#09 Biochemistry Hemoglobin II/Enzymes I Lecture for Kevin Ahern's BB 450/550

Uploaded by oharow on 16.10.2011

Captioning provided by Disability Access Services
at Oregon State University.
Kevin Ahern: Okay, folks, let's get started!
I can't hear, there I go!
You guys are the quickest-to-quiet-down class
I've ever had, and that's good.
Just think how much more biochemistry we can squeeze in
now when you quiet down quickly.
I hope everybody's doing well and ready for a big weekend.
We will have an exam in here a week from Monday, not today.
For those of you who thought we were having it today,
I know you'll be disappointed.
I will announce later where that stopping point will be.
I sort of decide on, it depends on where I get in my lectures,
and it varies a little bit from term to term.
Today I'm going to finish up hemoglobin
and start talking about enzymes.
I hope I gave you the impression
or the understanding that hemoglobin is a remarkable protein.
It has a remarkable number of functions built into it,
and those functions are directly related
to the structure of the protein.
We saw structural things about proteins,
in general, when I talked about primary,
secondary, tertiary, et cetera.
But here's a protein where you get a real live,
up close and personal look
at how those structures that we see in proteins
give proteins specific functions.
We'll see more of that when we talk about enzymes.
I want to emphasize, speaking of enzymes,
that hemoglobin is not an enzyme.
People commonly think that, but it's not.
Enzymes catalyze reactions and hemoglobin
isn't catalyzing anything.
So it's an oxygen-carrying protein.
That's really its only function.
We'll see that the way that it binds oxygen
is not unlike the way that enzymes bind their substrates,
but hemoglobin is not an enzyme.
Last time I finished by talking about fetal hemoglobin
and fetal hemoglobin has the very interesting property
of having the slightly different subunits.
It has the two gammas instead of the two betas,
and that sort of makes a structure
that doesn't have that doughnut hole
that fits the 2,3BPG in the same way.
As a result of that, as I had noted,
the fetal hemoglobin stays in the R state almost all the time,
and that's why the fetal hemoglobin
has greater affinity for oxygen than adult hemoglobin.
There are yet other things
that we need to understand about hemoglobin.
I always like to think about hemoglobin as having,
obviously, structures that give it the functions that it need,
that those structures correlate very well
with the needs of the body.
They correlate very, very well with the needs of the body.
You saw 2,3BPG was being produced by cells
that were actively metabolizing,
and it was providing a signal that,
"Hey, here's the place where I need the oxygen.
Let go of the oxygen."
And they cause hemoglobin to let go of the oxygen.
There are other signals that hemoglobin can respond to
with respect to actively respiring cells.
One of these is pH.
When we talk later about,
it'll actually be next term,
but when we talk about actively respiring cells next term,
one of the things we'll discover
is that actively respiring cells
have a higher concentration of protons around them
than non-actively respiring cells,
which means that the pH around an actively respiring cell
is lower than that of a non-actively respiring cell.
Again, when I think of an actively respiring cell,
you can almost always think of muscle.
Muscles really change a lot.
When muscles are contracting,
they're really needing energy a lot,
and things that need energy need oxygen.
They go hand in hand.
A scientist named Bohróand no, that's not B-O-R-E,
that's B-O-H-Rómade a very interesting observation
about hemoglobin many, many years ago.
The observation was, if he took hemoglobin
and he did this oxygen-binding curve that we did before,
where we see the percent of the hemoglobin
saturated with oxygen and the concentration
of the oxygen on the x-axis,
what he saw was, he did the plot
and he saw that nice sigmoidal plot that we did before
and if he dropped the pH the curve actually shifted downwards.
Well, that shift downwards corresponds to less affinity,
meaning that the hemoglobin is releasing oxygen,
and it's releasing oxygen as a function
of the pH environment in which it finds itself.
Now, again, this is a functionality
built into hemoglobin that is directly responding
to the body's needs: pH drops around actively respiring cells,
hemoglobin will tend to give up more oxygen
around actively respiring cells.
It's a very cool phenomenon.
Now, the chemical basis of the effect is not too surprising.
When we look inside of the hemoglobin molecule,
we see that there are various amino acid side chains
that can be charged.
In this case, we see a lysine up here
that is attracted to a portion of a histidine,
and here's the functional part of the histidine
where it can gain or lose a proton.
If it gains a proton, it is positively charged
and it will attract a negatively charged side chain,
in this case, of an aspartic acid.
If that proton is off of there,
then it will not attract that,
and we could imagine there would be
some slight shape changes that would happen,
whether it's being attracted or not being attracted.
