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>>One day closer to the exam.

One day closer to showing me
off how much you've learned.
That's good, right?
Nobody likes that one.
I have decided
that we will finish
the material for this exam
with where I finish here,
talking about
noncompetitive inhibition.
So very early in today's
lecture, that'll stop.
And I'll mark that on
the highlights, as well.
So you'll see, in
the highlights,
where the material stops.
I have also scheduled
a review session.
And that will occur tomorrow
evening at 7:00 PM in ALS 4001.
So ALS 4001.
I will videotape
that and, barring
any technical difficulties,
get that posted as quickly
as I can.
Usually that would be posted by,
certainly– what is tomorrow?
It's Wednesday.
Sometime on Thursday.
I might even get it posted
by sometime tomorrow night.
We'll see.
OK?
All right, any questions?
Concerns?
Thoughts?
Anything?
No?
OK.
Well, let's finish up
talking about the inhibition
of enzymes.
And yesterday I talked
about competitive.
And so just to remind
you a little bit
about these different
types of inhibitions,
let me just step you through
it, as I promised I would do.
So competitive inhibition
occurs when a molecule
resembles a substrate
and competes
with the substrate for the
binding site on an enzyme.
So competitive
inhibition, the inhibitor
will resemble the
substrate, and it
will compete with the substrate
for binding to the active site
on the enzyme, all right?
They both are trying to
get to the same place.
And so as I talked
about yesterday,
if you have a lot of
substrate, then the substrate
is going to win
that competition.
The substrate is going to bind.
If I have 50 million
molecules of substrate
and I have 500
molecules of inhibitor,
the likelihood that the
substrate is going to get there
is very, very high.
The likelihood that the
inhibitor is going to get there
is very, very low.
OK?
As a consequence of that, we
see that the maximum velocity
for a comparatively-inhibited
reaction does not change.
Because it means that
we can effectively
outcompete the inhibitor.
If we add enough substrate, we
will outcompete the inhibitor.
And the inhibitor
will, therefore, not
be able to do its thing.
That's not the case with
noncompetitive inhibition.
That is not the case with
noncompetitive inhibition.
With noncompetitive
inhibition, what we see
is that there is no competition.
It doesn't matter how much
substrate I have, I add.
I'm always going to have a
fixed percentage of the enzyme
that is going to be inhibited.
That percentage
was changing when
I had a competitive inhibitor,
because the more substrate
I added, the less the
effect the inhibitor had.
So for noncompetitive
inhibition,
I've got a fixed
percentage of enzyme
that's going to be inhibited.
Doesn't matter how
much substrate I add.
Does that make sense?
So let's say I have 50,000
molecules of enzyme.
And I add 20,000
molecules of inhibitor.

Effectively, that
means that every time,
every reaction I do,
20,000 molecules of enzyme
are going to be inhibited.
I'm only going to have
30,000 molecules of enzyme
in every reaction.
Right?
Doesn't matter how
much substrate I had.
Every reaction is going to
have only 30,000 molecules
of enzyme.
All right?
Everybody buy that?
What happens if I reduce
the amount of enzyme
that I'm using in a reaction?
What happens to Vmax?
Does it stay the same,
or does it go down?
It goes down, because
Vmax is a function
of how much enzyme I've got.
Remember that?
So that's what a noncompetitive
inhibitor is doing.
It's effectively reducing
the amount of enzyme
I have in a reaction.
Therefore, Vmax for a
noncompetitive inhibitor
decreases.
Vmax for a noncompetitive
inhibitor decreases.

That's not the case for
a competitive inhibitor.
Competitive inhibitor,
Vmax stays the same.
OK?
Now, the last point
I'm going to tell you,
I will explain to you later if
you would like to understand.
But a lot of
students just decide
they want to memorize
it, and that's fine, too.
Because it's a little
bit hard to understand.
noncompetitive inhibition,
the Km does not change.

