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OK.  So we're going to continue with
the discussion about biochemistry,

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and specifically focus on enzymes
today.  Professor Sive introduced

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those to you briefly in
her last lecture.

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I'm actually covering for her today.
This is one of her lectures but she

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has given me her material,
so hopefully it will go fine.

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She wanted me to remind you a
little bit about energetics,

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specifically that a negative Delta G
in a reaction implies that the

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reaction can occur spontaneously,
that is if the products have lower

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energy than the reactants.
And so given enough time this will

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happen in that direction.
But importantly for many reactions

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an activation energy might be
necessary to get that reaction

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started.  And if the activation
energy can be sufficiently high then

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in a reasonable amount of time the
reaction will actually never proceed

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forward.  And it's the job of
enzymes actually to deal with that

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problem, as we'll discuss.
So this is the third of three

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lectures in biochemistry.
We're going to transition next week

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to genetics, and I'll be actually
teaching that section on genetics.

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Before we get into today's lecture
proper, Professor Sive wanted to

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quiz your knowledge from previous
lectures.  So firstly in this

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structure, this polypeptide
structure, the question is was the

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valine amino acid,
the Val, added last or first in this

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reaction?  Specifically true or
false, was it the last amino acid

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added?  False.  Very good.
It was the first.

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Because amino acids are added in
polypeptides from the amino end to

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the carboxy end.
And in this case Val is next to the

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amino ends.  So the second question
is in this reaction shown here,

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the generation of sucrose from
glucose and fructose,

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which has a positive Delta G,
does this release energy or consume

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energy?  Specifically true or false,
does this?  False.  Good.

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So the Delta G is positive,
which means to get this reaction to

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go you need to add energy.
And there are various ways of

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adding energy.
In a synthetic case a chemistry

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example you might add heat or you
might link this to another reaction

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that actually produced energy.
And finally is activation energy

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required only to initiate reactions
with a positive Delta G?

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No.  Very good.
So, as we just talked about,

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activation energies can be important
both for reactions that consume

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energy and give off energy.
And so even in situations where

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reactions give off energy,
it may be necessary for an enzyme to

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deal with this activation energy
problem in order to speed up the

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rate of the reaction.
OK.  So Professor Sive gave you

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examples previously comparing the
cell to a factory.

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And I think it's a very apt analogy.
And it's particularly apt with

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respect to the subject of today's
lecture which are enzymes.

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There's a great deal of interest
these days in the MIT community and

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around the research community around
the world in nano technology,

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building tiny little machines that
can do work.  Well,

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I would say that proteins are the
consummate example of nano

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technology.  These are small
entities, five to ten nanometers in

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diameter, which carry out very
specific and very powerful

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reactions.
So proteins are already nano

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particles to the nth degrees.
And the cell is itself remarkable

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in this respect because the cell,
which is only ten to thirty microns

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in diameter not only has all these
nano particles doing all this work,

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but it also contains the blueprint
information for building those.

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It has the synthetic capability for
making the raw materials that they

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act on.  And it has the micro
machining capability to build the

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nano machines.
And this all takes place in a very,

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very tiny space.  So the cell is an
unbelievable example of engineering

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that we can only just aspire to
replicate.  OK.

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So, again, the subject of today's
lecture is enzymes as biological

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catalysts.
And I want to introduce this by

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putting some of the earlier stuff
into perspective.

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So Professor Sive told you in her
last lectures about

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macromolecules --

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I'm not going to talk about all of
them that she's told you about,

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but she's talked about DNA,
deoxyribonucleic acid which is

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responsible for information
storage.

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She also told you about RNA,

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ribonucleic acid, a related polymer,
nucleic acid.  RNA actually has many

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functions in the cell.
Information transfer is an

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important one,
but it's not limited to that.

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RNAs can also function structurally.

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When we talk about protein synthesis,
for example, you'll see that RNA

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molecules surface scaffolds for the
ribosome which is the machine that

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makes proteins.
And they can also be catalysts.

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This was not appreciated for a long
time.  We now know that in biology,

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in biological systems today RNAs can
function as catalysts.

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And we actually believe,
in an evolutionary sense, that RNAs

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were the first catalysts.
Before there were polypeptides and

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proteins, life was made possible
through the catalytic properties of

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RNA molecules.
And your book actually talks about

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those in the chapter that you just
read.  They're called ribozymes,

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for RNA ribose-based enzymes.  And
then there were proteins.

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And we'll talk about proteins as
catalysts today,

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but importantly proteins also serve
structural roles.

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They compose, for example,
the long cables that extend

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throughout your cells to give them
the proper shape and allow them to

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move.  They're important carrier
proteins.  For example,

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hemoglobin which carries oxygen to
different parts of your body.

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There are proteins that bind to
iron, for example,

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and deliver it from your diet to the
relevant parts of your body that are

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protein specific to that function.
And then there are catalysts.

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And it's the catalysts that we'll
focus on today.

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Now, the way that proteins carry
out these diverse functions is that

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by virtue of their amino acid
sequence, their primary sequence of

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amino acids, they fold up into a
particular shape.

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Proteins have very specific
three-dimensional shapes.

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And these shapes then dictate their
function.  Depending on what the

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protein is supposed to do,
it will have a different shape.

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So, for example,
there are proteins that,

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as I mentioned, function as
structural elements within the cell

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like long cables.
They form polymers.

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And these proteins have shapes a
little bit like Lego blocks that

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link together,
one to the next,

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and as such line up into these long
polymers.  So some proteins have

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structures that just allow them to
bind to one another and

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form long polymers.
The proteins that we'll talk about

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today, catalysts,
usually have, maybe always have a

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specific shape.
And I'm taking a three-dimensional

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structure here and simply cutting a
slice through it,

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so you're looking at sort of the
middle of the protein cut in half.

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Here's the overall
three-dimensional structure of the

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protein.  And in this portion here,
which we call the active site, is

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where the chemical reaction
takes place.

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Now, we know a lot about many
enzymes, as well as other proteins

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in the cell, based on the primary
amino acid sequence that we can

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determine from the genes sequence.
But we also have seen what these

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look like using methods in
x-ray crystallography.

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We won't actually review the methods

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of x-ray crystallography with you,
but basically sufficed to say that

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you can take a pure protein,
allow it to form into a crystal,

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shine x-rays through it, the x-rays
get diffracted based on the position

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of the atoms in the protein.
And you can then interpret that

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diffraction pattern to tell you what
the structure of the protein was.

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So these are not just cartoons.  We
actually know what these proteins

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look like in great detail.
Not only do we know what the

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proteins look like in many instances,
but we also often know what the

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protein looks like in complex with
some reactant to which its bound.

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And these we call co-crystals.
Crystals that occur between the

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protein and the substrate molecule
that it's going to act upon.