And that, actually, that very subtle difference there,
is the molecular basis for the change
in affinity of hemoglobin for oxygen.
So a slight shape change happening
according to whether or not we put a proton onto a histidine,
and that histidine changes its interaction
with another side chain of aspartic acid,
and, a result of that,
causes the protein to actually change its configuration
and its affinity for oxygen.
Another thing that we see around actively respiring cells,
and it's actually one of the causes of the drop in pH,
is carbon dioxide.
Carbon dioxide is the final oxidative product of metabolism.
When we go and we burn sugar,
or we go and we burn fatóI'm on a diet right now,
so that burning fat is really on my mind.
If you see me exhaling a lot of carbon dioxide, that's good.
I wish I could do that.
Carbon dioxide is an end product of those kinds of processes.
So, not surprisingly, if we examine the environment
around actively respiring cells,
we discover there's more carbon dioxide there.
It turns out that carbon dioxide also affects hemoglobin.
I thought I had a graph there.
That's not the thing.
If we look at this now, what we see is,
now we're going back to these plots that we did before,
the first plot was the, this is not getting my voice.
The first plot was the pH 7.4, no CO2.
Then if we take no CO2 and that same hemoglobin
and we drop the pH, this is what we saw before,
the affinity for oxygen drops.
But now look at the bottom line.
If we have a pH 7.2 and we add carbon dioxide,
the affinity drops even more.
That means, therefore, that hemoglobin is releasing oxygen
in response to both protons and to carbon dioxide.
Again, these are things that are both present
in higher concentrations around cells
that are actively respiring.
Questions about that?
Question over here?
Student: Could you go back to the figure real quick with the,
where you had the histidine structure?
Kevin Ahern: Okay.
Student: I had a question about that.
Kevin Ahern: Okay.
Here, yeah.
Student: When you say "add a proton,"
Kevin Ahern: So, as the pH changes,
protons will come on or come off.
So that's the variable that's there.
That's always true, yeah.
Student: So it's the pH drop, then?
Kevin Ahern: A pH drop, so a pH drop would be more likely
to put a proton on there.
Yes, sir?
Student: When we're talking about CO2
contributing in the same way
that a more acidic environment
around the respiring cells contributes,
is that actually in the form of CO2 or as carbonic acid?
Kevin Ahern: Good question.
His question is, "How does CO2 manifest its effect?"
I'm going to show you that in just a second.
Is that your question?
Student: My question was similar.
Kevin Ahern: Okay, so CO2 exerts its effect.
How does CO2 exert its effect?
Well, one of the ways it exerts its effect
is by forming a covalent bond with amine side chains.
CO2 can be carried in the blood in two ways.
One is it can actually be dissolved in the blood,
and we'll talk about that later.
The other way it can be carried
is by this covalent bond to hemoglobin.
We could imagine, looking at this structure here,
that we have a carbon dioxide.
We've got an amine,
in this case that is shown with no charge on it.
We put a CO2 on it and we develop something
that has a negative charge.
Again, we're introducing a charge
where there wasn't one before.
We could expect that we would, in fact,
see some changes that would happen in structure of hemoglobin.
Again, that's the basis for the change in affinity.
Very, very subtle changes that are happening to the protein,
but they're having big effects on its affinity for oxygen.
In this case, it actually, you'll notice it releases a proton,
and that actually enhances the effect that we saw before,
because, more protons, of course,
now we're going to affect hemoglobin in its own way, as well.
So these really work together
to make hemoglobin give up oxygen
at the places where it's needed.
So that's what I want to say about the Bohr effect.
The last thing I want to talk about
are some genetic considerations.
It's a disease that we hear a lot about,
and there are some interesting,
or at least one very interesting aspect of it,
and that disease is known as sickle cell anemia.
Sickle cell anemia is a disease.
It's a genetic disease where there are mutations
in one or more of the subunits of hemoglobin,
and there are different forms of sickle cell anemia
that correspond to, of course, different mutations.
Some may affect alpha, some may affect beta, et cetera.
So sickle cell anemia is not just one genetic mutation.
What happens when an individual has sickle cell anemia
is that the hemoglobin,
and by the way,
the changes that can happen,
the mutations that happen,
can change a single amino acid,
and if that single amino acid is in the appropriate place,
it will cause the disease.
So why do the cells get sickle shaped?
The reason that the cells get sickle shaped is,
under low concentrations of oxygen,
the hemoglobin inside the cells will actually form a polymer.