The Km does not change for
noncompetitive inhibition.
Now, as I said, I'm not
going to explain that here.
It's not complicated,
but it's more time
than I want to take here,
and it's not a major point.
And not a major point, in
terms of explaining how.
So we've seen two
types of inhibition.
I'm going to
summarize them here.
Competitive inhibition,
we saw no change in Vmax,
but we saw an increase in Km.
That's what happened with a
competitive inhibitor, right?
With a noncompetitive inhibitor,
we saw exactly the opposite.
We saw of Vmax decrease, and
we saw Km did not change.

Very different effects, these
two types of inhibitors have.

OK, questions about that?

None?
Yes?
>>Sorry, I was just [INAUDIBLE].
>>You're just like
me. [LAUGHTER]

>>[INAUDIBLE]
So competitive, the Vmax doesn't
change, but the Km increases?
>>Yes.
>>OK, got it.
>>So for competitive,
Vmax does not change,
but the Km increases.
For noncompetitive,
the Vmax decreases,
but the Km stays the same.

All right folks, that's
the end of the material
for the first exam.
Woo!
Celebrate, right?
OK.
Excellent.
What's that?
That's actually what
the Lineweaver-Burk plot
looks like.
Maybe I should explained
that Lineweaver-Burk to you.
Students get confused by
about a Lineweaver-Burk plot
for noncompetitive.
Here's the uninhibited
reaction right here.
Here's the
competitively inhibited.
You'll notice they both
have the same Km over here,
the same minus 1 over Km, which
means they have the same Km.
Why is it the top line
instead of the bottom line?

Why is the noncompetitive
inhibition the top line?

Anybody know?
What's the y-intercept?
What's the value
of the y-intercept?
>>[INAUDIBLE]
>>It's 1 over Vmax, and it's
because it's the reciprocal.
That's right.
So you take the reciprocal
of a small number,
you get a larger number.
You take the reciprocal
of the large number,
you get a smaller number.
So Vmax goes down, but 1
over a decreasing number
means an increasing
value on the y-axis.
OK, so that's the end of
the material for Exam 1.
OK.
Well, I told you that Exam
1 was pretty calculation,
blah, blah, with that.
So hopefully you won't get too
snowed by the calculations.
If you're having difficulty
understanding the calculations,
come see me.
I've had a couple of
you already do that.
I'm more than happy
to work with you
and to help you to
better understand
how the calculations are done.
All right.
So for the new
material here, I want
to talk about the
control of enzyme action.
And the control of enzyme
action is very important just
like controlling that Maserati
going to WinCo is important.
If we don't control it
with the speed limit,
we're going to probably run
over some things on the way.
And if we don't
control enzymes, we're
probably going to
break or make something
that we don't want to
break or make, right?
Because they work
so fast, we've got
to be able to have some
controls on them so they
don't go overboard.
If they start going
overboard, we're
going to have some troubles.
And we're going to see that
cells have some very, very
good controls and some
very clever controls
on enzyme action.
So we're going
spend a little bit
of time talking about those.
One of those, I've
already talked about.
Let me show you something that
will just kind of go, wow,
all right?
I talked about
ATCase the other day.
I said that it was an example
of an allosteric enzyme.
And ATCase catalyzes
this reaction
that you can see
going over here.
I don't care if you
know that reaction.
Doesn't matter, OK?
I do care that you know
something about that reaction,
all right?
So let me give you
some background.
ATCase catalyzes
the first reaction
in the synthesis of CTP.
CTP is a nucleotide.
It catalyzes the first reaction
in the synthesis of CTP.
Now you'll notice I
said the first reaction.
When we make nucleotides, we
start with very simple things,
and it takes a series of
reactions– it actually
takes 10 reactions– to get
the final product, which
in this case is CTP.
There's an extra C
in there, isn't it?
Cytidine triphosphate, I
guess, whatever that says.
All right.
Anyway, it takes a series
of reactions, 10 reactions,
to get here.
Why do I tell you that?
Well, it turns out that ATCase
is a really interesting enzyme.
There are several things
that can bind to it
that affect ATCase's activity.
One of these is the end
product of the pathway.

I'm going to repeat that.
One of the things that can
bind to ATCase and affect it
is the end product
of the pathway.
The end product of
the pathway is CTP.

Now I'm going to show you, in a
minute, why that's significant,
all right?
But for the moment, you can just
say, OK, ATCase binds to CTP.
And when it binds to CTP,
the enzyme is inhibited.

