[ Silence ]
>> Welcome back Chem-bio fans.
Today we're going to be talking
proteins and then we're going
to go on to much sweeter topics
as we transition
to glycobiology.
So I've got some really cool
stuff and story for you today
and I can't wait to get to it.
Specifically, we're going to be
talking about glycan structure.
These are the carbohydrates
found
on the surfaces of your cells.
The things that make
life taste sweet.
We'll be talking about
their reactivity.
How enzymes can hydrolyze them.
And then we'll talk about what
is it that they're doing before
and we talk about why they're
ultimately going to kill us.
So why is it that carbohydrates,
sugars taste so sweet?
Yet cause so much disease is
something that fascinates me.
I love to understand that
better and let's get started.
Hopefully, we will
at the end of this.
Oh a little picture,
I have a picture
of the great Hermann
Emil Fischer
in his laboratory around 1900.
This guy is a superhero
of chemistry
and he was doing things
like working out structures
of carbohydrates just
years after people figured
that carbohydrates actually--
that carbon actually
had stereochemistry.
So this guy is a tour de force
chemist, a giant in the field
of glycobiology and
chemical biology.
And he's one of my heroes.
OK. Some quick announcements,
I'd like you to read Chapter 7,
work the odd problems.
And then you're going
to be receiving back your
journal article reports,
sub time either today or
maybe in discussion section.
They are graded out
of 75 points.
I went for something
like a bell curve.
But I'll be honest.
They were really good.
I've got some fantastic
journal article reports.
And they were tough to great.
It was hard to come up with
the standard distribution.
I like to see that.
So nice job on this
book report--
on this journal article reports,
you're all to be
commended, excellent job.
All right.
So here's what we saw last week.
Last week I was telling
you about how it is
that proteins function.
What it is that makes
them so great
when they do the things they do?
And what we saw was
that enzymes work
by lowering the transition
state energy for reaction.
Doing this where you take a
transition state energy it's
up here and push it down
here has the need effect
of accelerating the
reactions, speeding it up,
pressing the go pedal
to the floor.
This is really cool.
This is powerful.
This makes biology possible.
So because these enzymes evolved
to bind to transition states,
analogs of these transition
states are very effective
as inhibitors of the enzyme.
They stick in there and they
plug it up like a whole,
like plugging up a whole in a
door or something like that.
They are very effective
at shutting
down the enzymatic reactions
and in fact we're going
to see a few more
examples of that today.
Indention, we saw last week
how enzymes take advantage
of their diverse functionality
of the amino acid side chain.
I'll show you for example,
examples of metal ions,
magnesium providing Lewis
acidity in the case of kinesis.
We talked about how lysozyme
has both bronsted acid
and bronsted base
functionalities.
And the two of them were like a
one-two punch where the acid was
up here and then the
base was down here
and then they switch places.
This is truly remarkable
chemistry in action and it's one
of those power tools in biology.
The next thing we saw is that
or this is what we're going
to see today is that
additional functionalities
from the vitamins,
co-factors can expand the range
of chemistry.
So we start with the easy
stuff, the bronsted acid,
the bronsted bases
but just you wait.
I've got more functionality
for you
and this other functionality
is super cool
because it allows a whole new
range of reactivity and access
to reactions that otherwise
were not be possible in water.
And then finally, the
major theme really
of our last lecture is
this concept of dynamics.
And this is one of
the challenges
that has tormented
me really for years.
I've always-- always
is too stronger word,
I've wanted to for the last
10 years understand why is it
that enzymes flap
around the way they do?
And how is that flapping
aid in their catalysis.
And what I showed you where the
sort of cutting edge theories
about how is it that
these things work?
And I showed you examples where
that flapping is both beneficial
and deleterious for
enzyme function, right?
We saw an example where the
enzyme protein kind they say was
munching away in a neat
waltz and we even called
that a waltz right because
I had three steps, one, two,
three just like a waltz.
And then sometimes, it got
stock on step one, two.
And I was doing almost
like a rumba,
it was going backwards
and forwards.
One, two, one, two.
I'm not going into the waltz
like it should have been
and that's a disaster for the
cell because when that happens
or it's a disaster
for the enzyme.
When that happens, the enzyme
is not going all the way
to make its product.
It's getting stock and
that's stockness is inherit
to the function of
a protein kinase
like protein kinase
A. This is a protein
that evolved not necessarily
to be a monster of catalysis.
It evolved to be a highly
regulatable molecular switch
where you can switch it on
and you could switch it off.
And that ability to get it
stock doing one, two, one,
two or it should be
doing one, two, three,
one, two, three is useful.
That's the kind of ability that
allows the protein kinase A
to be a useful tool
in cell biology.
OK. So hopefully, you're getting
some idea of how enzymes work.
I want to pick up on a
topic I kind of scheme over,
but its super important
and I feel--
I felt guilty frankly
for scheming over this,
this is just too cool
and too interesting.
So let's go back and
take a quick look.
I want to start by
talking about the class
of enzymes called
serine-based proteases.
This is a class of enzymes
like the cysteine proteases
that we saw last
time that relies
on having a serine functionality
in its active site.
And that's serine functionality
has an analogs role
to the cysteine found
on the active site
of the cysteine proteases.
In both cases the
serine or the cysteine--
in both cases those
functionalities act
as a nucleophile to attack
the amide bond that's going
to be hydrolyzed.
And so let's take a
quick look at this.
In the active site of
a serine proteases,
there is a catalytic
triad consisting
of the serine functionality
together with the histidine
and together of the
aspartic acid.
And this have the
remarkable ability to act
as a protein relay system where
the protein gets handed off
from one functionality to
the other and by doing that,
that actually allows the serine
to be a much better nucleophile.
So check this out.
In this case, what
we're seeing is
that the serine is getting
deprotonated by the nitrogen
of this imidazole of histidine
and it turn the imidazole is
passing off its other proton
to a nearby carboxylate
functionality
of an aspartic acid.
So again, it's a relay
system, proton comes of here,
get pass through an
intermediate, pass through over
to the center guy over here.
This is powerful stuff.
OK. So now he result in
alkoxide is a superb nucleophile
that can then attack
the carbonyl
of the amide functionality
and it turned this tetrahedral
intermediate can collapse
to give us a hydrolyzed
amide bond.
But wait there's more.
Of course, we now have
a covalent intermediate
where the serine of the enzyme
is stuck as an ester to fraction
to a half of the
hydrolyzed amide bond.