Multiple subunits will start joining,
joining, joining, joining together.
Now, normally hemoglobin doesn't do that,
and, in fact, regular hemoglobin,
that is, unmutated hemoglobin,
does not form polymers like that.
But sickle cell, people who have sickle cell anemia
will have their hemoglobin do that,
and the polymers actually cause
the shape of a blood cell to change.
Normal blood cells look like this.
Sickled cells look like this.
Now, I want to emphasize,
if you have sickle cell anemia,
all of your blood cells don't look like this.
They only look like this when the cell encounters
low concentrations of oxygen.
So if you are, for example,
exercising heavily and you have sickle cell anemia,
you will find, people who do that find
that their muscles will get excruciatingly sore.
In some cases, it can be life threatening,
because what's happening is,
the regular blood cells are starting to sickle.
They get in the muscle, in the tissues,
where they're dumping all their oxygen.
They get in the muscles and when these are dumping their oxygen
they're actually in little, tiny capillaries.
Most of the exchange of oxygen occurs
in capillaries of the body.
Normally the rounded blood cells
go through those capillaries very smoothly
like Teflon and don't have any problem.
But when they form sickle cells,
they don't, and they get stuck there.
Not only do they get stuck there,
but they stop the blood flow in that capillary,
which is one of the reasons
that people get this very intense pain,
because their muscle cells are starving for oxygen
and they can't get any because it's all blocked there.
You might wonder why it's called "anemia."
The reason it's called anemia is because our body
has a way of recognizing damaged blood cells,
and when it sees misshapen blood cells
it takes them out of action.
So even though we might be able to get this to revert
to some extent,
and by the way,
I can't tell you that that happens,
but if we might get this to revert to this form,
before that happens our body
can take this out of action and say,
"That's a damaged blood cell.
"I don't want to have that floating around.
"It's going to cause a problem."
So the more your blood sickles,
the more blood cells you lose,
and of course that's exactly what anemia is,
a lowered concentration of blood cells in your body.
People have studied sickle cell anemia for a long time.
Sickle cell anemia actually
has a very interesting historical component.
It was the first disease
that was proposed as a genetic disease,
the first disease proposed as a genetic disease.
Does anybody know who made that proposal?
Student: Linus Pauling.
Kevin Ahern: Linus Pauling made that proposal,
a very cool thing.
So there's an Oregon State connection there, again.
One of the questions people ask
when they see a genetic disease
that persists in the population for a long time,
and there's been evidence that this
has been around for a long time,
is that we think that there must be a reason why it persists.
Why doesn't it just die out?
Why don't people who have sickle cell anemia
eventually have trouble reproducing or don't reproduce as well,
and it would die out of the population?
But sickle cell anemia persists
and it has persisted over human evolution for a long time.
There's a very interesting observation
that people made about the distribution
of the genes in sickle cell anemia.
If you overlay the incidence of sickle cell type
with the prevalence of malaria in the world,
you'll see a disproportionate amount
of sickle cell genes present in locations
where there's very high incidence of malaria.
People have done epidemiological studies and found that,
in fact, there is an advantage for survival
for people in malarial infected areas
to have the heterozygous form of sickle cell anemia,
that is one normal and one mutant.
They have an increased incidence of survival
compared to people, for example,
who have both wild type or both mutant.
So there is, apparently, a genetic basis
for why sickle cell anemia persists in the population.
That's the next-to-last thing I want to leave you with.
The last thing I want to leave you with is,
one of the things that we're interested in
with sickle cell anemia
is what kind of treatments can we offer.
It's a disease that is being investigated very intently.
I've had actually several of my own students
who've gone off to summer internships
working on sickle cell anemia.
One of the interesting treatments
that has been experimented with
and I think is still being experimented with,
is actually trying to get around
the mutant component of hemoglobin,
whether it's the alpha or the beta.
What they do,
this thing keeps popping out on me,
what they do is they treat patients with a drug
that will induce the fetal hemoglobin to start being expressed.
Now, the fetal hemoglobin, of course,
didn't have that mutation.
The fetal hemoglobin is perfectly good,
and so by doing this, they flood the blood
or the blood cells with a normal hemoglobin
and in some cases it appears to help alleviate the disease.
That fetal hemoglobin, of course,
normally stops being made around the time
we're one or two years old.
But with proper drug treatment you can actually induce
it to be made again and for some people that provides relief.
So it's, again, another connection that
we have to one of the hemoglobin genes.
That said, I will take any questions that you might have.
Yes, Shannon?
