HAZEL SIVE: Let's move on
with Biochemistry III.
And we have, as you recall,
being exploring the
fascinating and complicated
set of
macromolecules inside a cell--
inside the cell that we're
using, in a way, analogous to
a factory that is a production
line for synthesis of various
components that are required for
function of the cell, for
its replication, and for
perpetuation of the organism.
Today I want to talk to you
about some parameters that
surround the chemical reactions
of biochemistry.
We'll talk briefly about
thermodynamics and energy
considerations.
And then I want to introduce
you to a huge and pivotal
class of proteins,
the enzymes.
And then we'll talk very briefly
about the currency of
energy in the cell.
Let's start with thermodynamics,
which we will
define as the rules
underlying energy
usage in chemical reactions.
Rules underlying energy use.
And really, it's the first law
of thermodynamics that we're
interested in.
We're interested in the fact
that energy is neither created
nor destroyed, that it is
converted from one form to
another and that there is
conservation of energy.
And the other laws are embedded
in what we'll talk,
but these are the ones that
are really important for
biochemistry.
Let's think about a basic
chemical reaction, some kind
of substrate.
That's a biological term
which we'll use.
It's equivalent to the term
"reactant." And I could use R,
but I'm going to use S, because
that's what we'll use
in biochemistry, in life.
And that gives rise
to a product.
And chemical reactions can
go in both directions.
One reaction may predominate
over the other.
But the reaction
is reversible.
And the questions that we're
going to ask is, does a
chemical reaction require
or release energy?
How fast does the
reaction go--
which is referred to as the
rate of the reaction.
And how far, which refers to
the equilibrium point.
Equilibrium, where equilibrium
is defined as the place where
the forward rate and the
back rate are equal.
And at equilibrium, even though
the forward and the
back rate are equal, it's not
that you have equal amounts of
substrate and product, you have
a particular ratio of
substrate to product that is
characteristic for the
chemical reaction.
So you get a specific substrate
or reactant to
product ratio.
Let's make this S a little
larger, to match
the P. There we go.
Good.
The thing that you need to
know is called Gibbs free
energy and the change in Gibbs
free energy with a reaction.
So in any chemical reaction,
there are a couple of kinds of
energy considerations.
The one important for us is G,
the Gibbs free energy, which
is the usable or, the convention
is, free energy.
The other term you need
is H, which is the
total energy, or enthalpy.
Entropy, S, the unusable
energy.
And you also need T, which is
the temperature in absolute
degrees Kelvin.
And the reaction that you may
be familiar with or the
equation you may be familiar
with is delta G equals delta H
minus T delta S. And the thing
that's important here is what
this means.
Delta G is the change
in free energy
with a chemical reaction.
And every chemical reaction has
a characteristic change in
free energy.
And depending on what that is,
one can say certain things
about how likely the reaction
is to proceed.
If delta G is negative for a
particular chemical reaction,
then as the reaction proceeds,
energy is released, the
reaction is termed exergonic,
and it is sometimes
"spontaneous." I'm going
to put that in quotes.
You'll see why in a moment.
The flip is true if delta
G is positive.
This means that the reaction
requires energy in order to
proceed, and it is referred
to as endergonic.
And if delta G is 0, the
reaction is at equilibrium.
The notion that's quite useful
to think about this is balls
rolling up and down hills.
For a reaction that is
exergonic, a ball rolling down
a hill will decrease its delta
G. It will release energy as
it rolls down the hill.
And you can actually
plot this.
And you'll see lots of plots
like this where, if you plot
energy over the course of the
reaction, you will see that
the reactants start
off at a higher
energy than the products.
And as they are rolling
down the hill,
there is energy released.
And the flip is true for an
endergonic reaction where
delta G is positive.
You have to put in energy
to get the product out.
Now--
and this is where it
gets interesting.
And this is where we get back
to "spontaneous" in quotes--
chemical reactions can be
exergonic, but they may not
proceed spontaneously.
If you imagine a ball sitting
at the top of a hill, if the
ball is sitting right on the
slope of a hill, and you let
the ball go, it'll go zipping
down the hill, right?
But if the ball is sitting on
the top of the hill, but it's
in a little hollow, it's not
going to go anywhere unless
you give a push.
That's very important.
And that kind of notion is
embodied in chemical
principles.
So let's write this out.
And then we'll formalize this.
Delta G can be negative, but a
reaction does not proceed.
And that's because you've got
to get the reactants out of
that little hollow at
the top of the hill.
And in order to get the
reactants out of the little
hollow at the top of the hill,
you need something called
activation energy.