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And so we have a very clear idea of
what the chemistry is that's going

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on inside the enzyme's active site.
And this information is not just

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useful for biological purposes,
but the more we understand enzymes

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and the specific structure of
enzymes the more we

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can do about it.
So in disease,

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for example, where you have an
enzyme that's causing some

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pathogenic problem,
if you want to inhibit that enzyme,

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the more you know about what's
happening here the more precise you

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can be in your design of an
inhibitor.  And that's now happening,

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and I'll tell you an example of that
later in the lecture.

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Now, I'm just curious to know
whether you have a sense for why the

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protein has its particular structure,
and also what distinguishes one

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active site from another?
There are thousands of enzymes

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inside this cell.
Do they all look like this or does

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each one look different?
Are they all the same?

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No.  They're all different.
They're different in shape, as I

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indicated earlier because their
primary amino acid sequence causes

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them to fold up based on local
interactions between amino acids

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into helices and beta pleated sheets.
As I told you,

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they interact with each other.
And that gives them that large sort

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of three-dimensional structure.
But it's the nature of the amino

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acids in this active site
which ones are there.

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Is it a glutamic acid,
a valine, a methionine?

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Which specific ones are present
there and how are they oriented

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which allows them to bind to
particular molecules,

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substrates, and do chemistry on them?
And the reason that proteins are

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much better catalysts,
or much more powerful catalysts than

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RNAs, is that our RNAs are fairly
boring.  They only have four

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subunits.  They don't have that
great diversity of chemical reactive

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groups that you find in proteins.
Proteins, as you heard,

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are composed of 20 distinct amino
acid subunits.

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They are all differently chemically
in those R groups that Professor

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Sive told you about before,
and they can do different chemistry

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in the active site.
So different enzymes are different

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by virtue of their overall structure
and the particulars within the

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active site that allows them to do
what they do.

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I wanted to mention briefly that we
often use the suffix ìaseî to

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designate an enzyme,
polymerase, DNA polymerase,

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sucrase.  You'll see those terms all
the time.  Whenever you see it,

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it reflects that the protein is an
enzyme, the suffix A-S-E.

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OK.  Again, just for perspective,
where do the proteins come from in a

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sense?
How does the cell know what to make?

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We're going to get into that in
later lectures,

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but just so you have a sense of it,
the information to produce a

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particular protein with a particular
amino acid sequence,

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and therefore shape and therefore
function, is encoded in the genes,

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which are in the DNA.  This
information is transferred into an

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intermediary molecule,
which is RNA.

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Again, you're going to learn about
these details.

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You don't have to worry about them
so much right now.

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You'll learn about these details
later in the class.

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And the particular RNA molecule
that carries the information from

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the DNA is called the mRNA,
messenger RNA.  And that mRNA is

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then translated into the protein
itself.  So the reason that we have

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proteins of particular sequence and
particular shape and particular

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function is that we have different
genes that carry the information to

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make those specific proteins.
OK?  And we'll see that again in

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detail in later lectures.
OK.  So very importantly we talked

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last time, you talked last time
about the energetics of reactions,

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as illustrated here, that in many
reactions there is the energy of the

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reactants themselves,
the energy of the products,

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as well as so-called activation
energy.

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That is the energy that's required
to make that reaction go,

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which can be greater than the energy
of the reactants.

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The important function of enzymes
is to lower the activation energy to

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reduce the threshold that these
reactants have to go over in order

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to carry out the reactions that lead
to the products.

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The enzyme's function is to lower
the activation energy.

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And the way that enzymes do that is
several-fold, as we'll review.

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The most important thing I can
imagine you can take away from this

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lecture is the understanding that
what enzymes do as catalysts is to

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lower the activation energy.
They don't change the nature of the

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reactants, they don't change the
nature of the products,

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they actually don't change
themselves in the course of the

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reaction, but what they do is to
facilitate the reaction by lowering

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the activation energy.
And that is the nature of catalysis.

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So enzymes are biological
catalysts.

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Their function is to increase the

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rate of a reaction.
As I said, reactions that release

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energy will happen spontaneously,
but it might take a very long time.

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Enzymes function to increase the
rate at which those reactions can

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happen.  And they can do so in
impressive ways.

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They can increase the rate by a
million-fold.  So they really can

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change whether a reaction will take
place in the lifetime of an

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individual compared to whether it
would take place in microseconds.

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00:16:37,000 --> 00:16:41,000
OK?  And many biological processes
have to happen within the course of

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microseconds or seconds.
And, therefore, without enzymes

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those would not be possible.
Importantly, as illustrated on this

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slide, enzymes do not change the
Delta G.  They don't change the

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Delta G of the reaction.
Delta G is the same.

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00:17:01,000 --> 00:17:07,000
What's being changed here,
in the presence of an enzyme,

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a catalyzed reaction, is the
activation state.

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The energetics of the products and
the energetics of the reactants

225
00:17:19,000 --> 00:17:24,000
don't change, so the Delta G does
not change.  So,

226
00:17:24,000 --> 00:17:30,000
again, they do so by lowering the
activation energy.

227
00:17:30,000 --> 00:17:36,000
And this is often done by combining
the reactants with portions of the

228
00:17:36,000 --> 00:17:42,000
protein to create what's called a
transition state complex.

229
00:17:42,000 --> 00:17:50,000
And it's the nature of that

230
00:17:50,000 --> 00:17:54,000
transition state complex,
the protein bound to the substrates

231
00:17:54,000 --> 00:17:58,000
that allows the activation energy to
be reduced, as you'll

232
00:17:58,000 --> 00:18:04,000
see in a moment.
I want to emphasize that the enzyme

233
00:18:04,000 --> 00:18:12,000
itself is the same at the end of the
reaction as at the beginning.

234
00:18:12,000 --> 00:18:20,000
The enzyme does not change.  It
goes through one reaction cycle.

235
00:18:20,000 --> 00:18:29,000
It's exactly as it was when it
started.

236
00:18:29,000 --> 00:18:33,000
And that's important because it
means that the enzyme can be reused.

237
00:18:33,000 --> 00:18:38,000
This is not a single reaction
process.  The enzyme can be used

238
00:18:38,000 --> 00:18:43,000
over and over and over again,
which is another part of the

239
00:18:43,000 --> 00:18:48,000
definition of a catalyst.
OK.  When we talk about enzymes,

240
00:18:48,000 --> 00:18:52,000
reactions, reactants and products,
we use slightly different

241
00:18:52,000 --> 00:18:57,000
nomenclature, and so it's important
that you see that and

242
00:18:57,000 --> 00:19:03,000
get to recognize it.
The enzyme, often denoted as E,

243
00:19:03,000 --> 00:19:10,000
combines with substrates, one or
more substrates,

244
00:19:10,000 --> 00:19:18,000
described as S, to form a complex
which is designated ES.

245
00:19:18,000 --> 00:19:25,000
That's where this transition state
is taking place.