And so what the enzyme does
is it turns on this machinery.
And this time, it operates
to hydrolyze this ester
functionality giving you
in the end, a returning us
to a serine functionality
and giving us a carboxylate
as the second half
of the hydrolyzed amide bond.
This is pretty call chemistry.
I'm sure your chemistry were
seeing a charge relay system,
there's a lot of subltties here
that I'm kind of glossing over
but in the end, this is a very
effective cutting machine.
This guy gets up there and it's
like Edward scissor
hands going to town.
This thing just start
chopping a part,
and in fact it's
working right now
in your stomach,
maybe not my stomach.
I'm kind of hungry.
But if you have something
in your stomach
that involves proteins,
these guys are
at work chopping a part
those proteins as we speak.
All right.
Now, this is the part
that absolutely amazes me,
astounds me and, you know,
keeps me dreaming at night.
This is the catalytic triad
up close so check this out.
In this case, I shown
you the arrows,
that's the same thing I
showed on the last slide.
The truth is though,
these arrows are kind
of an approximation
that we used.
And instead a better
picture, a better depiction
of this would be to have
these hydrogen bonds
between the serine
functionality to the histidine
and to the carboxylate acid.
The problem is these
hydrogen bonds take all
of the joy out of arrow pushing.
They suck out the mirror
of what it makes it so fun
to push arrows and
send up protons
and electrons flying around.
And so, this is an
accurate depiction,
this resident structure
is an accurate depict
or this equilibrium is
an accurate depiction.
But I like drawing it like this.
And so the convention that we're
going to use is an understanding
that when we draw it like this.
And we have one step that
one step is occurring
in a consorted mechanism with
one fell swoop, swoop, OK?
So this isn't going
bonk here, takes a step,
waits a little while, bonk
here, no, instead, this thing is
with one fell swoop
stepping over
and doing the whole
reaction all at once.
All right.
Yeah. We'll skip
the zinc proteases,
it pains me to do this.
They're fascinating.
I'd like you to read about
this topic in the book.
These are really cool too.
All right.
Something that we saw is
that may proteases had a
proenzyme arm that protects them
from turning on until
in appropriate time.
And this is a specially imported
for the proteases implicated
in the blood clotting cycle
because you sure as heck,
don't want your blood
clotting before, you know,
at random times during
the day, right?
That would be a disaster.
And so, we saw that
peptides, the--
in the proenzyme can
be very effective
at blocking active site.
For this reason, proteins in
general are very interesting
as blockers proteases active
sites and these are found
in the blood sucking
animal of the world.
These are things like
leeches, ticks, vampire bats
and when this little blood
suckers grabbed on to you.
There, you know,
they got the fangs.
They will grab on.
They punctured.
They're sucking the blood.
They want to prevent
blood coagulation, right?
They're trying to
prevent blood clotting.
If the blood clots, they
don't get a tasty dinner
and so they've involved
inhibitors like this one
that block the active site.
The paradox here is not of
course proteases equally evolved
to chop a part proteins.
And so the real trick of
these types of inhibitors,
this protein-based protease
inhibitors is that they get
into the active site
but they stay away
from the catalytic triad that
I showed earlier which is sort
of highlighted here,
a little hard
to see perhaps, but
it's in there.
And so by keeping back,
they manage to avoid
getting hydrolyzed, yet,
they take advantage
of the abundant molecular
recognition opportunities
that span this protein and
zoom all the way around it.
And doing that, gives
it a very tight binder
and that's critical, right?
If these things aren't really
tight binders of the proteases,
its game over and the blood
sucking animals is going
to go hungry.
All right.
So this is the kind of thing
that interest chemical biologist
because it gives us control
over enzyme activity.
We're really interested
in developing inhibitors
of proteins and enzymes that
allow us to add the inhibitor
at a specified point
shuts off its activity,
and then study what happens.
This gives us a powerful tool
for figuring out the function
of that protein inside the cell.
And so, I'll give you
some examples, covalent
or mechanism-based protease
inhibitors are often
irreversible, not always.
And here's one example, this
is a chloromethyl ketone, OK.
Those ketone or chloromethyl
functionality,
and it positions beautifully
this electrophilic chloromethyl
substituent right up close
to the nucleophilic
histidine functionality.
And it also has the ketone
which can act as an electrophile
to attract the serine alkoxide.
So the serine alkoxide doesn't
look to carefully at things,
thinks this is an amide bond
and then gets in there and goes
to town and attacks because
that's what it was evolved
to do.
And it attacks there giving
it a tetrahedral intermediate
which binds very
effectively to the enzyme.
But at the same time, this
chloromethyl is position neatly
for SN2 reaction, an SN2 attack
on this methyl functionality,
this methylene functionality
over here giving us
another covalent bond.
And so in the end, this
enzyme is shutdown.
It can't do anything.
It's got a covalent
bond to this site
and a covalent bond over here.
And it has the enzyme
in a bear hug grip.
It's totally stuck.
It's not going anywhere.
This is game over
for that poor enzyme.
OK. So it turns out that
because these enzymes have
such nucleophilic functionality,
there are many ways
of inhibiting enzymes
that take advantage
of this nucleophilicity
and do these using
superb electrophiles.
OK. So its electrophile
meets nucleophile and it's
like the yin and the yang
of chemistry, magic happens,
and we see inhibition.
So let's take another
look at this.
Here is a related reaction.
This is a serine esterase
that hydrolyzes this
acetylcholine, OK.
So here's acetylcholine
over here.
Acetylcholine is found in the
synapses between your nerves.
It's a junctures between nerves.
It's one of the ways that nerves
talk to each other and it's one
of the ways that
you can actually,
you know, move it around, OK.
So the fact that I
could move around,
it's due to acetylcholine.
And it turns out that
the enzyme that breaks
down acetylcholine hydrolyzes
this ester bond right here.
OK. So little enzyme
comes in sniffs this of,
shuts down the acetylcholine
from signaling.
Oh and I should say
that the mechanism
for this is very similar to
what we saw when we talked
about serine proteases.
It's everything I've shown you
on the previous slides
still it lies here.
But check this out,
there is a series
of incredibly toxic really,
new electrophiles that get
into this enzyme active site
and permanently cap
it and shut it off.
And the problem there is
that now, you have no way
of hydrolyzing the
ester of acetylcholine
and the effect is paralysis.