Activation energy is required.
I'm going to abbreviate
that AC.
You can abbreviate it
whatever you like.
And the thing about chemical
reactions that makes them
proceed in chemistry and makes
them proceed in biochemistry,
in life, is that you need
something to overcome that
activation energy.
Back to the ball analogy--
I think of this because
I have a dog.
His name is Archer.
He's a black Labrador--
in case you're interested--
and he's very fond of balls.
And they sit at the top of
the hill in my yard.
And there's a whole
line of them .
And they will sit there forever
unless Archer either
intelligently or by mistake
pushes one.
And then it goes rolling down
the hill, and he can get it.
So you have to do something
to start the reaction.
You have to give energy.
Where does that energy
come from?
Well, it comes from somewhere.
And you can decrease that
activation energy and really
get a reaction to proceed
quickly by the use of
chemicals called catalysts.
So catalysts decrease activation
energy and allow a
reaction to proceed.
Catalysts do not change delta G
of the whole reaction or the
equilibrium point.
So you're not going
to get more.
You're not going to really
change the reaction.
You're just going
to get it to go.
Catalysts do increase the
rate of reaction.
That's the point.
And they do this, as we'll
explore, by promoting
something called a transition
state, which is a high-energy
form of the reactants.
If we go back to our ball
analogy, if you kick the ball
so that it comes up out of the
hollow, that kick that you
give the ball, the energy you
put in there, is going to get
the ball rolling down
the hill, and the
reaction will proceed.
So catalysts promote a
transition state, which is a
high-energy form of
the reactants.
Let's look at a couple of slides
that I drew for you.
So here is a blank chart.
And I've got delta G on the
y-axis and the reaction course
on the x-axis.
And here's a reaction curve.
The reaction overall is going
to have a delta G that's
negative because you see that
the products are at the bottom
of the hill, relative
to the reactants.
But there's also this hump that
they have to get over.
And that is what requires
activation energy.
In an uncatalyzed reaction or a
catalyzed reaction, you have
to go through this high-energy
state.
So here is a catalyzed reaction
that I put on as
blue, the uncatalyzed as red.
And you can see what happens to
the little hill that has to
be climbed before the reaction
can proceed.
The amount of energy you need to
move that chemical reaction
decreases relative to the
uncatalyzed reaction.
So you decrease the little
hollow in which the ball is
sitting, in order to get the
reaction to proceed.
But at the end, you'll end up
with exactly the same reaction
as you would have if it
were not catalyzed.
All right.
What does this have to
do with biology?
Well, it has everything.
Because there's a class of
proteins that we'll talk about
for the rest of the lecture that
are biological catalysts.
And without them, there
would be no life.
So number two, we'll
talk about enzymes.
And enzymes are biological
catalysts.
They are usually protein.
And all of the ones that we'll
talk about are proteins.
But they can also be RNA.
And a Nobel Prize was given
some years ago for the
discovery of RNA enzymes
that are catalysts.
And Dr. Sinha has posted on
the website a video of the
Nobel lecture of Professor
Sidney Altman, who got the
Nobel Prize for the discovery
of RNAs as enzymes.
But we're going to focus
on proteins.
We're going to rewrite the
chemical reaction that I have
on that board in a slightly
different way, which is the
conventional way for thinking
about enzymes, where we're
going to have an enzyme,
abbreviated E, plus the
substrate that is
going to form an
enzyme-substrate complex.
And this is the transition
state, so called
transition-state complex.
And that, as I'll draw in a
moment, is going to give rise
to the enzyme plus
the product.
So let me just draw
this up there.
And then we'll talk about
what these mean.
E is enzyme, S is substrate,
as previously.
This enzyme substrate, ES, is
the transition-state complex.
And what it is in biological
terms is some kind of
high-energy state of the
substrate that is linked to
the enzyme.
So it's a high-energy substrate
linked to the enzyme.
This transition-state complex
then resolves to release the
enzyme, which can
be used again.
As in the case of all catalysts,
they can be reused
to give rise to the enzyme
and the product.
So why is this interesting?
It's fascinating because --
Actually, let me before I tell
you why it's so fascinating,
let me show you something.
So I drew this for you.
Here is a representation of an
enzyme and something I'll
explore on the board in a moment
called the active site,
where the substrate fits
into the enzyme.
Here is an enzyme-substrate
complex, a high-energy
transition-state complex, that
undergoes reaction, catalysis,
to give rise to the product.
The product is released,
and you start
the whole thing again.
The thing about biological
catalysts that are really
different from chemical
catalysts is their
specificity.