Student: So I'm not sure if I understand
the correlation between someone having one allele
for sickle cell and their survival in malaria areas.
Is that implying that someone with sickle cell
survives malaria better, or...
Kevin Ahern: So her question is,
does the person who's heterozygous for sickle cell anemia,
are they more resistant to malaria?
And the answer is exactly that.
They are.
Yes, sir?
Student: Does the increased affinity of fetal
hemoglobin affect the person?
Kevin Ahern: That's a really good exam question.
Did you hear what he said?
He said, "Does the presence of that fetal hemoglobin
"change anything for that person?"
What do you guys think?
Describe to me what you think might happen.
Student: They will have a lower net oxygen capacity,
as far as the ability to dump it off.
Kevin Ahern: Yeah.
Student: But their overall capacity,
because the functional gamma units will probably
be increased as compared to anemia.
Kevin Ahern: Yeah.
So they'll have less capacity, basically, is what'll happen,
because, if we think about it,
the more of that fetal we have,
the more hemoglobin is going to be in the R state.
And the R state's really good for binding oxygen,
but it's not so good for giving it up.
But it's probably better than not being able to get any at all.
But you're exactly right.
Maybe we should sing a song to summarize all of this.
Let's do that.
Lyrics: Oh, isn't it great what proteins can do,
especially ones that bind to O2, hemoglobin's moving around.
Inside of the lungs, it picks up the bait,
and changes itself from T to R state.
Hemoglobin's moving around.
The proto-porphyrin system, its iron makes such a scene,
arising when an O2 binds, pulling up on histidine.
The binding occurs cooperatively,
thanks to changes qua-ter-nar-y.
Hemoglobin's moving around.
It exits the lungs, engorged with O2,
in search of a working body tissue.
Hemoglobin's moving around.
The proton concentration is high and has a role,
between the alpha betas it finds imidazole.
Kevin Ahern: That's histidine.
Lyrics: To empty their loads, the globins decree,
"We need to bind 2,3BPG".
Hemoglobin's moving around.
The stage is thus set for grabbing a few cellular dumps of CO2
Hemoglobin's moving around.
And then inside the lungs it discovers oxygen,
and dumps the CO2 off to start all o'er again.
So see how this works,
you better expect to have to describe the Bohr effect.
Hemoglobin's moving around.
Kevin Ahern: Thank you.
Okay, So you better expect to have to describe the Bohr effect.
That's a hint there, right?
We turn our attention from hemoglobin now to enzymes.
Enzymes, of course, are proteins that catalyze reactions.
Hemoglobin, as I said, didn't catalyze any reaction,
but enzymes do.
We're going to spend a fair amount of time thinking
about how enzymes act as catalysts and what they do.
Enzymes are remarkable, and they are remarkable
especially when we compare them to chemical
or other chemical catalysts.
If I have a chemical catalyst
that I use to catalyze a reaction,
it might not be unreasonable
for me to expect a hundred or a thousandfold
enhancement using a chemical catalyst.
When I use an enzyme,
an enzyme can provide
up to 10 to the 17th enhancement.
I believe that's 170 quadrillion.
Now we start to see that enzymes are catalysts,
but they're really incredible catalysts,
absolutely incredible catalysts.
How in the world can something work like that?
Well, let me just give you some,
maybe some things that you can think about
or feel the magnitude of this.
If we look at this top enzyme
we'll actually talk about this next term,
the enzyme has a half-life,
meaning if...
not the enzyme, the reaction that this enzyme
catalyzes has a half-life of 78 million years,
meaning that if we took a mixture of it
and we let it sit in a test tube,
it would take 78 million years for half of it to react.
If I treat this with an enzyme,
I make this enzyme catalyze the conversion of
39 molecules per second per molecule of enzyme.
Now, that's pretty incredible.
The rate enhancement, if you do the math,
corresponds to this 140 quadrillion that I told you about here.
Now, that's mind boggling, okay?
That may sound very rapid, and that is, in fact, very rapid.
But there are other enzymes that are even more
incredible in terms of what they do.
We're going to spend a fair amount of time
talking about this enzyme, right here, carbonic anhydrase.
Carbonic anhydrase does something that, to me,
I can't get my head around.
It only does things by about 7.7 millionfold greater.
That's nowhere near the 140 quadrillion.
But when I look at how many molecules
of product each molecule of enzyme makes per second,
it's mind boggling.
Each molecule, I take one enzyme
one, a single enzyme,
and I put it with its substrate
the substrate's what an enzyme acts on,
and I discover it makes
one million molecules of product per second!