OK. So here's what
I'm taking about.
I'm taking about a
series of nerve agents.
These are things like
sarin, tabun, VX.
These are insanely
toxic electrophiles
that get into the active site.
And here's the alkoxide, here's
the electrophile and in the end,
we have a covalent bond
to that serine nucleophile
that fact is death.
OK. So that's why these
nerve agents caused death.
You're paralyzed.
You can get your
muscles to move.
You can't get your
heart beating.
And it's game over for you.
That's why this compounds
are so dangerous and scary.
All right.
So this sort of-- these
are quick overview
of a serine proteases
and for that matter,
catalyzes of the
hydrolase catalysis
by the hydrolase
class of enzymes.
I want to switch gears.
I want to talk to you next about
enzymes that use co-factors.
These are sometimes
called vitamins.
This is the way that proteins
expand their functionality
beyond the 20 naturally
occurring amino acids.
If we had to rely nor if
the enzymes had to rely
in the 20 naturally
occurring amino acids,
yeah you know, life
would be boring.
They would-- maybe they
do it, but the truth is,
this other functionalities
that act as co-factors bind
to the enzymes, participating
catalysis.
These co-factors, these
vitamins dramatically extend the
abilities of enzymes
to catalyzed reactions
that the otherwise would have
be capable of catalyzing.
And many of these are
familiar to you especially
if you take a multivitamin
tablet everyday.
And, you know, these
are the vitamin Bs.
Here I have a-- so these
are bunch of vitamin B is
that I'm showing on
this slide over here.
And why don't we zoom
in, and take a quick look
at an example of this.
Here's one example,
this is vitamin B3,
this is nature's
sodium borohydride.
This thing works great.
Why does in nature
you study borohydride?
Well, you know, Sigma and
Aldrich didn't exist back
when nature was working
the stuff out.
But equally importantly
borohydride, aluminum hydride,
those things are
an exactly stable
in water unless it's
sodium borohydride.
OK. So for the most part,
hydrides not so stable in water.
So these evolved
as a hydride source
that still stable
in good old H2O.
All right so check this out.
What this enzyme does
is breakdown alcohol,
ethanol down to acid aldehyde.
The enzyme is called alcohol
dehydrogenase and it relies
on this vitamin B
derivative called NADH
or this is actually NAD plus.
OK. And in doing
this, this breakdown
of alcohol is absolutely
critical.
By the way, this is the
same alcohol that you find
in Mickey's big mouth,
malt liquor.
OK. That's the stuff.
This is the stuff that your
fraternity friends are drinking
while you're out studying
on Saturday night.
This stuff works, you know,
it is toxic to humans,
it causes all kinds of protons
we talked about that before.
And so this is the reaction
that detoxifies alcohol,
the first step in the
campaign to detoxify alcohol
and make the stuff go away,
so the headache disappears.
But in order to do this,
the enzyme has a
remarkable transformation
that I want to share with you.
OK. And all these
experiments worked
out by the great Frank
Westheimer about 50 years ago.
And back in the day, when
he's working the stuff out,
it wasn't exactly clear whether
enzymatic catalysis should
be stereospecific.
There's no reason really
for reaction like this one.
A trivial reaction really to
involved to be stereospecific,
OK, but it turns out
it is stereospecific.
And let me show you
what I've mean.
So if you feed the
enzyme alcohol
that has these two
deteriums next, you know,
[inaudible] deuteriums
next to each other.
You'll end up with a deterium
atom placed stereo specifically
in the NAD.
OK. So now this is NADH
but it has one deuterium.
And notice the deuterium
is coming out towards us.
OK. So just imagine that.
Deterium coming out
towards us right now.
If you take these stereo
synthesize NADH and feed it
to this enzyme, lactate
dehydrogenase.
Lactate dehydrogenase uses
this as a starting material
to do this reaction on lactate
this molecule over here.
And so what's happening is
the deterium gets stereo
specifically inserted into
the one it reduces this
ketone functionality.
So what ends up happening is you
get perfect stereospecificity
for the second reaction
over here.
And this is beautiful stuff.
OK. This makes my
heart go pitter patter.
Here's one, what we're seeing in
this very elegant experiment is
that enzymes all pulling
off hydrides in one way
and then delivering
them in another way.
And they're doing this
with perfect stereo
chemical fidelity every time,
getting better.
OK. Let's take a closer
look to try to understand
that when we look very
closely at the atomic details
of what's going on, actually
everything I just told you
totally make sense,
more peter patter.
The origins here, this
is the active site
of alcohol dehydrogenase
that's the enzyme I showed
on the previous slide, there is
a zinc ion in the active site,
Mr. zinc act as usual as a Lewis
acid and here it is neatly,
you know, tie together with two
cysteines, a histidine up here.
And so this zinc grounds
on to the alcohol.
So it's going to form a
nice Lewis acid arrangement
with the oxygen of
the alcohol over here.
And the vitamin B3
derivative, NAD plus is present
but stationed below
the zinc and below
where the ethanol
molecules is going to fit in.
OK. So this is a plain down
here, up here we have the zinc,
and then in between the
two, we have the ethanol.
And so when the hydride hops off
the ethanol, it has no choice.
It only has one route
available to it.
It's going to go hopping
down here and down here
and it's going to attack
specifically every time the top
base of this NAD plus.
Check it out.
It's going down here.
It's like, you know, this is
on you know, a water slide
at ranging waters or something.
It doesn't have any choice to
go in some other direction.
It's stuck in the shoot.
And so, because it's
directed, it's stationed
in a particular arrangement,
the geometry and such
that it only gives you a hydride
that's introduce over here.
The reverse reaction
is equally cool.
The reverse reaction has
an also stereo specificity,
it is going to every
time pick off the hydride
from the top base.
Only the top base is available,
bottom base not available,
top base available.
And that's really key to
understanding the observations
that Frank Westheimer made.
And the beauty of this, to
summarize, the beauty is
that this gives us an atomic
detail precise mechanism
for understanding why it is
that enzymes catalyzed reactions
was stereo chemical fidelity.
And again, this enzyme doesn't
have to be stereo chemical,
you know, perfect every
time but it involves to be.
And that's more or
less what we're going
to see time and again.
OK. So again, here we see
the vitamin B3 extending the
functionality of native
enzymes that's pretty cool.
Here's another example,
this is a friend of mine,
called vitamin B6,
tasty little bugger,
this guy does all
kinds of reactions.