Now, you may have heard of
platinum as a catalyst.
Platinum is an incredibly
powerful catalyst that
promotes almost any
chemical reaction
that involves hydrogen--
but any chemical reaction.
The difference between platinum
and enzymes is that
enzymes are incredibly specific
for a particular
chemical reaction.
Let's write that.
Enzymes are highly specific,
exquisitely specific, for the
particular reaction.
And they maintain this
specificity by fitting the
substrate into what I've got
on the screen there, the
active site, which is a
particular part of the enzyme,
a particular part
of the protein.
And the deal is that the
substrate fits into a part of
the protein called
the active site.
The active site is a very small
part of the protein.
And it's this interaction with
the protein and the active
site that promotes the
transition state.
How does it promote the
transition state?
Well, you can think physically
it might change the shape of
the substrate to make
it higher energy.
You could imagine
it adds charge.
You could imagine it adds
particular orientation between
two substrate molecules
that happen to be
in the active site.
Or it might align the substrate
in the correct way.
So we can actually say that the
substrate is promoted to
undergo catalysis
by controlling--
let's just list these--
orientation of the substrate
in the access site, adding
strain to the substrate, or
possibly adding a particular
charge to the substrate.
Let's look through a couple
of things here.
Okay, this is from your book.
Let's not dwell on that.
Let's go to this one,
which is not quite--
Ah, it's on that screen.
Good.
This is an example of enzyme
specificity that you see every
day, probably.
On Diet Coke, it says,
"warning, fennel
phenylketonurics, contains
phenylalanine." What's the deal?
There is a disorder called
phenylketonuria where people
are unable to digest the amino
acid phenylalanine.
This is an essential
amino acid, but too
much of it is bad.
Normally when you eat
phenylalanine, you use some of
it to build proteins.
That's good.
But then you convert a bunch
of it, using this enzyme
called phenylalanine
hydroxylase, into tyrosine,
which you may remember is
another amino acid.
And then you use tyrosine.
And tyrosine goes on to do
a bunch of other things.
If this enzyme is absent, then
you get a side product whereby
the excess phenylalanine
makes this stuff called
phenylpyruvic acid.
And that is a neurotoxin.
And it poisons the nervous
system, and people have very
severe symptoms.
People with phenylketonuria
generally have a single amino
acid change in an enzyme that
is more than 1,000 amino
acids, where there's an arginine
that has being
changed to a tryptophan.
And the single amino
acid change lies
in the active site.
It stops the phenylalanine from
binding the active site.
And it leads to this really
debilitating disorder.
And so that's an everyday
example of a single tiny
change that reflects
the specificity of
the particular enzyme.
But here's a puzzle.
This is a space-filling
model of an enzyme,
taken from your book.
Here's a substrate in red.
And you can see that this
enzyme has 3D shape.
Each of these bubbles is part
of one of the amino acids.
That's what a space-filling
model is.
And the substrate fits into
this active site.
I think this is called
hexokinase.
And you can see as the substrate
enters the active
site, actually the protein
changes shape.
And it kind of closes up and
contains the substrate in the
active site.
And catalysis takes place.
But the active site is tiny.
And there's this huge
rest of the protein.
And you can't get rid of the
rest of the protein.
The enzyme doesn't function
if you do.
So there's a fundamental
question that we need to ask,
which is, what do the rest of
proteins do, the part outside
the active site?
So let's ask that question.
What does the rest of the
protein, which is large, do?
And by the rest of the protein,
I mean "not the
active site," which is the place
where the transition
state complex is promoted.
What it does is to regulate the
activity of the enzyme.
So the answer we can give
is that it regulates, or
controls, activity
of the enzyme.
What do I mean?
Well, if you go back to this
notion of the cell as a
factory and enzymes as the
wheels and the machines that
get things done so that you
get particular components
synthesized, then you really
want to make sure that you
have got your production line
working at the right pace.
If you need more product,
you need to speed up.
If you need less product,
you need to slow down.
The enzymes are a place where
you control how much product
is being made, how much reactant
is being used, how
much substrate is being used.
So the rest of the protein
controls the
activity of the enzyme.
And there are a couple of ways
that this activity is
controlled.
The end result is that all of
these regulations affect the
active site, even if they
happen far, far away.
And so you can imagine that if
you are all lined up in a
line, and the active site was
right at the front of the line
of you that was the enzyme
prototype, and I pushed you,
and you all fell down, the
active site, the very front of
the line, would eventually be
affected although I'd pushed
the back of the line.