OK?
ATCase binds to CTP, and
when it binds to CTP,
the enzyme is inhibited.
Now, this turns out to be–
it's a very simple fact.
You can sit.
You can memorize that fact.
But the really cool
thing about that fact
is the fact that CTP is the
end product of the pathway.

Every reaction in this
series of reactions,
is catalyzed by a
specific enzyme.
Meaning that there are 10
enzymatic reactions that
occur in going from here,
at the top, down here to CTP
at the bottom.

If the cell were a total
control freak– and by the way,
cells do tend to be
control freaks, OK?
If the cell were a total
control freak, what would happen
is the cell would have a
control on every single enzyme
in that pathway.
And if you think about it,
that gets kind of complicated.
I've gotta control
Enzyme number 1,
I've got to control
Enzyme number 2,
I've got to control Enzyme
number 3, et cetera, et cetera.
And I have to control
the last enzyme, as well.

What if I just
controlled the first one?
If I just controlled
the first enzyme
and I saw the first
enzyme got stopped,
wouldn't that mean that there
wouldn't be any product?
So that the next reaction
wouldn't have anything
to work with, and the next
enzyme wouldn't have anything
to work with?
By shutting off the
spigot, I shut off the lawn
from being watered, right?
I'm turning off the spigot.
And the spigot is right here.
The cell can control one enzyme
and control an entire pathway.
That's a very powerful concept,
a very, very powerful thing.
It makes for
simplicity, but it also
allows for some
pretty cool stuff.
Why is this important?
Well, if we think
about CTP building
up, if the cell is
making too much CTP,
there might be consequences.
And it turns out that there are
two consequences if the cell
doesn't control this pathway.
One of the consequences
is whenever
you have an imbalance
of nucleotides,
you make mutation
much more likely.

So if I get too
much CTP, the cell
is going to become a mutant
zombie from hell, OK?
We don't want cells to be
mutant zombies from hell,
because they become
known as tumors, right?
The other reason that we don't
want this pathway going out
of control is this pathway
requires a lot of energy.
The cell is going
to waste energy
if it doesn't shut
this pathway off when
it's got plenty of CTP.
Well, the beauty here is that
this pathway shuts itself off
when the CTP concentration
gets too high.
It binds to the first
enzyme in the pathway
and shuts down
the whole pathway.
There's a beautiful
balance here.
This phenomenon I've just
described to you has a name.
It's called feedback inhibition.
Feedback inhibition.

What's happening
is the end product
of the pathway is feeding
back and turning off
the first enzyme in
the same pathway.

Perfect balance.
Too much CTP?
Pathway turned off.
Too little CTP?
Pathway turned on.
Now, this enzyme, I said, was
a really interesting enzyme.
I And this is one
of three reasons
that this is an
interesting enzyme.
There are three things
that control this enzyme.
CTP is one of them.
The second one is
another nucleotide, OK?
Another nucleotide can
bind to this enzyme
and affect this
enzyme's activity.
The other nucleotide is ATP.
Right?

Now, we're going to figure
out, in a minute, if it
turns the enzyme on or
it turns the enzyme off.
Let's think about DNA and RNA.
Everybody learns in high school
that A pairs with T and G
pairs with C, correct?
Does everybody remember
how the nucleotides
are categorized in terms
of purines and pyrimidines?

Purines are what?
A and G, OK?
And the pyrimidines are
C and U and T, right?
So CTP is a
pyrimidine nucleotide.
Pyrimidine nucleotides,
in nucleic acids,
are complementary to
purine nucleotides.
That's why T pairs with
A, or U pairs with A,
and C pairs with G, right?
OK?
If we have too
little pyrimidine,
that means we have
too much purine.
Because the comparison
of the two–
if we have too
little of something,
we don't have enough, we
have too much of the other.
All right?
If we have too much purine
and not enough pyrimidine,
we would want to turn on
pyrimidine synthesis, correct?
So you tell me.
What does ATP do?