246
00:19:25,000 --> 00:19:33,000
And then following that the enzyme
releases the products.

247
00:19:33,000 --> 00:19:41,000
And, importantly,
the enzyme can then be recycled to

248
00:19:41,000 --> 00:19:50,000
do this process again on new
substrates to produce new products.

249
00:19:50,000 --> 00:19:59,000
So the S in this is the reactant or
substrate, and P is the product,

250
00:19:59,000 --> 00:20:08,000
and the ES is the enzyme transition
state complex.

251
00:20:08,000 --> 00:20:21,000
As I said at the beginning,

252
00:20:21,000 --> 00:20:25,000
enzymes have very particular
specificities.

253
00:20:25,000 --> 00:20:29,000
They are designed to do specific
reactions.  They don't bind to every

254
00:20:29,000 --> 00:20:33,000
old molecule in the cell.
And this is determined,

255
00:20:33,000 --> 00:20:38,000
as I said, by the shape of the
enzyme and its complementarity to

256
00:20:38,000 --> 00:20:43,000
the substrates to which it binds.
And that's illustrated here on a

257
00:20:43,000 --> 00:20:48,000
slide from your book where you can
see an enzyme with its active site.

258
00:20:48,000 --> 00:20:54,000
And here are three potential
substrates.  Based on the shape of

259
00:20:54,000 --> 00:20:59,000
the active site and the particular
side chains on those amino acids,

260
00:20:59,000 --> 00:21:04,000
the yellow one and the red one will
fit into the active site,

261
00:21:04,000 --> 00:21:08,000
but the green one will not.
So specificity,

262
00:21:08,000 --> 00:21:12,000
in this case, is determined by the
complementarity between the shape of

263
00:21:12,000 --> 00:21:16,000
the substrates and the shape of the
active site.  And then,

264
00:21:16,000 --> 00:21:20,000
by virtue of their positioning
within the active site,

265
00:21:20,000 --> 00:21:24,000
the enzyme is now catalyzing the
binding, covalent attachment of the

266
00:21:24,000 --> 00:21:28,000
yellow one to the red one to produce
the product, as shown here.

267
00:21:28,000 --> 00:21:35,000
So specificity is achieved by the
complementarity between the active

268
00:21:35,000 --> 00:21:42,000
site and the substrates.
The transition state is achieved by

269
00:21:42,000 --> 00:21:49,000
a variety of conditions that the
enzyme places upon the substrates.

270
00:21:49,000 --> 00:22:02,000
So the substrate fits in much the

271
00:22:02,000 --> 00:22:08,000
way a key fits into a lock into the
active site.  This then promotes the

272
00:22:08,000 --> 00:22:15,000
formation of this transition state.
And that is done by three distinct,

273
00:22:15,000 --> 00:22:22,000
sometimes related but, distinct
mechanisms.  One is the fixing of

274
00:22:22,000 --> 00:22:29,000
the orientation of the two
substrates to one another.

275
00:22:29,000 --> 00:22:33,000
They're not just randomly floating
around in solution anymore.

276
00:22:33,000 --> 00:22:38,000
They're literally aligned next to
each other in a way that will

277
00:22:38,000 --> 00:22:43,000
promote the chemical reaction.
And that's one way that the

278
00:22:43,000 --> 00:22:48,000
activation state is lowered because
now you don't have the problem of

279
00:22:48,000 --> 00:22:53,000
kinetic energy of the molecules
floating around.

280
00:22:53,000 --> 00:22:58,000
A second is what's referred to as
induced fit, and in your book

281
00:22:58,000 --> 00:23:03,000
referred to as strain.
And I'll show you a slide of this in

282
00:23:03,000 --> 00:23:07,000
a second.  And this is important
because often times the activation

283
00:23:07,000 --> 00:23:12,000
energy is due to the fact that the
molecules have to get contorted.

284
00:23:12,000 --> 00:23:16,000
It's not a native confirmation of
the molecules during the chemical

285
00:23:16,000 --> 00:23:21,000
reaction. They actually have to get
bent in ways that they don't like to

286
00:23:21,000 --> 00:23:25,000
be bent.  The enzyme helps this by
adding chemical groups around it

287
00:23:25,000 --> 00:23:30,000
which promote the bending process,
promote the sort of straining of

288
00:23:30,000 --> 00:23:35,000
chemical bonds that allows
additional reactions to take place.

289
00:23:35,000 --> 00:23:38,000
So the enzyme produced is this
so-called induced fit.

290
00:23:38,000 --> 00:23:42,000
And, finally, the enzyme can,
depending on the nature of the

291
00:23:42,000 --> 00:23:46,000
chemistry, apply charge.
We know that there are charged

292
00:23:46,000 --> 00:23:50,000
amino acids, both positively and
negatively charged amino acids.

293
00:23:50,000 --> 00:23:54,000
There are acid-base reactions that
often take place within the enzyme's

294
00:23:54,000 --> 00:23:58,000
active site.  So the presence of
charges can facilitate those

295
00:23:58,000 --> 00:24:02,000
acid-base reactions.
They can donate positive charge or

296
00:24:02,000 --> 00:24:06,000
donate negative charge to allow the
reaction to take place more rapidly

297
00:24:06,000 --> 00:24:10,000
than it would do spontaneously.
And those are illustrated in a

298
00:24:10,000 --> 00:24:14,000
following slide,
actually.  This just shows you an

299
00:24:14,000 --> 00:24:18,000
example, a specific example of a
reaction that is catalyzed by a

300
00:24:18,000 --> 00:24:22,000
particular enzyme.
Here we have the substrate.

301
00:24:22,000 --> 00:24:26,000
It happens to be a sugar, a
disaccharide sugar,

302
00:24:26,000 --> 00:24:30,000
sucrose, and is made up of the
subunits glucose and fructose for

303
00:24:30,000 --> 00:24:34,000
you to use sucrose,
which you can eat.

304
00:24:34,000 --> 00:24:39,000
To produce energy you need to break
it down into glucose and fructose.

305
00:24:39,000 --> 00:24:44,000
And this reaction is catalyzed by a
particular enzyme called sucrase.

306
00:24:44,000 --> 00:24:49,000
And you can see in diagrammatic
form what happens here.

307
00:24:49,000 --> 00:24:54,000
Here's sucrase.  Here is its active
site.  You can see that its

308
00:24:54,000 --> 00:24:59,000
structure is exactly complimentary
to the substrate.

309
00:24:59,000 --> 00:25:03,000
So the substrate now floats in,
binds to this active site.  There's

310
00:25:03,000 --> 00:25:07,000
then a chemical reaction,
which is basically the addition of

311
00:25:07,000 --> 00:25:11,000
water to break this bond,
which is catalyzed by the enzyme.

