Voiceover: So, we're gonna
talk about Cooperative Binding, which is a very interesting topic when discussing enzyme kinetics. But first let's review the
idea that we can divide enzyme catalysis into two steps. First, the binding of substrate to enzyme, and second, the formation of product. In using this idea we can derive
the Michaelis Menten equation, which is very useful for quantitatively looking at enzyme kinetics. Also remember that as you
increase substrate concentration, the speed of product
formation will level off at it's maximum value
as shown on this graph. Now the first thing that
I want to talk about is that some proteins can
bind more than one substrate, and not all enzymes have
just one active site. So, E plus S can form ES through what I've called reaction of one, but some enzymes can react with another molecule substrate to form ES two, through what I've called reaction two. And again, to form ES three, through reaction three, and so on. Now, these enzymes can form product at any stage of this process no matter how many molecules of substrate are bound. Now, you would expect
the rate of reaction one to be faster than the
rate of reaction two. If we're looking at the
example of an enzyme with three substrate binding sites, there are three empty sites available for substrate to bind
through reaction one, and only two available for reaction two. So, you would expect
rate one to be faster. Similarly, rate two would be faster than rate three for the same reason. And the idea is that the
active site saturation does not increase with substrate
concentration linearly. Now I get that that can be a mouthful so let's look at it graphically. But, we're also gonna show
an exception to this rule. So in this first graph, I've
plotted substrate concentration against the percent
saturation of the enzyme. And as you can see the curve levels off as substrate binding
sites become occupied. It becomes difficult to bind
more substrate molecules as you have more
substrate molecules bound. Next, I'm going to draw a different curve that you also might see
in some enzymes and, that's where substrate binding happens more quickly as binding
sites become occupied. Substrate binding changes
substrate affinity. And we call this Cooperativity. Now with respect to cooperativity, we can define three new ideas: Positively Cooperative Binding occurs when substrate binding
increases the enzyme's affinity for subsequent substrate. Negatively Cooperative Binding occurs when substrate binding decreases the enzyme's affinity for subsequent substrate more than you would normally expect. And Non-Cooperative Binding is the same as the first example where
substrate binding does not affect the enzymes affinity
for substrate molecules. So, let's look at this graphically. If we have a protein with
let's say five binding sites, and plot the Fraction Occupied versus the Substrate Concentration, you would come up with
three possible curves. The green curve, which
takes on a sigmoidal shape, would represent an enzyme
with Positive Cooperativity. The blue curve, with a hyperbolic shape, would represent an enzyme
with Non-Cooperative Binding. And the red curve would represent an enzyme with Negatively
Cooperative Binding. Now remember that the effects of cooperative binding are only seen after some substrate has already bound. Which is why the
difference in the fraction occupied between the three
curves is much smaller, it's smaller values, like the one-fifth that I've shown here, than
it is at the higher values. So, let's look at a specific
example of a couple of proteins. So haemoglobin, or Hb, is
the oxygen carrying molecule that you find in human
blood, and it can bind up to a total of four oxygen molecules, and it exhibits Positively
Cooperative Binding. Myoglobin, on the other hand, which is the oxygen carrying molecule that
you find in muscle tissue, can only bind one oxygen
molecule in total. And since it can only bind one, it must exhibit Non-Cooperative Binding since there's no subsequent
substrate to speak of. Now, if we make a graph where we plot the fraction of active sites bound in each of these proteins
versus the pressure of oxygen, remember oxygen is our substrate here, and since it's a gas we're gonna use pressure instead of concentration, you can see that the red
sigmoidal curve associated with haemoglobin's positive cooperative binding looks different from the
blue hyperbolic curve associated with myoglobin's
non-cooperative binding. So, what did we learn? Well, first we learned that some proteins can bind more than one
equivalent of substrate. And next, we learned that
there are three different types of Cooperativity: Positive,
Negative, and Non-Cooperative. Finally, we learned about
proteins that exhibit two different types of cooperativity, which were the oxygen binding molecules haemoglobin and myoglobin.