen here.
The third compound is the nucleotide CTP or the cytidine triphosphate
And that's cytidine triphosphate has the effect that you see on the screen.
The more cytidine triphosphate is added
the lower the velocity of the enzymatic reaction catalyze by ATCase.
Now CTP is the end product of the pathway that's initiated by the ATCase reaction.
If the cell is making too much CTP
what happens is CTP starts to bind to the enzyme
and it turns the enzyme into the T-state and when it puts into the T-state
the enzyme is less able to bind to substrate.
Increasing quantities of CTP make this happen. Now, this turns out to be important; because
the cell doesn't wanna be churning out too much CTP; because if it makes too much CTP
a, it’s wasting energy and then here is one of things, another reason cells don't wanna make too much of nucleotide.
If the nucleotide gets out of balance then the cell is much more prone to having mutation.
So balancing nucleotides and having the controls that I have described here
are important not just for energy purposes but also for maintaining the integrity of the DNA.
You can see that the relationship here between the velocity
and the concentration of CTP is not exactly linear.
But the most important point is that it decreases as the concentration of CTP increases.
Now, aspartate was a substrate but neither ATP or CTP
was a substrate. However, all affected the enzyme.
Aspartate and ATP turns the enzyme into the R-state and CTP turned it into the T-state.
Well, if we look at the enzyme we can see that the enzyme contains the
big blue balls called catalytic units.
And these are the places where the reaction that the enzyme catalyzes is catalyzed.
The C-units, the catalytic units, are the place
where a aspartate binds to the enzyme.
By contrast the green balls shown with Rs on them are regulatory subunits
and these balls are the things that bind either the ATP or the CTP.
And when this happens we see that the enzyme can flip its state, right?
So ATP, as I said, favors the R-state,
CTP favors the T-state, and aspartate favors the R-state as well.
So the flip to the R or the T-state can happen as a result of binding to any
of these proteins that you see on the screen.
Now to refresh what I have said here and show it on the graph, I wanna say a little bit
about what these different parts of the curve mean.
At low substrate concentrations, ATCase is low in activity
and that means it's in the T-state.
Well as the substrate concentration increases for aspartate,
what happens is that the enzyme starts flipping into the R-state. So we see that flipping happening
as a result of the change of the slope of that curve.
And finally at high substrate concentrations ATCase is mostly in the R-state.
Meaning it's ready to start churning out nucleotides in the form ultimately of CTP.
So, that explains the ATCase is a structure an activity as it relates to aspartate
and you know how the enzyme changes
with respect to ATP and CTP. But there is yet another thing to consider here
and that is that ATP, as I noted earlier, is a purine
and CTP is a pyrimidine.
So when ATP concentration is high, that means that the purine concentration is high.
And when we look at nucleic acids, purines pair with pyrimidines.
So in addition to having a high energy activating this enzyme
we have a purine activating an enzyme that makes pyrimidines.
This is a very nice way of bringing balance to the nucleotides for the reasons I mentioned earlier.
ATCase is least active when the pyrimidine concentration is high.
And when the pyrimidine concentration is high then its likely that it is higher than it is in the purines.
So that balance is important and the enzyme has all of these different considerations built into its structure.
Now another thing about the ATCase system that I'd like to point out to students
is the fact that it's a prime example of a phenomenon we call feedback inhibition.
So feedback inhibition occurs when the last molecule
in a metabolic pathway comes back and inhibits the first enzyme in the pathway.
Now this figure illustrates that principle clearly.
We can see that ATCase doesn't catalyze the formation of CTP
It catalyzes the formation of things that ultimately become CTP.
So CTP which is the end product of that process inhibits the enzyme.
Now that's a very efficient way of controlling an entire metabolic pathway;
because by controlling the very first reaction
and the formation of the very first product
one controls the entire pathway, because if the first product isn't available
then the second product won't be available, etc all the way down to CTP.
So, feedback inhibition provides a very efficient way of controlling a pathway.