When we saw the image of the reaction occurring, we
saw these various states that you see on the screen.
The enzymes plus the substrate bounds
together to make the ES complex,
which converted upon the change
in the enzyme to the ES* complex,
which created the EP or
the enzyme product complex,
which ultimately resulted in the release of the
enzyme and product. Now I comeback to this,
because, we are going to need to consider
somethings about the kinetic parameters that is the
speed of parameters of the reactions
that we are going to study.
Now this rate of formation of product
is really what we are interested in.
When we talk about how fast an
enzyme an make a reaction occur,
this is the guts of what we are after. We wanna
know how fast is the enzyme able to do this.
Well, if to do this we need to make
some simple assumptions. So we assume
in the simple case that the enzyme substrate complex
proceeds directly to enzyme plus product, okay?
So when we simplify this more complicated
equation above to a simpler
equation below and this is done to
help us better understand what's
going on in the overall mechanism.
Now these constants that are here won't
really enter into our consideration.
But they kcat that you see
in the enzyme going to E plus P
will, in fact, be an important consideration
for us as we talk about the kinetic parameters.
The kcat, as we shall see, is the
rate with which product is forming.
Now let's consider what's happening
inside of a couple of different scenarios of a reaction.
We can imagine that we have enzyme, for example shown in yellow.
And we have substrates as little red color balls that are there.
We can have a situation, first of all, where we have
a reaction going on in the condition of low substrate.
And if we have a low amount of substrate in
a solution, we could imagine that there is
very few enzymes that are going to be bound a substrate; because,
the chances of encountering a substance are reduced.
In the middle, of course, we have an intermediate
state where we have a little bit higher concentration
of substrate than we did before.
And so we can see here that there are more
enzyme molecules bound to and engaged
in the process of making the product.
And the third scenario we could imagine is high substrate.
And we have a situation of high substrate.
We notice here that every enzyme is bound to a substrate.
And that's important because at high substrate concentrations
we have enzymes that are what we call saturateable substrate.
Meaning that once it is bound to substrate
made a product and released it,
almost instantaneously it grabs another substrate.
It's not sitting around and waiting for things.
Now, so enzymes interestingly have some
kinetic considerations which is of course what
we are interested in studying here.
But we are seeing now for the first time a projection of the way that
the enzyme is working. So I need to explain
somethings on the graph that you see.
First of all, we are plotting on this graph a reaction.
The reaction is plotting the velocity of the reaction on the y-axis
verses the substrate concentration
that's used in the reaction on the x-axis.
Now you notice the V has a little 0 beneath it. And
the 0 beneath it, I will explain later. But it's
called the initial velocity for our purposes.
The velocity of a reaction is measured
as the concentration of product made, divided by time.
The concentration of product made per time. But we measure
concentration in molar, mini molar, micro molar, etc.
So that will be some morality per time.
That's how the velocity is measured.
The substrate concentration varies because,
to generate a curve like this,
I do not one reactions, but I do series of reactions.
So let me set that up. We could imagine
for example that I am setting up
a series of 20 reactions, 20 different test tubes.
I want to measure the velocity in each one of those test tubes.
And what I do is I take into that test tubes, I place
the buffer that holds the substrate.
I place the substrate and I place the enzyme.
Now when I am doing an experiment I wanna
have one variable; because, one variable
is the only thing I can really manipulate
and measure the effect of that.
The variable I have here is substrate concentration.
I used the same amount of enzyme in every tube.
All 20 tubes have the same amount of enzyme.
They all had the same amount of buffer
and they have varying amounts of substrates,
starting from small amounts to very high amounts.
I take and I let each one react for an exact same time
and then I measured the amount of product.
So by doing that I can see the effect
of changing substrate on the velocity and then I plot it.
So what you see on the screen is the sum of those plots, that is,
each point on that dot came from a series of reactions that I did.
And each one of those individual reactions
have a specific substrate concentration
and a specific velocity that was reached.
Well not surprising as we look at this what do we see.
Well, on the far left we are at low substrate
concentration. What's the velocity? It's very low.
And that's what I showed on the original image.
Low substrate concentration, enzyme is
sitting there waiting for substrate
there is not gonna be much velocity.
When I get to a high substrate concentration such
as I see on the right side of the screen
I have got a high velocity. Make sense.
Okay, low substrate, low velocity;
high substrate, high velocity.
I want you to remember that.
Now I am showing another plot here to
illustrate a principle of a reaction.
On the y-axis, I have the concentration of product,
we can think of that again as the velocity.
But on the x-axis now, I am plotting
the time of a reaction.
So I can take one of the tubes that I used in the previous one.
And for example, look at how fast
the product is being accumulated and what
happens with that product over time?
We can see on this plot that over the early range of the reaction,
there is a linear relationship between the production of product
and time, okay? But after a while what happen is
that curve flattens on.
Now what that means is that the
longer that we let a reaction go
it doesn't stay linear forever. And the reason
it doesn't stay linear forever because remember
enzyme catalyze reversible reactions.
So the more we let product accumulate
the more likely product will start
being converted back into substrate.
Well, that's not what we are interested in studying.
We wanna study how fast the enzymes makes product.
So for going to study an enzymatic reaction
we have to study what's called initial velocity.
We don't wanna wait too long in order to study the
concentration of product, because if we wait too long,
we are actually starting to study the reverse
reaction and that's not what we are after.
So that's why we used Vo or the
initial velocity in our measurements. Okay.
Now this is kinda complicated so I wanna step
you through it. But these are considerations