It turns on this enzyme.
ATP turns on this enzyme.
Now, ATP is a purine.
It's used to make nucleic acids.
But ATP, we also
remember, is important
because it's a measure
of the cell's energy.
The more energy a cell has,
the more ATP it will have.
Now, energy turns out to
be really, really important
for cells.
The reason it's
important for cells
is because cells need it to
do all the things that they're
going to do, including division.
Cell division, if
you think about it,
is a very
energy-requiring process.
You have to completely
duplicate the chromosomes.
You have to go through a pretty
elaborate process in order
for this to happen,
and that's going
to require a lot of energy.
It's also going to require
a lot of nucleotides.

When cells have a lot of
ATP, look what they're doing.
They're turning on the
synthesis of pyrimidine genes.
They're getting ready
to divide, folks.
Pretty cool, OK?
We see– in fact,
we'll talk later
in the class about
nucleotide metabolism.
But we see great
balance of nucleotides,
because nucleotides
are intimately
tied to every cellular process.
ATP is a very common
energetic one,
but we'll see GTP is
also involved in energy.
We'll see CTP is
involved in the synthesis
of glycerophospholipids.
You don't need to memorize this.
And you'll see UTP is involved
in the synthesis of glycogen.
All of the nucleotides have some
cellular role, besides being
in RNA, for example.
OK, any questions
about what I've just
been talking about here?
Yes?
>>You said that ATP
turns on [INAUDIBLE]?
>>ATP turns on ATCase.
>>[INAUDIBLE]?

>>So ATP is a purine.
And so if I have
a lot of purines
but not enough pyrimidines, I
want to make more pyrimidines
to balance, right?
So that's why turning
that on makes good sense.
On the other hand, if I
have too many pyrimidines,
they turn themselves off.

You look puzzled.
>>It just needs to sit there.
>>It needs to sit
there. [CHUCKLES] OK.
Well, think about that.
If you have questions,
let me know.
How's that?
Other questions on that?

OK.
Now, I said there are three
things that– oh, here's
a good representation.
This shows the entire pathway.
There's actually–
I think it's 10.
It depends on where
you start counting.
But there are several
reactions here
that are important for
the cell to control,
because they're
going to waste energy
if they're running reactions
they don't want to run.
They're going to favor
mutation if they make too
many pyrimidine nucleotides.
Now, the third thing
that affects this enzyme
is actually the
substrate, aspartate.

All right?
So you see that aspartate
is one of the substrates.
And by the way, remember now
that CTP is not the product
of the enzymatic reaction.
It's the product of the pathway.
OK?
The product of the
enzymatic reaction
is this mouthful guy over here.
A common mistake I see
students make on their exams
is they want to tell me
that CTP is something
that the enzyme makes,
and it doesn't, OK?
The pathway makes CTP.
All right, but anyway, this
guy right here, aspartic acid,
is also something that
affects the enzyme's activity.
It turns out that aspartic
acid turns the enzyme on.

Aseptic acid turns
the enzyme on.
What do we use
aspartic acid for?
What do we make
with aspartic acid?
What's that?
>>[INAUDIBLE]
I'm sorry.
I still didn't hear you.
>>Artificial sugar?
>>Artificial sugar?
Well, actually, that's
true, aspartic acid
and phenylalanine.
But in terms of our
cells, what are our cells
making with aspartic acid?
Proteins, right?
It's one of the amino acids.
A cell has got to divide.
A cell wants to have
enough energy before it
starts the division process.
Do you suppose a cell would
want to have enough amino acids
before the division process?
Absolutely.
And how does it measure those?
With one of the amino
acids right here.
If a cell has plenty of
amino acids, one of the signs
will be that it has a
good amount of aspartate.

And aspartate is
telling the enzyme,
hey, guys, let's make some
pyrimidine nucleotides,
because we're going to have fun.
We're going to go divide.
Happy hour, right?
Make sense?
OK.
All right, so aspartate
turns ATCase on.
CTP turns it off.
ATP turns it on.
OK.
Three things.
Really remarkable enzyme.
I showed you earlier that
ATCase has a sigmoidal plot.
And we talked about
the significance
of that sigmoidal plot.
The significance was that
this meant, first of all,
that binding of one substrate
was affecting another, just
like hemoglobin.
And that meant that we had
to have multiple subunits.
All right?
ATCase is pretty remarkable.
It has 12 subunits.