It's a silly example, but it
gives you a sense of how you
can change one thing and it
has an effect far away.
And the way that this is done
is through the use of
inhibitors.
I'll go through some
slides in a moment.
And there are two kinds
of inhibitors.
There are reversible inhibitors,
and there are
irreversible inhibitors.
These inhibitors may be
competitive inhibitors.
If they are competitive, they
actually will bind to the
active site as opposed to
the rest of the protein.
But if they are all of a class
that is non-competitive, they
will bind to the protein
elsewhere.
I'll show you a slide
in a minute.
But let's finish doing
some board work
here before I do that.
There is also a very important
and interesting class of
enzyme regulators called
allosteric regulators.
There are both allosteric
activators of enzyme function
or inhibitors, things that will
increase or decrease the
rate of the reaction.
And these allosteric activators
or inhibitors will
bind to a particular site that's
not the active site.
It's called an allosteric
site--
not the active site, but another
particular place on
the enzyme.
Other things that can change
enzyme function--
pH, the enzymes that work in
your stomach to digest your
food work at a pH of 2.
That's the pH in your stomach.
It's very acid.
Those enzymes will not work in
your small intestine, which is
at a pH of about 6 or 7.
So pH, physiologically, can
affect how enzymes work.
Temperature affects
how enzymes work.
And all of these things are
somehow changing the structure
of the protein.
And changing the structure
of the protein is
changing its function.
We'll explore this in more
specifics as the semester goes
by, but this is what you
need to understand now.
There are also classes of
molecules called cofactors,
coenzymes, and prosthetic
groups.
I'll go through one of
these in a moment.
But all of these things
have the outcome--
all change the structure of the
protein so they'll change
enzyme structure.
And with that, they will
change enzyme function.
So all of these things--
I'm going to put it in a box--
change the structure
of the protein.
They will change the structure
of the enzyme,
of the active site.
And they will change
its function.
And they're very complicated
to think about.
Let's go through some slides
that will help us with this.
Here's the notion of a
competitive inhibitor.
Here's the substrate
and an active site.
And here's an inhibitor.
And you can see I've drawn it so
that it kind of looks like
a substrate, but it's got
some extra stuff.
And so when it binds to the
active site, it binds
specifically.
But it's not actually a
substrate, because it's got
this extra chemical moiety
which does not make it
available for catalysis.
And that stops the enzyme.
That competitive inhibitor may
or may not be able to come off
the enzyme and allow
it to work again.
It depends on the inhibitor.
Here's another one, a
noncompetitive inhibitor that
doesn't look like a substrate
and binds somewhere else on
the enzyme and, in doing so,
changes the shape of the
active site.
So the active site now can't
bind the substrate anymore
because its shape has
being changed.
Here's the notion of an
allosteric activator.
Here's the enzyme, and here's
the enzyme with its substrate.
And here's its allosteric
site.
And it can't work until an
allosteric activator, which is
not the substrate--
it's a different molecule--
binds to the allosteric site and
then makes the active site
the correct shape to fit
the substrate so that
the enzyme can work.
And you can get a cycle here
again, so that you get
catalysis, and the whole thing
can take place again.
I want to show you, to give
you a sense of the kind of
complexity of what this all
looks like, a particular
example of an enzyme reaction.
And I'll show you an animation
that goes with this, to give
you a sense of how these
things work.
The enzyme I'm going to
focus on is called
dihydrofolate reductase.
That's an essential enzyme.
It's required for nucleotide
synthesis, DNA modification.
And the reason you take folate,
or folic acid, is to
allow dihydrofolate to
actually work --
dihydrofolate reductase
to work.
The substrate is
dihydrofolate.
It's a complicated molecule.
But I've circled the
important part.
You can see there's a double
bond here which is going to be
reduced when the enzyme acts.
And here is the reduced form
or tetrahydrofolate of
dihydrofolate reductase.
You don't need to take
notes on this.
Just look and listen.
Here is a structure
of the protein.
You can see the alpha helices
in brown and the beta sheets
in blue and this kind
of unstructured
loopy stuff in between.
And here is the substrate
dihydrofolate binding to its
active site.
Dihydrofolate reductase requires
a coenzyme, something
called NADPH, which is a very
important coenzyme.
It's also part of the energy
production cycle.
And dihydrofolate reductase is
not only essential for life
but it's a target of the
anticancer drug methotrexate
and of trimethoprim, which
is an antibiotic.
This is an animation which
will show you how
dihydrofolate binds to
dihydrofolate reductase, how
NADPH comes in, and how NADPH
donates the hydrogens that
will allow dihydrofolate
to be reduced.