Now, I don't know about you,
but I can't think of something happening that rapidly.
One million molecules of product per second
every enzyme is making.
Imagine that I was running a factory,
and a factory has an assembly line,
and the assembly line is putting products out the end.
I don't care how fast or how many people
you have working in that factory,
you are not going to make a million products per second.
This tells us that the nanoscale world,
"nanoscale" being the level of molecules,
the world that exists at the level of molecules
is very different than the world we know out here.
The nano world is very different than the macro world.
There's no way I can do a million things a second.
No matter how hard I try, I'm not going to do that.
Yes, sir?
Student: As long as it's not high enough to denature them,
would a higher temperature increase these reaction rates,
as in inorganic chemistry?
Kevin Ahern: His question is,
will temperature affect enzymatic rate?
And the answer is, yes, it will, to a point.
You could imagine that if we raise the temperature
we may favor the reaction,
and then we'll actually see it fall off rapidly.
Any ideas why it falls off rapidly?
We denature the enzyme.
Yes, sir?
Student: Is this one million per second only
when the substrate is in excess?
Kevin Ahern: So, yes, and it's a good question.
His question is, does the substrate have to be in excess?
And the answer is, yes, it does.
So you're actually getting a little ahead of me,
but I will address that directly in what
I'm going to say in just a little bit,
but it's a very good question.
So, pretty cool stuff.
This sort of sets the stage for enzymes.
Enzymes are proteins, and what you've seen in a protein
so far is its structure is critical.
Proteins have very, very specific structures,
and, consequently, they have at least fairly specific
molecules that they will bind to and catalyze reactions on.
Notice I said "fairly specific."
Some enzymes are more specific than others.
Some are really rigid,
they only want one thing and that's it.
But enzymes have a specificity.
They don't catalyze a reaction on everything
because they can't bind to everything,
and the reactions that would be catalyzed
would differ from one molecule to another.
This shows a reaction that we're going to spend
a fair amount of time talking on,
and it's a reaction that involves the cleavage
of a peptide bond.
To cleave a peptide bond,
you have to add water across it,
and that adding water causes the bond to split.
It's called proteolysis,
Proteolysis breaks peptide bonds,
and enzymes that catalyze proteolysis are called proteases,
P-R-O-T-E-A-S-E-S, proteases.
We're going to spend some time talking about those,
but before I talk too much about those,
I'll come back to that later,
I want to say a few words about energy.
I'm going to talk about delta G.
You guys have heard the change in Gibbs free energy
in your basic chemistry classes.
I'm going to introduce it here only in very general terms,
and I will tell you right now that you're not,
underline "not,"
going to do delta G calculations on this exam.
Student: Yay!
Kevin Ahern: You will, later.
Students: Ohhh.
Kevin Ahern: But I'm not,
I figure you've got enough for this exam.
They're actually not very relevant for us right now,
except for to understand the beginnings of enzymes.
But later we'll see that they actually will be important.
But on this exam you will not have to do delta G calculations.
But I'm going to say a few words about delta G
because it's relevant for understanding
how enzymes do what they do.
We know that there's a standard Gibbs free energy.
The change in the standard Gibbs free energy
is known as "delta G."
Delta G tells us the direction of a reaction.
If the delta G is negative,
the reaction proceeds forward as it's written.
If the delta G is positive,
the reaction proceeds backward as it's written.
If delta G is equal to zero,
the reaction is at equilibrium.
Equilibrium does not mean equal concentrations
of products and reactants.
Get that in your head, okay?
That's the number one mistake that students make.
They didn't learn what equilibrium meant, back when.
It does not mean equal concentrations
of products and reactants.
It means that they're unchanging over time.
You might have ten times as much of one or than the other.
But it doesn't mean equal concentrations.
There's a related quantity called "delta G zero prime."
Delta G zero prime is called the "standard Gibbs free energy."
The prime is on there for biologists,
like biochemists, because,
for a regular delta G zero that one would calculate,
that would correspond to everything being present
at a concentration of 1 molar.
Well, if we have a reaction that involves protons,
we don't want that being at 1 molar because
it'll kill our enzyme.
It would have a pH of zero.
That would not be good.
So the delta G, the prime on there indicates
that everything is at 1 molar,
except for the protons.
So we've got a pH 7, basically, that we're doing this at.
So what is the standard Gibbs free energy?
The standard Gibbs free energy is the standard
Gibbs free energy change under standard conditions.
That's all it is.
Under standard conditions, that's what it is.