312
00:25:11,000 --> 00:25:15,000
And then the products are released,
the enzyme remains as it was at the

313
00:25:15,000 --> 00:25:19,000
beginning of the reaction,
and it can go through the reaction

314
00:25:19,000 --> 00:25:23,000
cycle one more time.
This is the picture that shows you

315
00:25:23,000 --> 00:25:27,000
the various ways that the active
site can promote the transition

316
00:25:27,000 --> 00:25:32,000
state complex.
One, as I mentioned,

317
00:25:32,000 --> 00:25:36,000
is orientation.  Again,
two substrates here are positioned

318
00:25:36,000 --> 00:25:40,000
next to each other in the way that
we want them to react.

319
00:25:40,000 --> 00:25:44,000
So that's helpful.  A second is the
straining process,

320
00:25:44,000 --> 00:25:48,000
the fact that the protein can impose,
in a sense, stress on the molecules,

321
00:25:48,000 --> 00:25:52,000
the substrates, change their shape,
and in that way facilitate the

322
00:25:52,000 --> 00:25:56,000
chemical reaction,
and finally, as I mentioned,

323
00:25:56,000 --> 00:26:01,000
to charge.
In the case of acid-base reactions

324
00:26:01,000 --> 00:26:06,000
there are positively and also
negatively charged amino acid side

325
00:26:06,000 --> 00:26:11,000
chains that can contribute their
positive or negative charge to

326
00:26:11,000 --> 00:26:16,000
facilitate the reaction.
And this is one final example.

327
00:26:16,000 --> 00:26:21,000
Here we're talking about an enzyme
that adds a phosphate group onto

328
00:26:21,000 --> 00:26:26,000
glucose in an early step in glucose
metabolism.  And,

329
00:26:26,000 --> 00:26:31,000
in this case, the enzyme actually
changes its shape as a consequence

330
00:26:31,000 --> 00:26:37,000
of the substrate binding to it.
This happens [UNINTELLIGIBLE PHRASE].

331
00:26:37,000 --> 00:26:43,000
At least he didn't point to me.
[LAUGHTER]  What was that all about?

332
00:26:43,000 --> 00:26:50,000
I was actually prepared to have
those sorts of interruptions on

333
00:26:50,000 --> 00:26:56,000
Monday which we always have.
I don't know what that was all

334
00:26:56,000 --> 00:27:02,000
about.
So what was it all about,

335
00:27:02,000 --> 00:27:08,000
pretty boy?  [LAUGHTER]  And who is
pretty boy anyway?

336
00:27:08,000 --> 00:27:14,000
Somebody back there.
OK.  Well, that was fun.

337
00:27:14,000 --> 00:27:20,000
Anyway.  Well, we were talking
about enzymes.

338
00:27:20,000 --> 00:27:26,000
So, again, some enzymes,
as indicated here in the example of

339
00:27:26,000 --> 00:27:32,000
hexokinase, will actually change
their shape in response to the

340
00:27:32,000 --> 00:27:38,000
binding of the substrate,
here glucose.

341
00:27:38,000 --> 00:27:41,000
And this is maybe a more interesting
example of something I'm going to

342
00:27:41,000 --> 00:27:44,000
come to later,
which is that proteins are not

343
00:27:44,000 --> 00:27:48,000
static in their shape.
They actually do change a little

344
00:27:48,000 --> 00:27:51,000
bit, and the ability of them to
change can tweak,

345
00:27:51,000 --> 00:27:54,000
can tune their activities so that
positioning the exact structure of

346
00:27:54,000 --> 00:27:58,000
the active site can change based on
other things that are happening

347
00:27:58,000 --> 00:28:02,000
in the protein.
And that's a useful thing with

348
00:28:02,000 --> 00:28:06,000
respect to regulation.
You can turn up the activity of an

349
00:28:06,000 --> 00:28:11,000
enzyme.  You can turn down the
activity of an enzyme based on the

350
00:28:11,000 --> 00:28:15,000
changes that the overall structure
can make.  OK.

351
00:28:15,000 --> 00:28:20,000
And then just to bring the subject
of specificity of enzyme function

352
00:28:20,000 --> 00:28:24,000
home, here is an example from real
life.  This is a packet of Equal,

353
00:28:24,000 --> 00:28:29,000
which you may use.  It's an
artificial sweetener.

354
00:28:29,000 --> 00:28:33,000
And you may have noticed on the very
back of the Equal packet it says

355
00:28:33,000 --> 00:28:38,000
phenylketonurics: contains
phenylalanine.

356
00:28:38,000 --> 00:28:42,000
And you might have wondered,
what the hell is that all about?

357
00:28:42,000 --> 00:28:47,000
Does anybody know?  Is anybody a
phenylketinuric?

358
00:28:47,000 --> 00:28:51,000
You don't actually have to say if
you are or not,

359
00:28:51,000 --> 00:28:56,000
but does anybody know what this
means?  Yes.  It's close.

360
00:28:56,000 --> 00:29:00,000
You're actually thinking of a
similar disease called

361
00:29:00,000 --> 00:29:05,000
alkaptonuria.
But you're on the right track.

362
00:29:05,000 --> 00:29:09,000
Right.  You cannot break it down.
And specifically what you cannot

363
00:29:09,000 --> 00:29:13,000
break down is phenylalanine.
This is a disease that affects only

364
00:29:13,000 --> 00:29:17,000
about one in 12,
00 individuals.  It's a so-called

365
00:29:17,000 --> 00:29:22,000
metabolic disease.
We'll talk about metabolic diseases

366
00:29:22,000 --> 00:29:26,000
later.  And the important point here
is that the enzyme that's

367
00:29:26,000 --> 00:29:30,000
responsible for breaking down
phenylalanine is altered in these

368
00:29:30,000 --> 00:29:35,000
individuals in one residue,
one amino acid out of 451.

369
00:29:35,000 --> 00:29:39,000
Protein has 451 amino acids.
One of those amino acids in the

370
00:29:39,000 --> 00:29:43,000
active site is not what it's
supposed to be.

371
00:29:43,000 --> 00:29:47,000
And therefore it cannot bind
properly to phenylalanine.

372
00:29:47,000 --> 00:29:51,000
And that causes a defect in the
breakdown of this and the build up

373
00:29:51,000 --> 00:29:56,000
of a toxic substance,
as I'll show you in a second.

374
00:29:56,000 --> 00:30:00,000
This is NutraSweet.  You might not
have known that it is a dipeptide

375
00:30:00,000 --> 00:30:04,000
composed of aspartic acid and a
phenylalanine linked together with

376
00:30:04,000 --> 00:30:08,000
an extra group on it,
probably to make it more soluble or

377
00:30:08,000 --> 00:30:13,000
ability to pass through
cells more easily.

