Okay well let's now look at these parameters.
I have introduced the concept of Vmax
and we see that eventually the enzyme reaches
a place where it's not gonna make any more
product over time; because,
it's saturated with substrate.
Vmax turns out to be an interesting quantity
but Vmax as we will see has some limitations.
Nonetheless, Vmax allows us to study some things.
Now the quantity Vmax gives us a
maximum amount and we can say "Well,
if we wanna understand how much an
enzyme interacts with a substrate
maybe we should compare Vmaxes."
Well that doesn't really tell us very much.
It tells us how fast a reaction goes.
But it doesn't tell us how well an
enzyme interacts with a substrate;
because, any enzyme will reach Vmax
as we add an infinite amount of substrate
which is theoretically what Vmax
is occurring at, when it is completely saturated.
That doesn't tell us very much.
However the quantity Vmax/2
where we are getting an enzyme to a certain point of
velocity, but not the maximum amount of velocity,
actually allows us to measure that the affinity
that enzyme has for its substrate.
If we compare a variety of enzymes
and we compare how much substrate
it requires the enzyme to get to Vmax/2,
we get something very interesting.
We get a quantity called the Km.
And the Km is actually a measure
of the enzyme's affinity for its substrate. So
I say affinity is the desire to bind to.
How well does it bind to its substrate?
Now Km is interesting. If
we think about two enzymes
one enzyme that catalyzes a
reaction that has great affinity
for its substrate. It really likes that
substrate. It really grabs that substrate.
And we have another enzyme over here that
doesn't like its substrate as well, okay?
Well which of the two are gonna bind substance more readily?
The first one, of course; because, it has got greater affinity.
Which one is going to get to Vmax/2
with a lower substrate concentration?
Well the one that grabs
its substrate more easily.
So enzyme that have a greater
affinity for their substrate
are going to have a low Km.
And those that have less affinity for their
substrate are going to have a higher Km.
Okay? Greater affinity, low Km;
lower affinity, high Km. So
Km is inversely proportional
to the enzyme's affinity for its substrate.
Okay? So here we see high Km, low affinity.
We see low Km, high affinity.
A very important concept to remember with respect to Km.
And I would like to think about
an enzyme that has low affinity
we have to pounder on the head with
substrate before it starts to bind it.
And by poundering on to head, the way we do
that is by adding a lot more substrate.
Now Vmax, as I said, is a very
interesting and important quantity.
But it actually is not the
perfect quantity to measure
the speed of a reaction. It's good for the
reaction but it's not so good for the enzyme.
So what does that mean? Well it means that Vmax
when we a do a reaction, the way I described
doing a reaction, is we set up 20 tubes,
and we have in those 20 tubes, buffer,
we have substrate, and we have enzyme.
And when we are doing a V versus S plot, what we
are doing is we are having one variable.
The one variable that we have is substrate,
which means that all 20 tubes
have the same amount of enzyme.
That's great. We don't wanna
have variable amounts of enzyme.
But imagine I would
do the same set of reactions.
And instead of using the
amount I used in the first set
Let's say that I did the reaction and I used twice the
amount of enzyme for the second set of reactions, in each
case constant, however,
varying substrate but now
with twice the amount of enzyme.
What would I see with respect to Vmax?
Well if I go back to my factory analogy, and I think about what
happen with the factory, I said that the factory got to a point
where it's saturated. It made a maximum amount of
product that the workers are gonna put out per day
and it wasn't gonna make any more.
What if I had two factories?
Well if I had 2 factories I would say
"Well I'd probably expect that I will get
twice as much product per day".
And so I if used a set of tubes
that have twice as much enzyme,
the parallel follows. I would
get twice as much product.
So Vmax is proportional to the amount of enzyme
I use. It's not a constant for an enzyme.
But it's a constant only for a reaction
with a set amount of enzyme.
I would like to be able to compare enzymes
with a quantity that is independent of
the amount of enzyme that I used.
Well fortunately that's fairly easy to do.
Okay? Vmax is a velocity and
we measure velocity of a reaction
as the concentration of the
product produced divided by time.
If I take the quantity of enzyme
that I used in the reaction
and I divide Vmax by that quantity,
I said quantity in this case meaning concentration,
the concentration of enzyme that I used,
what will happen? Well, the Vmax was
measured as a concentration of product
and I divided by a concentration of enzyme,
as I long as I used concentration
and concentration consistently
the concentrations actually drop out.
And so what happens is, I get a number
and the units on the number are per time.
So I get something it says a 1000/second.
What does a 1000/second mean? Well, I have
taken the enzyme out of the equation
and now the number that I get corresponds to the
number of molecules of product per enzyme per second.
So a 1000/second means every enzyme in that solution
is making a 1000 molecules of product per second.
And that's the fastest it's gonna go,
because, remember we started with Vmax.
That quantity is called Kcat.
Kcat is a number that's also
called the turnover number.
But I can compare the Kacts of two enzymes
and have a much better understanding about the relative
speeds of production of product that those enzymes have.