So delta G tells us the Gibbs free energy
change under any conditions.
The delta G zero prime is what corresponds
to standard conditions.
There's a calculation that we're not going to do.
We will do it later.
But just to remind you from your delta G equation,
delta G equals delta G zero prime plus the gas constant R,
times the temperature in Kelvin,
times the natural log of the concentration
of products over the concentration of reactants.
That's a simplification of the actual equation,
but for our purposes that's fine.
At equilibrium, delta G equals zero,
so the delta G zero prime must equal
these two things must be the opposite sign of each other,
so they cancel out.
That's not really essential for us to understand
to understand to understand enzymes.
But we do understand and recognize that delta G
is a very important parameter to understand
for directions of reactions.
It tells us some very important things.
So I want you to keep that in mind
and I'll show you a couple of things here.
Enzymesóno surprise from that first table
I showed youóspeed reactions and
they can speed reactions immensely.
They're very, very important for speeding reactions.
In this case, we're calculating, we're determining
the concentration of product versus time on the x-axis.
We see there's more product being made with time.
When we measure velocities of reactions,
we measure the concentration of product per time.
That's what velocity is.
When we measure the velocity of a car,
we measure distance per time.
When we measure a reaction rate,
we calculate concentration of product per time.
Okay, everybody got that?
So this should be concentration of products going up,
and that's concentration of product over time.
This schematic introduces a sort of unusual delta G,
and we can think of it as an activation energy.
It doesn't really relate to the equation that we had before,
but we can think of the importance of this activation energy.
Activation energy is an energy that has to be put
into a reaction before the reaction will proceed.
It has to be put into the reaction before
the reaction will proceed.
If we look at an uncatalyzed reaction,
we see here is the free energy of the starting materials
and here is the free energy of the product.
The change in the Gibbs free energy is the difference
between this and this,
and we see that this will give us a negative delta G.
This reaction is favorable and it will go forwards.
Now, on the x-axis, we're plotting what's called
"reaction progress," and we're just sort of seeing
how this thing is going, what's happening
to the energy of this reaction over time.
Okay, well, the top line corresponds
to an uncatalyzed reaction.
For an uncatalyzed reaction,
I don't have an enzyme that's helping me out.
I've got a molecule A over here,
and I've got a molecule B over here.
They're in solution, and they're bouncing around
and they're bouncing around.
All of a sudden, they bounce and, if they hit the right way,
they will, in fact, react and give a product.
We could imagine that these two things
bouncing around in here,
if there's only one here and one over here,
the likelihood they'd bounce and hit each other
in the right orientation is low.
If we increase the concentration,
we've got a bunch of these.
The more concentrated it is,
the more likely it's going to go.
If we measure the energy it takes to put these guys together,
when they do work right,
that's what's being plotted here.
It's called the "transition state."
It's also called the "activation energy."
I'll take either one on an exam.
Once we get to that hump,
that reaction can either fall backwards
to where it started from,
or it can fall forwards and go down this hill.
There's our delta G for the reaction.
Now, what happens if I put an enzyme in there?
Well, I'll tell you something very important to remember.
Enzymes do not change the delta G for a reaction.
They do not change.
Notice, the enzymatic reaction starts with
a substrate at the same energy
and it creates a product with the same energy as
the uncatalyzed reaction.
The delta G is exactly the same.
So what did the enzyme do?
Well, the enzyme lowered the activation energy.
It lowered the activation energy,
and it made it much more likely that when two molecules
hit each other they would have enough energy
to make this thing go forward.
Now, we'll see enzymes do some other tricks,
as well, but the number one thing that enzymes
do to speed a reaction is they lower the activation energy.
Now the analogy I like to give for this is,
if I took the class and I said,"Okay.
"I'm going to give you guys extra credit.
"We're going to go out here and we're going to go up
"to this giant steel ball-bearing."
And we're going to go out the door
and Corvallis is at about 250 feet above sea level,
and we're going to push it towards the ocean.
In theory, it should go right over to the ocean,
because 250 feet higher, there's Corvallis, there's the ocean.
Duh, right?
Well, of course, it's not going to go.
Why isn't it going to go?
There is a coastal mountain range between us and them,
between us and the ocean.
There's our activation energy.
So we say,
"Okay, well, we've got a whole bunch of us.
"Let's take, and we want to make sure it gets there,
"so let's go take this ball-bearing
"and we'll push it to Marys Peak."
Marys Peak, of course, is the tallest peak
in the Coastal Range.
It's just to the southwest of Corvallis.
We work and we struggle very hard to get that up there.