378
00:30:13,000 --> 00:30:17,000
In your body, when you take in
phenylalanine from the diet like

379
00:30:17,000 --> 00:30:21,000
with respect to NutraSweet,
when NutraSweet gets into your body,

380
00:30:21,000 --> 00:30:25,000
the phenylalanine and the aspartic
acid get broken apart so you have

381
00:30:25,000 --> 00:30:29,000
increased phenylalanine,
but however you intake phenylalanine

382
00:30:29,000 --> 00:30:33,000
from your diet it's normally
converted enzymaticly by an enzyme

383
00:30:33,000 --> 00:30:37,000
called phenylalanine hydroxylase
which converts it from phenylalanine

384
00:30:37,000 --> 00:30:41,000
to tyrosine.
And the tyrosine is either used for

385
00:30:41,000 --> 00:30:45,000
stuff or it's broken down itself.
It's actually used for making

386
00:30:45,000 --> 00:30:49,000
melanin.  And so these same patients
who I have just mentioned,

387
00:30:49,000 --> 00:30:53,000
these phenylketonurics also have
lighter hair and lighter skin

388
00:30:53,000 --> 00:30:57,000
because they cannot make as much
tyrosine, and therefore don't make

389
00:30:57,000 --> 00:31:00,000
as much melanin.
But the real problem is not that.

390
00:31:00,000 --> 00:31:04,000
The real problem is that if you
have too much phenylalanine in your

391
00:31:04,000 --> 00:31:08,000
blood because you cannot break it
down properly.

392
00:31:08,000 --> 00:31:11,000
Then you build up phenylpyruvic
acid which is a natural byproduct of

393
00:31:11,000 --> 00:31:15,000
phenylalanine.
And this stuff is toxic when

394
00:31:15,000 --> 00:31:19,000
present in high levels.
And so the patients, these

395
00:31:19,000 --> 00:31:22,000
phenylalanine hydroxylase mutants,
which are now called

396
00:31:22,000 --> 00:31:26,000
phenylketonurics,
have a defect in this enzyme,

397
00:31:26,000 --> 00:31:30,000
cannot carry out this reaction
properly.  And therefore this

398
00:31:30,000 --> 00:31:34,000
spontaneous reaction happens
more readily.

399
00:31:34,000 --> 00:31:37,000
And therefore you have high levels
of this toxic compound that causes

400
00:31:37,000 --> 00:31:40,000
mental retardation,
actually.  It causes some sort of

401
00:31:40,000 --> 00:31:44,000
neuro toxicity.
And that's how it was originally

402
00:31:44,000 --> 00:31:47,000
defined.  And the reason we're
telling you about this is that this

403
00:31:47,000 --> 00:31:51,000
is an example of enzyme specificity
because this enzyme is different,

404
00:31:51,000 --> 00:31:54,000
as I said, in only one of 451 amino
acids.  The 408 amino acid is

405
00:31:54,000 --> 00:31:57,000
supposed to be an arginine,
and instead is a tryptophan.

406
00:31:57,000 --> 00:32:01,000
And as a tryptophan it cannot
properly bind to or carry out the

407
00:32:01,000 --> 00:32:05,000
chemical reaction.
And therefore the enzyme fails,

408
00:32:05,000 --> 00:32:10,000
levels build up, and the individuals
have a very, very severe phenotype.

409
00:32:10,000 --> 00:32:15,000
A very, very severe disease
presentation, I should say.

410
00:32:15,000 --> 00:32:20,000
Now, I indicated a little bit ago
that enzymes can be tweaked in their

411
00:32:20,000 --> 00:32:25,000
function.  Enzymes are not just
static in their ability to interact

412
00:32:25,000 --> 00:32:30,000
with substrates and catalyze
reactions.

413
00:32:30,000 --> 00:32:35,000
In fact, they are highly regulated.
And they're regulated by a number

414
00:32:35,000 --> 00:32:41,000
of different both external and
internal processes.

415
00:32:41,000 --> 00:32:47,000
So regulation of enzyme function is
critical with respect to producing

416
00:32:47,000 --> 00:32:53,000
the right sort of products at the
right sort of times and rates within

417
00:32:53,000 --> 00:32:59,000
your cells.  And one key factor that
determines the regulation of a given

418
00:32:59,000 --> 00:33:05,000
enzyme is the pH,
the pH of the solution that the

419
00:33:05,000 --> 00:33:11,000
enzyme finds itself.
Now, why would that be?

420
00:33:11,000 --> 00:33:18,000
Does anybody have a sense for why
that would be?

421
00:33:18,000 --> 00:33:26,000
Why does the pH of the cell's
environment determine the enzyme's

422
00:33:26,000 --> 00:33:31,000
function?  Yeah.  Say it again.
Right.  So proteins can denature at,

423
00:33:31,000 --> 00:33:35,000
extreme pHs in both directions,
actually.  But more importantly

424
00:33:35,000 --> 00:33:39,000
they're optimized based on the side
chains that are present within the

425
00:33:39,000 --> 00:33:43,000
active sites.  So,
as I said, there are charged amino

426
00:33:43,000 --> 00:33:46,000
acids which have different pKas.
And depending on the pH of the

427
00:33:46,000 --> 00:33:50,000
solution, they'll either be
protonated or not protonated.

428
00:33:50,000 --> 00:33:54,000
And their state of protonation,
whether or not that they have a

429
00:33:54,000 --> 00:33:58,000
proton bound to them,
will affect their ability to carry

430
00:33:58,000 --> 00:34:02,000
out the chemistry.
So enzymes are perfected to function

431
00:34:02,000 --> 00:34:07,000
in the pH conditions they find
themselves.  So,

432
00:34:07,000 --> 00:34:12,000
for example, salivary amylase,
which breaks down carbohydrates in

433
00:34:12,000 --> 00:34:16,000
your saliva, has a pH of around
seven because your saliva is around

434
00:34:16,000 --> 00:34:21,000
pH seven.  So it's been evolved to
function best at that pH.

435
00:34:21,000 --> 00:34:26,000
If you increase the pH or decrease
the pH it doesn't work so well,

436
00:34:26,000 --> 00:34:31,000
as indicated by this reduced
reaction rate at higher

437
00:34:31,000 --> 00:34:35,000
and lower pHs.
In contrast pepsin,

438
00:34:35,000 --> 00:34:39,000
which is an enzyme that is present
in your stomach acid and in your

439
00:34:39,000 --> 00:34:43,000
small intestine where the pH is very,
very low, works best at low pH.

440
00:34:43,000 --> 00:34:47,000
If you raise this to increase pH,
it doesn't work at all well because

441
00:34:47,000 --> 00:34:51,000
presumably at the increased pH
things that should be protonated are

442
00:34:51,000 --> 00:34:55,000
not protonated.
And now those reactions that should

443
00:34:55,000 --> 00:34:59,000
be taking place in the
active site don't.

444
00:34:59,000 --> 00:35:02,000
This is also another interesting
example of regulation in the sense

445
00:35:02,000 --> 00:35:06,000
that pepsin breaks down proteins.
And you actually don't want rampant

446
00:35:06,000 --> 00:35:10,000
protein breaking down enzymes
floating around in your body.