So let me start the animation,
and I'll point things out as
you're watching it.
And you'll see it's quick.
Here is the coenzyme
coming in.
And here is the substrate
dihydrofolate.
And if you look at it, you'll
see that the coenzyme is near
the active site.
And as it binds first, donates
its hydrogens to
dihydrofolate, which then
becomes tetrahydrofolate, is
released from the enzyme.
There are a number of things
you can see here.
You can see the enzyme cycle.
You can see this goes
over and over again.
You can see that the
enzyme, the protein
structure, is changing.
It's moving as the coenzyme
is binding, as the
substrate is binding.
You can see that there's a
chemical reaction going on
here as the NADPH is giving
its hydrogens to this
dihydrofolate.
And you can get a sense
of the kind of
animation that is enzymes.
This is an animation.
No one's actually seen an enzyme
work like this because
it's impossible to take
real movies like this.
But based on the structure of
the enzyme and the binding of
NADPH and the substrate to the
enzyme, these authors have
reconstructed this really
beautiful movie which gives
you a sense how enzymes
work in action.
Okay.
I'll post this on
your website.
And you can watch it.
The last two things I want to
tell you are that, firstly,
enzymes don't work
as one-off deals.
They work as part of
a production line.
And the production line
is called a pathway.
And like any good production
line, as I've mentioned
already, there are checks and
balances to make sure that the
production line is working at
optimal speed and making what
it needs to make.
So we can talk about pathways
and feedback.
And let's draw a simple pathway
on the board to give
you a sense of what I mean.
Let's start off with a substrate
that's converted
through the action of enzyme
one to product one.
And then there is also a
competing pathway where this--
Actually, lets make
it product one.
Or we can call it
substrate two.
But let's call it product one,
and then through the action of
enzyme two, to product one.
And here's a competing pathway
where the substrate, through
the action of enzyme three makes
product three, and then
through enzyme four action,
makes product four.
Okay.
This is a pathway.
It's a bifurcating pathway.
It may be that's if you've got
this branch, the E3-E4 branch
of the pathway active, you
actually want to shut off
production of the
other pathway.
And it may be that if you get
product four made, you
actually want to shut off
production of product two.
In that case, you could draw a
line like this-- you could
make it up, whatever you like.
But for the example I'm giving
you, I'm telling you that when
you've got lots of product four,
it's going to shut off
the other branch
of the pathway.
And this nomenclature
is as follows.
An arrow means activate.
And what's called a T-bar
means inhibit.
And these are very standard
nomenclatures in biology that
you should know.
So this T-bar here is inhibit.
It's also called negative
feedback.
At the same time, you might
get positive feedback.
It may be that if you have some
of product three, you
actually want more
of product one.
And in that case, product three
might activate enzyme
one so that you actually
activate the other branch.
This is a complicated pathway.
There's a lot of competition
going on here.
But in that case, that
activation would be positive.
And so for this example,
my star
would be positive feedback.
And my T-bar, which I'll make
a pound sign, would be a
negative feedback.
This is a really pivotal
concept in biology.
It's not just true of enzyme
pathways, it's true of
multiple chemical reactions.
And you really need
to understand
this aspect of biology.
The last thing that I want to
tell you has to do, very
briefly, with where the energy
comes from that cells use for
chemical reactions.
And what I'm going to tell you
very briefly, in about two
seconds, is that in the cell,
there are very controlled
amounts of energy that are
contained in a nucleotide
called ATP.
ATP is a nucleotide
triphosphate.
We talked about nucleotides
before.
The triphosphate is very
important here.
And this nucleotide triphosphate
is kind of like
the dollars and cents of energy
currency in the cell.
If a chemical reaction needs
energy, the cell
uses ATP to get it.
ATP is hydrolyzed to give rise
to ADP, adenosine diphosphate,
and PPI, inorganic phosphate,
plus an increment of energy.
This is an exergonic reaction.
This energy is then used for
chemical reactions, to build
or to break down various
molecules.
For this reaction, delta
G is negative.
That is why there's
energy released.
And this energy is used for
two types of reactions--
those that are catabolic, that
break down molecules, and
those that are anabolic, which
build up molecules.
We're not going to talk about
generation of ATP.
That is a separate topic.
And those of you who are go on
to do biochemistry will talk
about it in great detail.
But I do have some slides on
here that I will refer to.
Here is --
This is the last thing
I'll show you.
Please wait for it.
Here is adenosine
triphosphate.
Here is the high-energy
bond that is
broken to release energy.
And we'll stop there.