12 subunits.

Six of the subunits
catalyze the reaction.
The other six subunits
bind to either CTP or ATP.
So we break the subunits
into two categories–
catalytic subunits, that's
the first six that catalyze
the reactions, and
regulatory subunits, which
are the ones that
control the enzyme.

ATP and CTP bind to the
regulatory subunits.
Aspartate binds to
the catalytic subunit.

Now when I talk about turning an
enzyme on and turning an enzyme
off, we have a parallel,
another parallel
that we had with hemoglobin.
With hemoglobin, we talked
about two different states.
What were the two
states of hemoglobin,
in terms of structure?
R and T.
We see the same
thing with enzymes.
Enzymes that are more active
or in the R-state, and enzymes
that are less active
are in the T-state.
Binding of ATP or aspartate will
put ATCase into the R-state.

Binding of CTP by ATCase
will put it into the T-state.

Yes?
Say it one more time.
Yeah, a lot of names
and everything.
I'll stop here in a second, OK?
So binding of ATP or
aspartate puts the enzyme
into the R-state.

Binding of CTP puts the
enzyme into the T-state.

You guys look like
you're needing a joke.
A joke?

OK, let's see.
OK.
This is a joke that
a student told me,
and I kind of like this joke.
It's kind of cute, OK?
It's the crunch bird joke.
How many of you have heard
the crunch bird joke?
Be honest.
OK.
So there's this lady, right?
And this lady decides that
it's her husband's birthday,
and she wants to go get him
something cool and exotic
for his birthday.
And so she goes down
to the pet store,
and she tells the pet
store owner, she says,
"I want to buy something
for my husband's birthday,
but I don't want the usual gift.
I want something different."
And he says, "Piece of cake.
I got it for you."
He goes back, and he comes out,
and he walks out– on a leash,
he has this iguana,
about 6 feet long.
Giant lizard-like thing, right?
And she looks at
it and says, "Oh, I
don't think I want that
around the house, you know?
He says, "OK, no problem."
He takes the iguana back.
He comes out a bit later, and
he's wrestling this giant boa
constrictor.
Puts it down.
Everybody's going, oh.
And she says, "I
don't think I want
that around the house
either, you know?
"OK, ma'am."
So he goes back, and
he begins to think he's
a shoe salesman here, right?
And so he picks up another
animal and brings it out. "No,"
same story. "No," same story.
This goes on and on and on.
He's just about
running out of animals.
And he says, "I
know what you want.
You want a crunch bird."
And she says, "What's
a crunch bird?"
And he says, "Look over
there in the corner."
She looks over the corner, and
there's this little tiny bird
sitting on a perch.
And this little bird
is sitting there,
and he says, "Watch this."
He says, "Crunch bird."
He says, "Chair."
The bird jumps up
off of this perch
and flies over to
this wooden chair
and demolishes the chair
in front of her eyes.
It's sawdust in just a
very short period of time.
She says, "That's incredible!"
He says, "That's nothing.
Crunch bird– desk."
And there's this great big
wooden desk, and this bird
flies over, and the same thing.
And she's going, "That's
absolutely incredible!"
She says, "I've
got to have that!"
It's the exotic thing that
she wants for her husband.
So she grabs the bird, pays for
the bird, and takes it home.
And she walks in and says,
"Honey, I got something
for your birthday."
He goes, "Oh."
He said, "What is it?"
She says, "It's a crunch bird."
He looks at this little
thing on her finger
and he says, "Crunch
bird, my ass."
[LAUGHTER]

You get the idea.
[LAUGHTER]

OK.

OK.
Everybody awake now?

There's the exciting
structure ATCase.

At this point, who cares, right?
You got six regulatory subunits.
You've got six
catalytic subunits.
And that's what I've
already told you.
OK.

Let's think about this.
Tell you what.
Let's not think about
that, all right?

Trying to think of
a way that I can
make this– one of
the things I find
is when I teach this part
of the thing, students
frequently go, "Oh, man.
It's so hard to understand."
I'm trying to think
of a different way
that I can make you understand.
All right, so let's try
a little experiment here.