447
00:35:10,000 --> 00:35:14,000
Particularly, you don't want them
in the cells that make pepsin.

448
00:35:14,000 --> 00:35:18,000
You could imagine that the cells
that make pepsin run the risk that

449
00:35:18,000 --> 00:35:22,000
pepsin is going to eat all the
proteins in those cells.

450
00:35:22,000 --> 00:35:26,000
It doesn't happen because the pH of
those cells is around seven,

451
00:35:26,000 --> 00:35:30,000
and therefore the pepsin isn't
active.

452
00:35:30,000 --> 00:35:34,000
It only becomes active when it gets
dumped into the acid environment of

453
00:35:34,000 --> 00:35:39,000
the stomach and the small intestine.
So that's another justification for

454
00:35:39,000 --> 00:35:44,000
tweaking the activity of enzymes.
Another example is temperature.

455
00:35:44,000 --> 00:35:52,000
Again, based on the structure of the

456
00:35:52,000 --> 00:35:56,000
protein, which is strongly
influenced by the temperature,

457
00:35:56,000 --> 00:36:00,000
proteins have optima.  Most of our
proteins function best

458
00:36:00,000 --> 00:36:04,000
at what temperature?
37 degrees Centigrade.

459
00:36:04,000 --> 00:36:08,000
98.6, or whatever, Fahrenheit.
But that's not true of all

460
00:36:08,000 --> 00:36:12,000
organisms.  As I mentioned in the
first lecture,

461
00:36:12,000 --> 00:36:17,000
there are organisms that live in
thermal vents where the regular

462
00:36:17,000 --> 00:36:21,000
temperature is 80 degrees centigrade
or higher.  Their enzymes actually

463
00:36:21,000 --> 00:36:26,000
work like crap at 37 degrees,
but they work great at 72 or 75

464
00:36:26,000 --> 00:36:30,000
degrees.  They've been optimized,
based on their structure, to

465
00:36:30,000 --> 00:36:35,000
function best at the temperature in
which they find themselves.

466
00:36:35,000 --> 00:36:41,000
And finally, or almost finally,
covalent modification.

467
00:36:41,000 --> 00:36:48,000
And there are different ways that

468
00:36:48,000 --> 00:36:52,000
proteins can be modified after
they've been made in the translation

469
00:36:52,000 --> 00:36:56,000
process.  Other stuff can get added
to them covalently.

470
00:36:56,000 --> 00:37:00,000
And I'll give you an example of
phosphorylation --

471
00:37:00,000 --> 00:37:04,000
-- because it's going to come up in
later lectures,

472
00:37:04,000 --> 00:37:09,000
too.  Phosphorylation which means
that the protein gets an extra

473
00:37:09,000 --> 00:37:14,000
phosphate group added to it.
And if you imagine, for example,

474
00:37:14,000 --> 00:37:19,000
an enzyme, it has an active site
here --

475
00:37:19,000 --> 00:37:29,000
-- which is blocked at its front

476
00:37:29,000 --> 00:37:33,000
door.  Stuff cannot get into it
because this little arm is kind of

477
00:37:33,000 --> 00:37:38,000
hanging over the front of the active
site.  What can happen is that a

478
00:37:38,000 --> 00:37:43,000
reaction, another chemical reaction
that adds a phosphate group --

479
00:37:43,000 --> 00:38:00,000
Phosphorylation,

480
00:38:00,000 --> 00:38:06,000
which is another enzymatic reaction,
can cause the enzyme to open up and

481
00:38:06,000 --> 00:38:12,000
allow substrates to come through.
And this is a reversible process.

482
00:38:12,000 --> 00:38:18,000
Other enzymes called phosphatases
can come along,

483
00:38:18,000 --> 00:38:24,000
clip the phosphate off and return
the enzyme to its inactive state.

484
00:38:24,000 --> 00:38:32,000
And then, finally,

485
00:38:32,000 --> 00:38:36,000
there are partners,
other molecules that the enzyme

486
00:38:36,000 --> 00:38:41,000
binds that help the enzyme do its
thing.  And these are summarized on

487
00:38:41,000 --> 00:38:45,000
this slide, which comes from your
book.  There are three groups that

488
00:38:45,000 --> 00:38:49,000
are shown here.
You should probably be familiar

489
00:38:49,000 --> 00:38:54,000
with what these groups are and
examples from within them.

490
00:38:54,000 --> 00:38:58,000
For example, cofactors.  These are
usually small metal

491
00:38:58,000 --> 00:39:02,000
molecules, atoms.
Iron, copper and zinc are three that

492
00:39:02,000 --> 00:39:06,000
are shown here.
These participate in the chemistry.

493
00:39:06,000 --> 00:39:10,000
They actually participate in the
catalysis for enzymes that require

494
00:39:10,000 --> 00:39:13,000
them.  And, actually,
many enzymes in your bodies do

495
00:39:13,000 --> 00:39:17,000
require such cofactors.
And that's one of the reasons it's

496
00:39:17,000 --> 00:39:20,000
encouraged, you're encouraged to eat
zinc and stuff like that,

497
00:39:20,000 --> 00:39:24,000
because many of your enzymes need it.
Another class called coenzymes,

498
00:39:24,000 --> 00:39:28,000
this happens to be a horrible name
in my opinion, a really,

499
00:39:28,000 --> 00:39:32,000
really bad name.
It's one of these historical names

500
00:39:32,000 --> 00:39:36,000
that we're stuck with.
But these are, again, partner

501
00:39:36,000 --> 00:39:40,000
molecules.  They're really
substrates.  They're really

502
00:39:40,000 --> 00:39:44,000
substrates in the reaction,
but they're called coenzymes because

503
00:39:44,000 --> 00:39:49,000
they're used by many different
enzymes in coupled reactions.

504
00:39:49,000 --> 00:39:53,000
And an example here is NAD which is
involved in a hydrogen donating and

505
00:39:53,000 --> 00:39:57,000
receiving.  And this I mention
specifically because it's the

506
00:39:57,000 --> 00:40:01,000
product, or it's the byproduct of
one of the vitamins you

507
00:40:01,000 --> 00:40:05,000
eat, vitamin B.
And many of these coenzymes are the

508
00:40:05,000 --> 00:40:09,000
products of vitamins.
And so that's one of the reasons

509
00:40:09,000 --> 00:40:12,000
it's important to eat your vitamins.
And finally what are called

510
00:40:12,000 --> 00:40:16,000
prosthetic groups,
the same word as artificial arms and

511
00:40:16,000 --> 00:40:19,000
legs.  Prosthetic groups like heme,
which is present in hemoglobin,

512
00:40:19,000 --> 00:40:23,000
flavins, other things,
which are involved, again,

513
00:40:23,000 --> 00:40:26,000
in helping the enzyme or the protein
do its thing.  And the distinction

514
00:40:26,000 --> 00:40:30,000
between these and these is
that they are larger.