I'm assuming there's no trees in the way,
and, given clear cutting, that's not an unreasonable
thing to think, anymore.
[class laughing]
Bad joke, huh?
Bad professor!
We push this ball-bearing to the top of Marys Peak.
And we say, "Well, it's going to have some ups
"and downs along the way, but it's going to have enough
"energy to make it to the ocean."
And it will.
Again, assuming we do enough clear cutting, right?
Okay, well, then the smart person says,
"Wait a minute, this is the dumb way to go.
"We really don't have to go to Marys Peak to make it go.
"All we have to do is make sure we get over
"the highest pass."
As long as we get it to the point of the highest pass,
then it's still going to have enough energy
to get down because all of the other passes will be lower.
The enzyme is helping you to find the pass.
That's what it's doing.
The enzyme has found the pass.
It's found that.
You're not going to have to put as much energy
into getting it all the way up to Marys Peak.
You've only got to get it up to this pass to get it across,
over to the ocean.
So that is my metaphor of the day.
Questions on that?
You guys are looking tired.
Student: It's Friday.
Student: Yeah, it's Friday.
Kevin Ahern: You're still looking tired.
Yes, sir?
Student: Are there enzymes that can bring
that down to where there is no positive activation energy
and just make it spontaneous?
Kevin Ahern: Enzymes will help this reaction go.
The spontaneity of a reaction is really
determined by the delta G.
But the enzymes can lower that significantly.
They can lower the activation energy significantly, yes.
Question over here?
Student: You said earlier that there's molecule A
and molecule B and they bump into each other?
Kevin Ahern: Bang!
Student: Does the enzyme grab A and B?
Kevin Ahern: Oh, good question!
Does the enzyme grab A and B?
In fact, that's one of the other tricks the enzyme does.
It has a specific binding site for A,
it has a specific binding site for B,
and the enzyme is positioning them in exactly
the right way so we don't have to worry about
them hitting the right way and bouncing off.
It positions them in exactly the right way so they bond.
Kevin Ahern: So, what?
Student: [unintelligible] lowering the activation energy?
Kevin Ahern: That would also contribute
to lowering the activation energy.
That's correct.
Yes, Connie?
Student: What usually contributes to that activation energy?
Like, is it the heat of the general surroundings?
Kevin Ahern: What contributes to activation energy?
Well, activation energy is a function of the temperature,
certainly the heat of the surroundings.
It's a function of the concentration.
So those are two variables that can really affect what's there.
There are other things that can play into it, as well.
Concentration is a very important one.
Concentration is very, very important,
because the more concentrated something is
and this was the question that he had over here earlier today
when I want to see a million molecules of product per second,
do I have to have the enzymes saturated with substrate?
You betcha!
That really works well.
So I'm going to get to that in just a second.
But before I do that, why don't we stand up and stretch?
Just stand up and stretch.
You guys will get some oxygen in your system.
[indistinct talking]
Now you look alive, alert, refreshed, and ready for more,
yes, sir, more biochemistry.
Kevin Ahern: What's that?
Student: [unintelligible]
Kevin Ahern: Oh, yeah, it is distracting.
I wish I could just make this thing go away,
but it's part of the security system
and they will not let us fire that thing.
But whenever you see it,
let me know and I'll be happy to turn it off, yes.
About every hour it starts up.
Let's think about concentration.
This is a plot that you're going to see a lot of.
It's called a "velocity-versus-substrate concentration plot."
It's also called a V-versus-S plot.
So I want to introduce it to you and describe
to you what it tells us.
What does this plot tell us?
This plot tells usóI think the batteries
in this thing are just going badólet's imagine that,
instead of having an enzymatic reaction,
I have this factory that I'd talked about.
The factory is full of workers and the factory is try to make,
let's say, automobiles.
To make an automobile, you have to have a lot of materials,
they have to be assembled,
they have to be stuck together.
This group of workers in the factory assemble
the automobile from the parts, and that's their job every day.
If they have very, very few parts, or the parts come in very,
very slowly, what happens to their ability to make automobiles?
Well, it's going to go down.
They're not going to make automobiles so fast.
They're going to be spending a fair amount
of their time waiting on parts, waiting on parts,
waiting on parts, right?
Their velocity is going to be low when their amount
of raw materials is low.
As they get more and more raw materials,
they start making more and more cars,
and we see that go up fairly rapidly, at least at first.
And now, all of a sudden, the factory is starting
to get more and more raw materials,
and the employees are going,
"Oh, wow, I'm going as fast as I can, going as fast as I can.