OK. It makes cameos in all
kinds of different enzymes here
and there and in every time it's
providing crucial functionality
that equipped the
enzyme with abilities
that in otherwise would
not be able to acquire.
Let me show you some
examples of this.
This is a decarboxylation
reaction.
In this case, in the
enzyme active site,
this pyridoxal phosphate
forms a shift base
with the-- with an amino acid.
And that sets you up for
this decarboxylation reaction
with amino acid is decomposed.
Now, and this happens
for example
with glutamic acid deform
GABA, the neurotransmitter.
This is a sort of thing that
goes on that it takes place
in a lot of different cases.
Here's another one, this is
hydroxymethyltransferase,
this is aminotransferase.
Notice in every case,
the arrow is ending
up on this carbon over here.
OK. So we're going to build
up some negative charge
on this carbon that's
adjacent to the shift base.
And you're probably wondering
where are those electrons
got to go?
Really, really, I mean, are they
going to really hangout there?
What so special about something
it's alpha to a shift base?
Well, this shift bases are
analogous to carbonyls.
So check this out.
The electrons up here, that's
a negative charge that results
from each on these arrows, the
electrons up here can hop, hop,
hop, hop, hop, hop all the way
down to the positive
charge down here.
Check this out, this is
a resident structure.
Again, hop, hop, hop, hop.
And then the end they
bounced their way all the way
down to the positively charge
nitrogen which we know doesn't
like having positive
charge, its electro negative.
And the net effect
is that this has--
they stabilizes this
negative charge up there
and makes this reaction
possible.
Otherwise, this reaction
is not going
to go, there's no go there.
And so this is really powerful
chemistry and it's no surprise
to us that it should
be no surprise to us
that this makes multiple,
multiple appearances.
:Let's say, I want to show
you the decarboxylation
in a little greater detail.
Here we have-- this
is decarboxylation
of a hydroxylated tyrosine
called dopa to give as dopamine,
made famous of course
by the fantastic movie Awakening
starring Robert De Niro,
you haven't seen that movie,
you owe it to yourself
to rented especially when if
you want to go into medicine.
OK. So check this out.
Again, we had PLP, grabbed it
on to the amino acid using
the shift based handled
that we talked about earlier.
And now what happening is
we do the decarboxylation,
we get the negative charge
and electrons bounce,
bounce all the way down
here to be stabilized.
There's a whole resident
structure and I'm showing
but its happening and then
this can then get hydrolyzed
over here to give us
back the free amine.
OK. So shift bases recall our
reversible, the bond forms,
the bond breaks, the
forms, the bond breaks.
That sets you up
to do a reaction
and then release the
product over here.
OK. One more in the PLP world,
I can go on all day I loved PLP.
But I'm going to show you one
more, this is a really cool one.
This is an example
on a transamination.
So these are amino
acid, aminotransferases.
They take of the amino
from one amino acid
and then handed of to another.
They do that using
PLP as a co-factor.
And this makes pyridoxal
amine phosphate that's the
transcending intermediate
that grabs the amine
and makes this possible.
So here we are, we
have an amino acid.
It's bound up as a
shift base as usual.
In the enzyme active
site there an amine
that access a base deprotonates.
And we see this base that pulls
of the alpha proton over here,
we see it now acting
us an asset, OK?
This is [inaudible] kind
of stuff, right, base acid,
based acid, geez, it
cannot make up its mind.
But by doing this, this
kind of versatility
and reactivity equips the enzyme
with really powerful abilities.
OK. So here we go, with base
over here, acid over here
that gives us a new shift
base check this out.
Now, when the hydrolyzed
this guy.
We now have lost the amine that
use to be on the amino acid.
This gives us a new ketone.
OK. So again, shift base acting
as a reversible functionality,
and doing all kinds
of cool chemistry.
OK. Now you're probably
one you're in what happens
to this now weird PAP?
This PAP shown here can then
be used as an amine source
with the ketone, a
different ketone.
A Different Keto acids that
then becomes an amino acid.
OK. So in this case we see the
amine getting stored chance
transiently as PAP.
And then it hands off the
amine to another amino acid,
deform another amino acid.
All right.
At this point I usually ask
you if you have any questions.
I imagine you're
fussy slippers hanging
out drinking margaritas
or something.
I don't know what you're doing.
I don't want to think about it.
But if you have questions,
you e-mail them to me as usual
or you ask the TAs, please come
to my office hours, et cetera.
Let's change gears.
I want to change gear.
I want to talk you about
protein engineering.
This is a new field
relatively new field
and it has unfortunately
a terrible name.
The name is not a very accurate
description of what it evolves.
It does involved proteins,
but a very bizarre
type of engineering.
So, most of the time when
I think about engineering,
I think about, you know,
building, buildings or,
you know, engines for
cars or something.
And those cases, we understand
to an extraordinary
degree really.
The properties of the
materials that are being use
to build the stuff,
the buildings,
the engines, whatever.
Proteins in turns out, you know,
are made out of these
floppy materials.
And we're still don't understand
all of their aspects of folding
and all their aspect
semicircular ignition.
And so, it makes it very
hard for us to do atom
by atom, protein engineering.
It turns out that's
actually pretty nontrivial.
However, despite those
challenges scientist have been
doing this for several decays
and they've been doing this
with the goal of
improving protein function.
And also, understanding
how proteins work.
Why do we take a look at the
second example first, OK?
So, here's an example of
the kind of immunogenesis
that protein engineers do
to dissect the part
proteins work, OK.
So, in this case you start
with amino acid side chain,
and you convert it into
alanine, how do you do that?
You change around encoding
DNA, results in altered RNA,
was results immunoprotein
or protein variants as I
like to call them, because
mutants really should refer
to the DNA at the very top.
Now, here's what's
great about this,
if you do this mutation you
basically remove this hydroxyl
right here.
Notice that?
Notice we use to have hydroxyl.
Now we have a metal group, OK?
So, hydroxyl is gone.
It basically gives you
away of removing all
of those atoms pass
a bit of carbon.
And so now, you can
ask what function
if any the hydroxyl
group contributed
to this big complicated
protein over here?
OK. So, you're taking out 16
daltons of molecular weight
out of something it
might weigh, you know,
44 kilodaltons or something.
And you're asking, what is that
oxygen really doing for you?
This is a technique that I
like think of as the equivalent
of reverse engineering, you
know, about reverse engineering.