515
00:40:30,000 --> 00:40:34,000
They're actually synthesized by the
body, as opposed to these which are

516
00:40:34,000 --> 00:40:39,000
just taken up in the diet.
OK.  This is an example, and I'll

517
00:40:39,000 --> 00:40:44,000
go through it quickly for lack of
time.  This is an example of one

518
00:40:44,000 --> 00:40:48,000
chemical reaction.
What we're talking about here is an

519
00:40:48,000 --> 00:40:53,000
enzyme called dihydrofolate
reductase.  This is an important

520
00:40:53,000 --> 00:40:58,000
enzyme in producing molecules that
are required to build nucleotides in

521
00:40:58,000 --> 00:41:03,000
your body, both DNA and
RNA precursors.

522
00:41:03,000 --> 00:41:06,000
Without this enzyme you cannot make
those things, you would be dead.

523
00:41:06,000 --> 00:41:10,000
It's a very critical enzyme both in
biology and in medicine.

524
00:41:10,000 --> 00:41:14,000
Dihydrofolate reductase happens to
be one of the targets of an

525
00:41:14,000 --> 00:41:18,000
important chemotherapeutic agent
called Methotrexate.

526
00:41:18,000 --> 00:41:22,000
It's used because cancer cells grow
a lot.  You want to inhibit their

527
00:41:22,000 --> 00:41:26,000
ability to grow,
so you inhibit their ability to

528
00:41:26,000 --> 00:41:30,000
carry out this reaction to produce
this relevant product.

529
00:41:30,000 --> 00:41:35,000
And also in bacteria it's the target
for a particular antibiotic called

530
00:41:35,000 --> 00:41:40,000
Trimethoprim.  Folic acid you know
that you take in from your diet.

531
00:41:40,000 --> 00:41:45,000
And it's then broken down in a way,
or not broken down.  It's modified

532
00:41:45,000 --> 00:41:51,000
in a way that it becomes useful for
these synthetic reactions.

533
00:41:51,000 --> 00:41:56,000
And that's the job of dihydrofolate
reductase.  Dihydrofolate has two

534
00:41:56,000 --> 00:42:02,000
hydrogens positioned at the seven
and eight positions.

535
00:42:02,000 --> 00:42:06,000
And tetrahydrofolate has four
hydrogens at these four positions.

536
00:42:06,000 --> 00:42:11,000
And it's the tetrahydrofolate that
you want to use,

537
00:42:11,000 --> 00:42:16,000
that you need to use for these
subsequent reactions.

538
00:42:16,000 --> 00:42:21,000
So the enzyme then, dihydrofolate
reductase adds hydrogens.

539
00:42:21,000 --> 00:42:26,000
It reduces dihydrofolate to
tetrahydrofolate.

540
00:42:26,000 --> 00:42:31,000
And it utilizes a cofactor NADP
shown in green here to do that.

541
00:42:31,000 --> 00:42:35,000
It's the NADP,
NADPH which transfers the hydrogens

542
00:42:35,000 --> 00:42:39,000
to the dihydrofolate.
This is a little bit hard to see

543
00:42:39,000 --> 00:42:43,000
because it goes pretty quickly.
You might look at it on the Web or

544
00:42:43,000 --> 00:42:47,000
at home.  This is the dihydrofolate
coming in.  You can see in white the

545
00:42:47,000 --> 00:42:51,000
enzyme actually moving.
It moves in order to carry out the

546
00:42:51,000 --> 00:42:55,000
chemical reaction.
And you can see in green the NADP,

547
00:42:55,000 --> 00:42:59,000
initially NADPH coming in,
interacting with the dihydrofolate,

548
00:42:59,000 --> 00:43:03,000
transferring the hydrogens and
allowing it to become

549
00:43:03,000 --> 00:43:07,000
tetrahydrofolate.
OK?  So that's one example of an

550
00:43:07,000 --> 00:43:13,000
enzymatic reaction.
OK.  In the final eight minutes,

551
00:43:13,000 --> 00:43:19,000
and we do have a lot to get through,
so if you could just hang in there

552
00:43:19,000 --> 00:43:25,000
until five of that would be good,
I want to talk about another form of

553
00:43:25,000 --> 00:43:31,000
regulation, and that is specifically
the existence of inhibitors

554
00:43:31,000 --> 00:43:36,000
and activators.
So, again, the regulation of enzyme

555
00:43:36,000 --> 00:43:40,000
function is extremely important.
You want to make sure that the

556
00:43:40,000 --> 00:43:44,000
enzyme is working at optimal rates,
higher or lower, depending on the

557
00:43:44,000 --> 00:43:48,000
circumstances.
And this can be adjusted naturally

558
00:43:48,000 --> 00:43:53,000
inside the cell by other molecules
that can function to inhibit the

559
00:43:53,000 --> 00:43:57,000
enzyme or to activate the enzyme.
And, as I said, we can also do that

560
00:43:57,000 --> 00:44:01,000
medically by making inhibitors or
activators that change the activity

561
00:44:01,000 --> 00:44:07,000
of enzymes inside our cells.
Terminology.  These inhibitors and

562
00:44:07,000 --> 00:44:13,000
activators can be reversible or
irreversible.  They can either bind

563
00:44:13,000 --> 00:44:20,000
and come off and bind and never come
off.  This takes the enzyme out of

564
00:44:20,000 --> 00:44:26,000
play.  It goes to the bench.
It cannot work anymore.  This one

565
00:44:26,000 --> 00:44:33,000
can come back off and the enzyme can
continue to function.

566
00:44:33,000 --> 00:44:37,000
They can be competitive.
And I'll show you what this means

567
00:44:37,000 --> 00:44:48,000
in a second.  Competitive --

568
00:44:48,000 --> 00:44:52,000
-- versus noncompetitive.
And, again, I'll tell you what that

569
00:44:52,000 --> 00:45:01,000
means in a second.

570
00:45:01,000 --> 00:45:06,000
Competitive is illustrated here.
If this is the active site and this

571
00:45:06,000 --> 00:45:11,000
is the substrate which would
normally fit into that active site,

572
00:45:11,000 --> 00:45:16,000
a competitive inhibitor, like the
name suggests,

573
00:45:16,000 --> 00:45:21,000
competes with the active site.
It gets in there and prevents the

574
00:45:21,000 --> 00:45:26,000
substrate from binding.
Pretty simple concept, right?

575
00:45:26,000 --> 00:45:31,000
A noncompetitive inhibitor
functions by binding somewhere else

576
00:45:31,000 --> 00:45:36,000
on the protein and changing the
structure of the active site.