We talked about how
hemoglobin bound one oxygen,
and that favored the binding of
a second oxygen, and a third,
and a fourth, right?
And we call that phenomenon
cooperativity, right?
The binding of one favored
the binding of others, right?

When we think about an
allosterically-controlled
enzyme– and we've seen a lot
of parallels to hemoglobin–
we can think of a
same sort of thing.
The binding of one may favor the
binding of another, et cetera,
et cetera.
But we also now have to account
for inhibition, as well.

The binding of one might inhibit
further binding, et cetera,
et cetera.
And in fact, that does happen.
Because what's happening is
the binding of an inhibitor
converts the enzyme
into the T-state,
and the T-state was
just like hemoglobin.
In the T-state, hemoglobin
didn't bind oxygen very well.
In the enzyme, the
T-state doesn't bind
substrate very well.

The question, then, is how do we
explain this process happening?
And there's really two
ways of explaining.
One is what I will call
a cause and effect way,
and the other is, magic happens.
I'm trying something
different here.
Cause and effect
versus magic happening.
Cause and effect is
really easy to understand.
Cause and effect says,
I've got an enzyme
that's got multiple subunits.
Binding of one favors the
binding on the second,
favors the binding on the
third, et cetera, et cetera.
And so the first subunit causes
the second subunit to change
structure so that it more
favorably binds a k-i-n= one,
which causes the third subunit
to change structure so that it
more favorably binds something.
And that's the cause and effect.
The first one causes
everything else to happen.
Does that make sense?
Cause and effect is
fairly easy to see.
It's fairly easy
to conceptualize.
OK, so that's what happens
with cause and effect.
And that's what we call the
sequential model of catalysis.
It's cause and effect.
It's sequential.
Binding of the first favors
the second, favors the third,
favors the fourth,
favors the fifth,
favors the sixth,
blah, blah, blah.
Everybody with me?
That's one way of explaining
how these processes occur.
Now the magic happens.
If I call it something
magic, what does that
conjure up in your mind?

I don't know.
I'm just sitting here.
All of a sudden, there was
this rabbit in this box, right?
Poof!
There's a rabbit.
That's magic.
We don't ask how the
rabbit got in the box,
because we say
it's magic, right?
Everybody with me?

The other model of catalysis
is called the concerted model.
This is the magic model.
The magic model says
that enzymes flip,
all by themselves, from
R to T or from T to R.
Doesn't take a cause and effect.
In the case of the first thing
I told you, the binding of one
favored the binding of another.
Or the binding of one might
inhibit the binding of another.
We're going from T to R or
we're going from R to T.
We can go either
direction, depending
upon whether we're binding
an inhibitor or an activator,
whether we're binding
ATP or CTP, right?
The magic model says, I
don't know how it happens,
but the enzyme flips
from R to T on its own.
And then when it bind
the appropriate molecule,
it gets locked in that state.

So let's imagine
I've got ATCase.
It flips from the
R to the T-state.

If, when it's in the
T-state, it binds to CTP,
it gets locked in the T-state.

I might have ATCase that
flips from the T to the R,
and if it binds to ATP, it
gets locked in the R-state.
With me?
The magic is in the flipping.
T to R, we don't ask how
it happens, or R to T,
we don't care how it happens,
in the concerted model.
In the sequential model,
we say R to T or T to R
is caused by something, right?
Cause and effect.
The cause is the
binding of something.
If I bind ATP, it causes
it to go into the R-state.
If I bind CTP, it causes
it to go into the T-state.
The magic happens says R
and T happen on their own.
ATP locks in the R-state.
CTP locks in the T-state.
Make sense?
If you guys buy that,
that's really good.
Any questions about that?
Yes?
>>So ATP locks it in
the R-state and CTP–
>>Locks in the T-state.
That's right.
ATP locks in the R-state.
CTP locks it in the T-state.

So that's two ways of explaining
activation and inactivation
of an enzyme.