This is when Mercedes
buys up a BMW.
And then, you know,
proceed to check it apart,
maybe they remove some
wire and then asked,
how does the BMW performed under
still conditions or whatever?
OK. So, reverse engineering
is a powerful technique
and protein engineers have
been using it for years.
I've already shown you how human
growth hormone dimerizes the
human growth hormone
receptor, but--
and I also even showed you
pictures that look like this one
of the hotspot of
human growth hormone.
I did not tell you however, how
it is that we know what we know
about how growth hormone works.
So, here's the way this were
experiments done by Jim Wells
and coworkers at Union Tech.
And what they did was mutate all
of the very reduce
and growth hormone.
It turns out there 19 of this
side chains of growth hormone
and they systematically went
through and mutate it each
of those back to alanine, OK.
So, it's a mutation from--
let just say phenylalanine
to alanine.
And then they asked, what
is the contribution made
by that phenyl group?
When they do that, they find
that only these reds residue
or actually contributing binding
energy mutating this other
residue to alanine had
no effect on the binding
of growth hormone
for its receptor.
And furthermore, when they zoom
in they saw this beguiling
hotspot of binding energy.
These red residue look
like this and notice
that they have all this
hydrophobic stuff in the middle,
the green, and then
this is rimmed
by hydrophilic functionality
over here.
Its kind like a core
sample of a protein.
All right.
So, this teaches us stuff
about how proteins work.
I've been using it in
this class to tell you
about how protein work,
but it turns it also
as practical purpose.
And this is one of
the fun things
about protein engineering
trying to engineer protein
that do stuff that they
otherwise want to do.
And I mean this is kind of stuff
that you find in your house.
Its turns out that proteases
had been engineered using the
techniques of protein
engineering
to develop better proteases.
And I'm going to give
you one example of this.
So, subtilisin is a protease
that had some modern
specificity,
but it was pretty
broad spectrum.
And the goal was to engineer
a new variant of subtilisin
that could go out and
cleave apart any protein.
And the reason why you want
to do this is you'd want
to have a protease that can trap
apart proteins that form stains
on people's clothes, OK.
So, you've got a drop of blood
on your shirt or whatever.
You definitely want to
get that remove, right?
So, protease is bond
in these products going
in just literally Edward scissor
hand style start clipping apart
the proteins that would
otherwise stain the clothes.
So, the key was engineering
the pockets that bind
to the side chains
of these proteases
and basically, opening them up.
We giving it more space
that extra space means
that this subtilisin variant can
then accommodate a diverse array
of different proteins, OK.
So, you know, maybe this
is one day, you know,
chewing apart some pea soup
that lands on your jacket.
The next day its, you know,
I don't know, you know,
chewing apart some other
protein that it happens
to find the stain for.
So, this is powerful and this
is use in all wide variety
of different products.
This is the kind of
stuff you don't hear
about that's actually
superbly useful.
All right.
The problem is I told
you the good parts.
I've giving you the
greatest hits.
It turns out for
every greatest hits,
there's probably a dozen
total failure want to beast
that are lurking in the shadows.
And the reason for this is
that most mutations take
a perfectly good protein
and turn it into trash, OK.
So, most mutations make
proteins less functional
and here's our really
cool example of this.
In this example, this is
staphylococcus nuclease
and enzyme that digest DNA.
And in blue these
are amino acids
that can not tolerate mutations.
Every single one of
these blue residues
on totally resists
any substitution.
In yellow, those are the few
that allows some changes.
They can tolerate mutations.
And you'll notice, there aren't
that many yellow residues here,
the fast majority of blue.
So, random you mutagenesis
does not work so well.
It takes a lot of time
which is why, you know,
natural evolution doesn't
happen so, quickly either.
So, scientists have come up with
all kinds of more powerful ways
of introducing mutations.
We've talk about in class.
We've talk about for example
oligonucleotide directed
mutagenesis using quick
change, PCR, we've talked,
I believe about Kunkel-based
mutagenesis.
So, there are ways of
focusing the mutations
into particular regions
of proteins space.
And then, using evolution
as a powerful tool,
say using paid display for
example as a powerful tool
to evolve new functions
of proteins.
All right.
I'm going to skip this.
I want to talk to you
next about carbohydrates.
That's all I had to
say about proteins.
I can talk about it
for entire class.
Its one of my all time the
ever topics but, you know,
I have another things I
need to talk to you about.
So, we're going to
be switching gears.
We're now on Chapter 7.
We're going to be talking
about carbohydrates.
If you're not familiar
of carbohydrates,
if you were sophomore organic
chemistry class did not
cover carbohydrate.
I need you to go back and review
the chapter on carbohydrates
in that textbook that
you kept from Chem 51
or whatever sophomore organic
chemistry class you took.
Don't get to wrap up
in all the reactions.
I'm interested in reactivity.
And let me show you
what I mean by that.
So, carbohydrates are
hydrates of carbon.
We've already seen
for example ribose,
you've seen glucose before.
But they all have this
general formula of carbon
with the same number of waters.
OK. So, over here five carbons
and then five water
molecules despite
that rather beguiling
simplicity.
The truth is these things
are darn complicated.
These are, you know, battling
really to a chemical biologist,
not all chemical biologist,
but many chemical biologists
find this annoyingly battling.
Its really one of the
frontiers in chemical biology is
to better understand
carbohydrates, their properties,
their reactivity, their
function on the cell, et cetera.
And this is really a
challenge really for us.
They often have complex
structures that are difficult
to assign for example.
All right.
Before we go any further,
I need to introduce you
to some important nomenclature
that we're going to use
that you must memorize.
We're going to referring to
five-membered rings as pyranose
and six-membered rings,
six-membered carbohydrate
rings as pyranose.
This-- if there are five
carbons will be referring
to the carbohydrate as pentose.
Notice they are five
carbons here.
So, you can have both a five
carbon pyranose ring that's
pentose, and also, pyranose
ring that is also a pentose.
OK. I hope you now,
you're totally confused.
Here I'm going to rescue you.
You can have a six carbon hexose
that has five-membered
ring called the pyranose
or a six-membered ring, OK?
Make sense.
I hope so.
That's the nomenclature
we're going to be following.
All right.
This is one of those
extraordinary slides
that when I first notice this.
I was like, "I can't believe
this is true but it is."
OK? It turns out that
in the human body,
there's only nine carbohydrate
building blocks, that's it.