577
00:45:36,000 --> 00:45:40,000
So here again is the substrate.
It could bind to this active site,

578
00:45:40,000 --> 00:45:44,000
but when the noncompetitive
inhibitor binds over here it changes

579
00:45:44,000 --> 00:45:49,000
the active site.
And now the substrate cannot bind.

580
00:45:49,000 --> 00:45:53,000
So it's not competing with the
substrate directly,

581
00:45:53,000 --> 00:45:57,000
but it's affecting the ability of
the substrate to bind

582
00:45:57,000 --> 00:46:03,000
to the active site.
Now, these noncompetitive inhibitors,

583
00:46:03,000 --> 00:46:09,000
and you can also have molecules that
activate the enzyme at these other

584
00:46:09,000 --> 00:46:16,000
sites, are binding to portions of
the protein that we call allosteric

585
00:46:16,000 --> 00:46:22,000
sites.  A term you should be
familiar with,

586
00:46:22,000 --> 00:46:29,000
allostery, allosteric regulation.

587
00:46:29,000 --> 00:46:33,000
And what this is,
again, are molecules.

588
00:46:33,000 --> 00:46:37,000
Sometimes they're products of the
reactions.  Sometimes they are other

589
00:46:37,000 --> 00:46:41,000
things that bind somewhere on the
protein, not at the active site,

590
00:46:41,000 --> 00:46:46,000
which change the nature of the
active site.  So in this example we

591
00:46:46,000 --> 00:46:50,000
see an allosteric site next to an
active site.  The binding of an

592
00:46:50,000 --> 00:46:54,000
activator can lock this enzyme,
it happens to have four subunits,

593
00:46:54,000 --> 00:46:59,000
into a confirmation where the active
site is open.

594
00:46:59,000 --> 00:47:03,000
Or another small molecule,
an inhibitor can bind to the

595
00:47:03,000 --> 00:47:07,000
allosteric site and lock the protein
into an inactive confirmation.

596
00:47:07,000 --> 00:47:11,000
OK?  So allosteric regulation,
sensing some other molecule and

597
00:47:11,000 --> 00:47:15,000
changing the activity of the enzyme.
Why would you want to do that?

598
00:47:15,000 --> 00:47:19,000
Before I get to that,
let me give you one real-life

599
00:47:19,000 --> 00:47:23,000
example of inhibition.
It comes from my world.

600
00:47:23,000 --> 00:47:27,000
Cancer drugs increasingly are
becoming more specific,

601
00:47:27,000 --> 00:47:31,000
and this is the best example.
I don't have time to go into it in

602
00:47:31,000 --> 00:47:35,000
detail now for lack of time,
but this is a drug made by a local

603
00:47:35,000 --> 00:47:39,000
pharmaceutical company called
Novartis which binds to one of these

604
00:47:39,000 --> 00:47:43,000
kinases, these phosphorylating
enzymes, important in a particular

605
00:47:43,000 --> 00:47:46,000
type of cancer.
It is a competitive inhibitor of

606
00:47:46,000 --> 00:47:50,000
ATP.  ATP needs to bind to an active
site for this protein to function.

607
00:47:50,000 --> 00:47:54,000
The drug called Gleevec is a
competitive inhibitor of ATP,

608
00:47:54,000 --> 00:47:58,000
therefore ATP cannot bind, therefore
the enzyme cannot function,

609
00:47:58,000 --> 00:48:02,000
therefore the cancer cells cannot
stay alive, therefore the cancer

610
00:48:02,000 --> 00:48:06,000
patient is cured.
Great example.

611
00:48:06,000 --> 00:48:11,000
It happens to be true.
So this is not just theory,

612
00:48:11,000 --> 00:48:15,000
not just bench biology.  This is
real-life in pharmacy in this

613
00:48:15,000 --> 00:48:20,000
example.  And this is a
three-dimensional picture of Gleevec

614
00:48:20,000 --> 00:48:25,000
in green bound to the active site of
the kinase called able shown in the

615
00:48:25,000 --> 00:48:30,000
red and blue ribbons.
OK.  So, again, feedback regulation.

616
00:48:30,000 --> 00:48:34,000
The reason that we have allostery
and changes in regulation relate to

617
00:48:34,000 --> 00:48:38,000
a diagram like this.
And the important points here are

618
00:48:38,000 --> 00:48:43,000
that these enzymes that we've been
talking about almost never function

619
00:48:43,000 --> 00:48:47,000
in isolation.  They're almost always
in pathways.  Something produces

620
00:48:47,000 --> 00:48:51,000
product one, the next enzyme works
on product one to make product two,

621
00:48:51,000 --> 00:48:56,000
and so on and so forth.  And you
actually, the cell wants to

622
00:48:56,000 --> 00:49:00,000
coordinate the activity of these
enzymes so that the right amount of

623
00:49:00,000 --> 00:49:05,000
product is made at the bottom
of the process.

624
00:49:05,000 --> 00:49:08,000
It's like the regulation of an
assembly line in a factory.

625
00:49:08,000 --> 00:49:12,000
You don't want to make too many
tires if you don't have enough cars.

626
00:49:12,000 --> 00:49:16,000
So when you have enough tires you
feedback the tire generation and you

627
00:49:16,000 --> 00:49:20,000
bump up the car generation.
The same thing happens in biology.

628
00:49:20,000 --> 00:49:24,000
This pathway A goes to B goes to C.
C splits to D and F goes to G goes

629
00:49:24,000 --> 00:49:28,000
to E.  Depending on how much G you
have, you might feedback on this

630
00:49:28,000 --> 00:49:32,000
enzyme to make less F.
And you might feed forward,

631
00:49:32,000 --> 00:49:37,000
you might have positive feedback on
this enzyme to make more E.

632
00:49:37,000 --> 00:49:41,000
And this regulation is done by this
sort of allosteric process whereby

633
00:49:41,000 --> 00:49:46,000
the G product to an allosteric
reaction might inhibit the enzyme

634
00:49:46,000 --> 00:49:51,000
producing F and might activate the
enzyme producing E.

635
00:49:51,000 --> 00:49:55,000
And this example shown here from an
actual metabolic process,

636
00:49:55,000 --> 00:50:00,000
the generation of isoleucine is a
specific example whereby when you

637
00:50:00,000 --> 00:50:05,000
make enough isoleucine the
isoleucine will bind back to the

638
00:50:05,000 --> 00:50:10,000
enzyme way up here in the pathway to
shut it down.

639
00:50:10,000 --> 00:50:14,000
Once I have enough isoleucine,
isoleucine binds to this allosteric

640
00:50:14,000 --> 00:50:19,000
site on this enzyme,
which now slows down the production

641
00:50:19,000 --> 00:50:23,000
of these intermediates,
and therefore results in the

642
00:50:23,000 --> 00:50:28,000
production of less isoleucine.
I left off two slides on energetics

643
00:50:28,000 --> 00:50:31,000
and ATP, but I'll mention
those next time.