OK.
That's all the blah, blah, blah,
that you see on the screen.
All right.
Well, allosteric
control– oh, yes.
Question?
>>[INAUDIBLE]

>>Two different ways of
explaining a phenomenon.
And it turns out that enzymes
tend not to have only one.
They actually have a
sort of a blend of them.
But those two different ways
that I've described to you
are two different ways
of explaining activation
and inactivation of an enzyme.
OK?
Good.
Allosteric mechanisms–
and by the way,
when I say allosteric,
allosteric,
I'm going to give you
a definition here.
If there's one definition I ask
on an exam, more than almost
any other definition,
it's this one right here.
What does it mean to say
an enzyme is allosteric?

An allosteric enzyme
has its activity
affected by the binding
of a small molecule.

An allosteric
enzyme is an enzyme
that has its activity
affected by the binding
of a small molecule.

Depending on the molecule,
that might activate it.
It might inactivate it.
But an allosteric
enzyme is an enzyme
whose activity is
affected by the binding
of a small molecule.
Everybody with me?
It's a good definition to know.
Well, allosteric control is only
one way of controlling enzymes.
I'm going to talk
about some others.
Allosteric activity is only one
way of controlling an enzyme.
All right?
Another way to control enzymes
is to covalently modify them–
that is, to cause some chemistry
to happen on the enzyme.

Now, there are several
ways of doing this.
Several ways of doing this.
I'm going to talk
about two, all right?
The most common of these is
the first one you see listed,
phosphorylation.

The name tells you what it does.
Phospho- part telling you that
there's a phosphate involved.
The -ylation part means
it's getting put on.
So phosphorylation involves
putting on of phosphates.

The reverse of that
involves dephosphorylation,
which is the taking
off of phosphates.

Now we're going to
see what effect that
has in just a minute.

First of all, what does
phosphorylation mean?
I said it means
putting a phosphate on.
It turns out that there
are three amino acids that
commonly get phosphorylated,
meaning they gain a phosphate.
One is the side chain of serine.
Here's a serine residue.
Serine has a hydroxyl group.
In fact, all three that
get commonly phosphorylated
have hydroxyls.

The OH gets a
phosphate put onto it.

Look at the charge
on that phosphate.

Serine didn't have a charge.
The side chain
didn't have a charge.
Now, when we put
a phosphate on it,
it's got one, two
negative charges there.
We've changed the charge
of a part of a protein.
What happens when we
change charge in a protein?
We may change the shape.
And if we change the shape,
we may change the way
in which the protein functions.

Phosphorylation, for some
proteins, turns them on.

Phosphorylation, for other
proteins, turns them off.
It depends on the protein.
We'll talk about
different examples.

You should know the
name of the enzymes that
catalyze phosphorylation.

The name we give to enzymes
that catalyze addition
of a phosphate is kinases.
K-I-N-A-S-E-S. Kinases.
You're going to hear a lot
more about kinases in the class
as we go along.

So this reaction that
you see on the screen
would be catalyzed by a kinase.
And this reaction
is very common.
You'll see that here's
phosphate on ATP,
and ATP is donating not only
a phosphate, but also energy
for this reaction to occur.

OK?
Here's another amino acid
that gains a phosphate,
and it's threonine.
Threonine also has
a hydroxyl group.
And you can see a
kinase does that,
and we see the same
result. The oxygen
gets a phosphate put onto it.
We're going to
change the charge.
And when we change
the charge, we
are going to likely change
the way the protein acts.

Serine and threonine are two.
The third amino acid that
gains a hydroxyl is tyrosine.

Serine and threonine
are chemically similar.
Tyrosine is quite
a bit different.

But again, we see the
hydroxyl gets the phosphate.
Same thing as we saw before.
Change the charge.
May change the structure and
function of that protein.
Kinases catalyze all
of those reactions.
OK?
Well, I said earlier that
cells are control freaks.
Control freaks want to control
everything about something,
right?
So if cells have a way
of putting phosphates
onto something, they
also want to have
a way of taking phosphates
off of something.
In fact, they need to.
Let's say that we put
phosphate onto an enzyme
and it activated that enzyme.
And remember, were trying
to control enzymes here.
We said there may be times
the enzyme will be active
and we don't want
it to be active.
If we're going to have
a way to activate it,
we want to have a way to
inactivate it as well.