That's it, that's the sum total.
So it turns out that some of
the ones that you're familiar
with aren't really found in a
oligosaccharides that are found
in the surfaces of cell.
For example, ribose.
Ribose is not incorporated.
Ribose is not listed here.
OK. So although there's only
nine, you don't have to go out
and memorize them, OK?
So don't bother, you
know, learning all
of the carbohydrate building
box unless you're planning to go
into carbohydrate,
glycobiology or glycochemistry
which incidentally I recommend
it's a really exciting frontier
this across stuff going on.
Carbohydrate nomenclature,
unfortunately,
we're kind of stock with
the old tiny conventions.
There's just no way around it.
So and it turns out there's
actually those conventions make
a lot of sense, they
make our lives easier.
And so if you have
to convention,
that's kind of annoying but
it's make sense and it's easy
to use, so your stuck.
OK. So for example, we're
going to be referring
to the structure as D-glucose.
It has an R functionality
at this carbon over here
and also have more to
say about in a moment.
Check out how much better
it is to call it D-glucose
than to call it this crazy name
which would be the IUPAC name.
What is it with the
Ds and the Ls?
All right.
So straight up most
carbohydrates
on the nature are Ds, but
the truth is, we have some Ls
as well referring around.
And so we have to know what
this D and L business is.
The D and L nomenclature refers
to the carbon that's furthest
away from the anomeric carbon.
The anomeric carbon highlighted
with the gap here is the carbon
that has two oxygen's
attached to it.
Two oxygen's, oxygen
1, oxygen 2, anomeric.
And then if we go as far away
from anomeric carbon as we can.,
we look at the stereocenter
if it's at R stereocenter,
it will get the designation
D. If it is an S stereocenter,
it will get the designation
L. OK.
And, you know, here's an
example where it's not even next
to the oxygen over here so
this one the furthest away are
therefore it's the--
this one check this out.
OK. It's not even part of the
ring, the ring is all over here,
it's the way of on it
almost crazy side chain,
it doesn't matter.
OK. We still look at the stereo
center, that's furthest away
from the anomeric carbon.
In this case, it's R,
so therefore it gets the
D designation, make sense?
Good. That's what
we're going to using.
Now, here's the other
thing, the carbohydrates,
the anomeric carbon is
subject to some change.
OK. So this anomeric
carbon as we'll see
in the moment could either
have an alpha configuration
or a beta configuration.
The alpha and beta designation
refer to it's relationship
with this D, L set in carbon.
OK? Now, don't get
confuse, don't panic,
it's very straightforward,
alpha equals anti.
OK. So if this one is
up, and this one is down,
up, down, Egyptian style.
If these two are like this,
then we are going to have--
we're going to designate
this as alpha.
OK. The D again comes from
the carbon furthest away
from the anomeric carbon.
OK, so anti is alpha, same
side is beta, the stereo coming
out towards us that
both sticking up.
And therefore it's
going to be called beta.
OK. Very straightforward
designation,
it does take a little practice
so try it out, you know,
a maze your friends
at cocktail parties
or whatever it is you want
to do at this information,
it will be useful because
it's how we talk to each other
and you and I have
to be a little talk
to each other using
a common language
or else we won't know anything
about what we're talking about.
OK. I won't be listen to you,
you won't be able
to listen to me.
Som anyway, this is the
nomenclature we're going to use.
It is essential that
you learn it.
All right.
There is this notion
of an anomeric effect.
The truth is it's very modest.
So I'm going to skip that.
To really understand,
carbohydrate reactivity,
we first have to talk about
the reactivity of a hemiacetal.
And so it turns out that
carbohydrates often times are
found inter converting between
a hemiacetal configuration
and an open chain configuration.
And I can offer you two
different mechanisms
for this interconversion.
In one mechanism, we start
with acidic conditions
and protonate the
oxygen of the ring, OK?
So you protonate here and
then electrons bounce,
bounce giving us neatly
this aldehyde open chain
configuration of the ring.
Conversely, we do the same
thing under basic conditions.
But this time, we first
deprotonate the hydroxide taking
electrons bouncing, bouncing
opening up the ring, OK?
And there's a second arrow
in both cases that's
second arrow just refers
to a simple protonic exchange,
not even worth of talking about.
What this tells us that
is that no matter what,
all the carbohydrates are
susceptible deforming their
reactive aldehyde form.
OK. Notice the hemiacetal
has the aldehyde all bundled
up protectively, right.
The aldehyde is hidden away
but after under your acidic
or basic conditions, the
aldehyde gets exposed
and aldehydes are
simple for reactive,
they are electrophiles.
This is bad news, this is
why coke, you know, well,
I could say a lot about coke.
But this is why sugars in
general are not so good
to have floating around our
blood stream where aldehydes
like this one can find the
reactive functionalities wrap
up nucleophiles and start going
to town and forming all kinds
of uncontrolled products.
All right.
I'm getting off the topic.
Let's get back to the
topic, I want to talk to you
about stability in the ring.
I've shown you the ring can
come apart under both acidic
in basic conditions, there is,
however, a general rule of thumb
that tells us whether
or not the ring is going
to come part or not.
In general, the list
strained ring wins, OK?
So if it's a choice, a forming
a pyranose ring in the case
of glucose or furanose
ring on those ring,
this one is going to win.
OK. So the six-membered,
less strained,
then five-membered this
can form neatly a nice
chair configuration.
This one could form an envelope,
but still it's not
quite as good.
The seven-membered
ring can also form.
The truth is, we
never see this, OK?
That sugar stuff that you
ate with your, you know,
with your sugar puffs
this morning
on over your cereal or whatever.
None of it was in the
seven-membered ring.
We never see that.
This thing is super duper
strained and it's also
and tropically disfavored,
right?
This means that this carbon
over here that's flapping
around in the breeze has
to somehow get in to--
up close to the aldehyde carbon.
And it's just too far away.
So entropically disfavored,
thermodynamically disfavored,
all that adds up to bad news.
Let's zoom in and start
taking a look at examples
of carbohydrates
found in biology.
And no one is better at this
than the surface coatings
of the TB bacteria,
mycobacterium tuberculosis, OK?
So this is now a
little schematic view
of the outer surface
of these bacteria.
And check this out, this guy has
decorated itself like, you know,
Christmas in, you
know, some country
that really likes
Christmas and lots of whites.
Because this one is
[inaudible] gone to town
and has Christmas trees,
Christmas trees and
lots of whites.