"Whoa, there's more!
"I'm going to go as fast as I can!"
Eventually, we get to a point where the employees,
no matter how hard they work,
they can't work any faster.
They reach a maximum velocity of making cars.
In this case, the enzyme is reaching
a maximum velocity of making product, exactly the same thing.
Not surprisingly, enzymes have a maximum.
Now, if I take my automobiles, I take my factory, and I say,
"Whoa, this factory is working at maximum output
"of cars that it can get."
This is 400 cars a day.
But I can sell 1,000 cars a day.
Well, it doesn't matter how much more I pour into
that factory in terms of raw materials,
the employees can only do so much, right?
So what do I decide to do?
Well, the smart thing to do would be
to build another factory, right?
If I build another factory identical to the first one,
with the identical abilities of the workers to work,
instead of having 400 cars per day,
my two factories can make 800 cars per day.
You with me?
Now, I'm illustrating a point about factories
to you that's important to understand when
understanding how enzymes work.
The maximum velocity an enzyme can work at is called Vmax,
velocity maximum, Vmax.
Vmax is reached when an enzyme is saturated with substrate.
Substrate is the stuff that the enzyme works on.
It doesn't matter if I keep increasing the substrate anymore.
I'm not going to get any more product made per time.
Just like the factory, I'm working as hard as I can do.
I can't put out more.
But if I add twice as much enzyme,
what's going to happen to Vmax?
It's going to double, right?
I double the enzyme, I'm going to double the velocity.
That tells us something very important.
Vmax is an interesting quantity,
but it's not a characteristic of an enzyme,
because Vmax depends on how much enzyme I use in my reaction.
If I use twice as much enzyme,
I'm going to get twice as much Vmax.
That make sense, no, yes?
Student: Well, so, in theory, proportionally speaking,
it still could be a characteristic quantity, then, right?
It could be unique to an enzyme in terms of...
Kevin Ahern: Vmax is not a characteristic of an enzyme.
No, okay?
Vmax is not a characteristic.
And you will talk to many people who don't know that.
They'll go, "Oh, yeah, the Vmax of this enzyme is
blah, blah, blah."
And I'd say,
"And how much enzyme did you use?"
Now, Shannon is sort of thinking ahead.
There's gotta be something characteristic
about the enzyme that's here.
What could it be?
Well, I told you that the Vmax depended upon
the concentration of the enzyme.
I double the enzyme, I double the velocity.
What if I took Vmax and I divided it
by the concentration of the enzyme?
Now I've taken the concentration of the enzyme
out of the equation.
What do I get?
I get something called "Kcat,"
K, with a lowercase c-a-t subscript.
Kcat is equal to Vmax divided by the concentration of the enzyme.
Now I've gotten rid of the concentration out of the equation,
and I get something that's really interesting.
It's called Kcat, "Cat," c-a-t, yes.
It's also called "turnover number."
And you've already seen Kcat.
That very first table I showed you that had those big numbers?
When I said that carbonic anhydrase had made a million
molecules of product per molecule of enzyme per time?
That's Kcat.
So Kcat is a measure of the number of molecules
of product an enzyme makes per time.
A Kcat of one million means it makes a million molecules
of product per molecule of enzyme, per second, in this case.
That is a characteristic of an enzyme.
We can compare Kcats between two enzymes.
We can't compare Vmaxes between two enzymes.
Everybody got that?
If you get that, you will know as much as many
people know about enzymes, and I'm very pleased,
in this class, that students usually take
that message away with them,
and I think it's a very important message.
Kcat is a characteristic of an enzyme.
Vmax is not.
It depends on how much enzyme I use.
So if I want to compare two enzymes,
I've got to compare their Kcats, not their Vmaxes.
That's a lot of stuff for one day.
Let's finish a few minutes early.
I have a new clock to celebrate, over here.
It tells me I'm finishing early.
So let's do that and I will see you guys on Monday.
So, is that where you were headed?
Student: Yeah.
Kevin Ahern: I figured it was.
Student: I guess it must remind me of something.
So on Monday I have to miss class.
Kevin Ahern: Okay.
I'll give a pop quiz.
Student: Oh, okay.
Well, of course, I'll stay on top of the reading and so forth.
Is there anything else I ought to do?
Kevin Ahern: You'll be fine, you'll be fine.
Kevin Ahern: Take care, Shannon.
I've have nobody to pick on in the front row.
Hi, how're you doing?
Student: I have a question for you.
Kevin Ahern: Yes, sir.
Come on back.