There's another group of
enzymes that remove phosphates
from things.
Those enzymes have
their own name.
They're called phosphatases.
P-H-O-S-P-H-A-T-A-S-E-S.
Phosphatases.

So a phosphatase would
remove the phosphate off
of this tyrosine and
leave a hydroxyl.
In other words, just making
the reaction go backwards.

Everybody with me?
Now, one of the things that
we will see before too long
is that there's a
process that cells
have for talking to each other.
Cells actually
talk to each other,
and they communicate
information to each other.
And one of the ways
that they do that is
by putting phosphates
on or taking
phosphates off of
certain proteins.

Putting phosphates on
or taking phosphates
off of certain proteins, that
process is called signaling.
And signaling is really just
cells talking to each other.
That's really all it is.
OK?

OK.
Well, let's see an example
of an enzyme that is affected
by addition of a phosphate.
Now, this looks
pretty complicated,
and I'm going to
simplify it for you, OK?
What you see on the
screen is an enzyme known
as glycogen phosphorylase.

Glycogen phosphorylase.
It catalyzes the
breakdown of glycogen.
This enzyme is
regulated allosterically
and by covalent modification.
It has both regulatory
mechanisms that affect it.

I'll repeat that.
It's regulated allosterically
and by covalent modification.
And by the way, I should say
that putting phosphate on
is a covalent modification,
and so is taking phosphate off.
Any time I'm affecting
a chemical bond,
it's a covalent modification.
So putting a phosphate
on and taking it off
are both covalent modifications.

This enzyme is pretty cool.
It breaks down
glycogen really fast.
And glycogen is a
polymer of glucose.
We have a lot of
glycogen in our liver.
We have a lot of glycogen
in our muscle cells.
When you break down
glycogen, you get glucose,
and glucose is what we use to
make energy for our muscles.
So having glycogen in
our muscles is important.
And being able to activate
that enzyme that breaks it
down quickly is important.
We've all heard stories
about the parent that
sees the child in
danger underneath a car,
and their terror level goes
up, and they grab the car
and they lift the car up.
You've heard these
stories, right?
We said they've had adrenaline.
The stories are true.
They can do that.
They can do that.
And they can do that because
what's happened is– adrenaline
has another name, and it's a
hormone we call epinephrine.

Epinephrine is a
way of signaling.

It comes from our
adrenal glands.

It's released into
our bloodstream,
it goes to our liver cells,
it goes to our muscle cells,
and it favors the
phosphorylation
of glycogen phophorylase.

When glycogen phosphorylase
gets phosphate put onto it,
it becomes really active.
It starts breaking
down glycogen,
and that's what you
see going on here.
Active, active.
OK?
This is something else.
I'm not going to talk about
the T-state form of this, OK?
In summary here,
to keep it simple,
putting a phosphate onto
to glycogen phosphorylase
makes it active.
That's pretty cool.

And it's happening because
the bloodstream has gotten
epinephrine, and epinephrine
has told glycogen phosphorylase
to break down glycogen.
And by the way,
we're in a lot of danger, so
break down a lot of glycogen.
Make a lot of glucose and
get those muscle cells doing
what muscle cells can do.

Make sense?
All right.
I keep running over, so today
I'm not going to run over.
I will stop there, unless
there are questions.

I have a song for you today.
It's another one I can't sing.
Most of them I like
to sing, but this
is another one I can't sing.
It is– it's called
"The Way They Work."
[MUSIC – BARBRA STREISAND, "THE
WAY WE WERE"]

It's a hard one to sing.

>>(SINGING) Enzymes,
mighty powerhouse peptides,
cause reactions to go faster
in the cell's insides.

Tiny substrates bring
about an induced fit.

Enzyme structure is affected
by what binds to it.

Can it be that it's just simple
zen, how the enzymes activate?

If they bind effector,
will they go to an R-state?

T-state?

Folding gives the
mechanistic might
to three-D arrangement
of the active site.

Enzymes have a bias
they can't hide.

Hydrophobic side chains
are mostly found inside.

So it's the structure for
celebrating whenever there's
debating the way they work.

The way they work.
>>All right, see
you guys tomorrow.

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