The bacteria does this to escape
the immune system for one thing.
This stuff holds off the immune
system out of distance, OK?
But notice each one of these
little polygons is a different
carbohydrate, a different
monosaccharide.
And notice that they're linked
together into little chains
and then these chains
kind of branch off.
And the linkages of
these monosaccharides are
through glycosidic bonds that
I'll show you on the next slide.
But what we find is abundant
and highly diverse architecture.
This is doesn't look like, you
know, a smooth outer surface.
This is an outer surface
that's very rough that's
incredibly diverse.
There is all kinds of different
chains that are found here.
And we're just going to
have to do approximations
to describe these things.
This is going to make our
lives miserably complicated.
And it will make your
life miserably complicated
if you want to study
tuberculosis
because this gives the TB
bug a really potent weapon
for avoiding being tackled
by the immune system.
OK. So again, oligosaccharides
extremely complicated,
extremely complex.
Here's another example,
this is one example
from the cell surface,
check this out, OK?
So this guy has this
long chain over here,
all kinds of branchy points,
each branching point going off
in different directions.
But at the end over
here, there's a lipid.
The lipid sticks the thing
down into the plasma membrane.
This is a spike that
drives it straight
into the plasma membrane
and anchors it firmly.
So, this-- all these branch
stuff is like shrubbery.
It's kind of waving
around out there in space.
And it's anchored
firmly down here.
Its feet are stuck
firmly into the ground
because it has this lipid
tail that likes to be
down in the plasma membrane.
It has no choice but
to be down there.
All right.
So this is the schematic diagram
but the truth is we're chemist.
We're again a chemist.
We don't like thinking
about things
on this polygon representation,
instead, [inaudible].
We like to do things
much more complicated.
And instead, we like
to look at them
at the level of atoms and bonds.
And so, when we look closely,
we see this crazy complexity
where we have all kinds
of alpha glycosidic bonds,
beta glycosidic bonds,
and a very,
very complicated situation.
So, what's a chemical
biologist do?
I'm showing you the
worst case scenario.
Things are super complicated.
I want to step back
for a moment.
I will try to simplify
things so that
when you see a complicated
diagram like this,
you don't get all
daunted and scared.
Instead, I just want
to start off easy.
We're going to start off slow.
And then later when you
encounter these complicated
things, they won't
be as intimidating.
So, let's get started by
talking about formation
and breakage of glycosidic
bonds.
It's clear these glycosidic
bonds are important, right?
You know, this whole
thing is stitched together
by glycosidic bonds.
Here's one, here's
one, here's one.
Every single carbohydrate
here has a glycosidic bond,
so that should be
our first priority.
Glycosidic bonds are an either
a linkage between one saccharide
and another and one
glycan and another.
When we look at the mechanism
for the formation or hydrolysis,
they all take advantage of
the fact that the carbon,
this anomeric carbon is
adjacent to another oxygen.
This sets you up for forming
either an oxocarbenium ion
or an oxonium ion.
I think it's safe to say all
textbook show you this oxonium
ion type of configuration.
And it's not exactly wrong but
it's not exactly right either.
Instead, the truth is somewhere
between these two cases,
these two extremes.
On the one case, we
have a carbocation.
One the other case, we have
something that's even more
disgusting than the carbocation
which is an oxygen
bearing a positive charge
where oxygen being
electronegative and doesn't
like having that
positive charge.
In any case, this intermediate
sets us up for either hydrolysis
or for a new alcohol to
attack giving us formation
of either a hydrolytes
like acidic bond
or a new glycosidic bond.
OK. So in every case,
we're going to kick off either a
hydroxide down here or alcohol.
And that's going to set us
up with some possibly
charge intermediate
that can then be attacked.
OK. And it should make sense
to what this is going
to be attacked.
We've seen the mechanism
for this hydrolysis before.
And I'd like to remind
you of it.
We saw it when we talked about
lysozyme a glycosidase enzyme.
In that case what I emphasized
you was that the nuclei file
that was attacking
was going through kind
of an SN2 reaction, right?
It turns out it's somewhere
between SN2 and SN1,
in other words, the alcohol
that's getting hydrolyze--
the alcohol functionality
is getting hydrolyze kind
of steps a little
further at the door
than nucleophile attacking, OK?
So what I'm showing you is
I'm showing you how we chemist
like to represent intermediate
cases between SN1 and SN2
where we show a super long bond
here and then kind of implies
that we're going to have a
little more positive charge
down here which incidentally
makes it all the more attractive
for negatively charged
nucleophile
to come driving in, OK?
And notice too that I'm showing
you the substrate distortion
that was the hallmark for
lysozymes functionality.
Again, you start with this
wonderful little cozy chair
and the chair gets torqued
physically and doing
that sets you up for this
neat backside displacement
of the SN2 reaction.
OK so again, lysozyme, the
[inaudible] of chemical biology,
twist this chair and forces
it into this both confirmation
or twisted chair
confirmation setting
up this nucleophilic attack and
making this reaction possible.
I showed you one
example of lysozyme.
It turns out there
are many others.
There are many glycosyl
hydrolases.
And it turns out we
could classify them
as either inverting
or retaining.
In inverting enzyme--
Let's just start over here.
An inverting enzyme converts at
alpha anomer into a beta anomer.
And a retaining enzyme keeps
whatever stereochemistry
was there.
So, if it's beta
stereochemistry to start,
you finish with beta
stereochemistry, OK?
So, there's two possibilities
here.
They have two distinct
mechanisms.
Beyond saying that,
I'm not going
to get you wrapped up in this.
We've talked about this before.
All right.
Let's move on.
So we've talked about how the
fundamentals of forming a bond.
We've talked about the
fundamentals of breaking one
of these glycosidic bonds.
I now want to talk to
you about why it is
that this matters in
terms of diseases.
I have already shown
you tuberculosis.
Unfortunately, unfortunately,
I don't have a great way
that tuberculosis can be
cured using hydrolysis
of glycosidic bonds.
That's a frontier, maybe someone
at this class will be able
to solve that which
would be really cool.
Instead, I want to talk to
you about the common cold, OK.
Which I realized and
you realize is one
of those unsolved
challenges, right?
You know it's, you know,
practically a swear word to say,
"Why don't you get a
cure for common cold?
Why don't you do something
useful with your life
as though it would be so easy?"
So, here's the closest
that we've come.
There's an enzyme called
neuraminidase that is
that is a key enzyme in the
life cycle of influenza,
the virus that causes flu.
This enzyme helps to release
the virus from the cell surface
of flu infected cells, OK?
So here is the host cell, recall
that enzymes parasitically take
over the machinery of the cell
to produce new zombie copies
of themselves and then the
enzyme or the virus bugs
on the surface of the cell.
And after forms-- a fully
formed enzyme, it needs some way
of getting off of
being released.
And so this enzyme neuraminidase
cleaves the carbohydrate
that has it firmly
held in place.
So in ambition of neuraminidase
has been a key target
for influenza inhibitors
and therapeutics.
Every since I knew what the
term chemical biology means
which is a really long time.
Unfortunately, we don't have any
great solutions to the problem.
But let me show you the
best of what we have.
OK. So the best that we have
are things that kind of look
like the carbohydrate
that's getting cleaved
by neuraminidase.
These are substrate mimics.
We've seen substrate
mimics before, right?
AMP was like ATP except
that it wasn't, OK?
So here it is a compound
called Zanamivir
that actually looks a lot
like sialic acid especially
if you squint like this.
When I squint at it,
it really does kind
of look like sialic acid.
And that's good because
by looking sialic acid,
it can fit neatly into the
enzyme active site that have off
to bind to sialic acid.
And then here's another one,
another one called oseltamivir
that is actually given
to patient as a prodrug.
OK. So earlier, we
talked about proenzymes.
I don't mean earlier today,
I mean back on last
Tuesday's lecture.
We talked about proenzymes
as concept
of an enzyme that's
saying cleaved apart
to expose its active fragment.
Here we're seeing a prodrug.
We have to hydrolyze
or the cell has
to hydrolyze this ethyl
ester functionality and free
up the carboxylic acid in order
to have a functional drug.
In the absence of the esterase,
the enzyme does not work, OK?
But fortunately esterases
are dime and dozen.
And this strategy is
a very effective one
for hiding away negatively
charge functionalities that need
to be present to make
the drug function.
Negatively charged
functionalities, however,
affect things like the
ability of the drug to pass
through the hydrophobic
plasma membrane.
And so, this is the
way of making
that carboxylate a
little more greasy
and a little bit more
readily able to pass
through hydrophobic passages.
OK. Here we see a zoomed in view
of the active side
of neuraminidase.
And I'm not going to get
through the mechanism
because it has a
mechanism similar
to other glycosyl hydrolases
that we've talked about.
But check this out.
This is the-- this
compound over here
that a carboxylic acid
oseltamivir downed
in green to the active site.
And look at how beautifully
positioned it is.
OK. I mean I just want to
take a moment just to gaze
on all at this beauty.
Sorry, I can't help myself.
Check this out, there's this
positively charged arginine
precisely poised above the
negatively charged carboxylate
that's this after the ester
is hydrolyzed exposing it.
And you could see that's
absolutely crucial, right?
We have one, two possibly
charged functionalities,
two quantity functionalities
from arginine
that are perfectly
poised to grab on to
that negative charged.
So if you don't have
negative charged here,
the thing is not going
to bind, beautiful stuff.
OK. So chemist working on
this in a while-- for a while.
Unfortunately, our best shots
are on drugs that you take a day
or two after you get infected.
And they shorten the length
of time if you have influenza.
The real problem is, we're
not so good at recognizing
when you have influenza.
If we had a way of knowing,
yeah, you have a coupe
of viruses in influenza,
they're going to expand
and then give you a full
blown, you know, it's not
yet flu in a day or so.
We would be really effective
at treat again but we don't.
And to be that suggested
need for better diagnostics.
All right.
Let's change gears.
I want to talk to you more
about oligosaccharides.
We have to talk more
about nomenclature.
These things are getting
complicated really fast.
What we're going to be doing
is we're going to be referring
to the attachments, the carbons
that are attached to each other
on parenthesis over here.
And then we we'll have a
three letter abbreviation
to designate the monosaccharide,
the glycan that's
being attached.
So for example, this
is sialic acid.
Over here, that has an
alpha configuration.
Notice it is anti,
alpha being anti.
And it has an alpha
configuration and then linkage
between carbon 2 and
carbon 3, carbon 2,
carbon 3 to a galactose
functionality linked
to a glucose functionality
or actually this one
in an acetyl glucose
functionality.
So things are going to
get complicated quickly.
Don't panic, don't
get all worked
up about this especially don't
spend anytime memorizing all
this stuff.
Rather, I want you just to be
comfortable with the concept
and familiar enough
with the concept
that it doesn't throw
you a curve ball.
All right.
I want to talk to you about how
it is that these long chains
of carbohydrates of
oligosaccharides get formed.
Typically, the
glycosyltransferase,
class of enzymes uses
a diphosphate base
as a glycosyl donor.
OK and that by base, I
really mean like a DNA base
or actually an RNA kind of base.
In fact, they use a UDP--
Sorry, it's a GDP variant of
the starting material as a way
of activating the
starting material.
Recall that phosphates are
nature's tosylate or mesylate,
it's nature's living group.
And so this enzyme,
fucosyltransferases
starts with the fucose.
The fucose is now is attached
covalently to this diphosphate.
The diphosphate is going
to be the living group
and that sets you up performing
neatly this glycosidic bond
over here.
OK. So what's going on?
Hydroxide is attacking
this anomeric carbon
and then the diphosphate
is stepping out the door.
This again is that kind of
hybrid that we saw earlier,
hybrid SN2, SN1 reaction
where the GDP functionality
is starting to step
out the door a little
more quickly
than the hydroxyl is acting as
an SN2 nucleophile to attack.
But it's kind of a
hybrid of the two.
Similar transition state
to what we've seen earlier.
Let's get in to the
mechanism a little bit more.
Here is a picture of the enzyme
that actually does
this reaction.
And then here's what it looks
like in the active site,
pure beauty isn't it?
In this case, we have the
hydroxyl neatly placed
above this anomeric carbon.
Notice that this
guy is set up neatly
for backside displacement,
you know,
all the orbitals
are neatly in line.
This sigma star orbital
is precisely positioned
to have a lone pairs in
here, wind they're way
in nucleophilically and
attack this anomeric carbon.
I love this kind of stuff.
Is this pure beauty in action.
OK. Now, what this is good for?
What this is good for
is that it sets us
up for building really complex
structures out of carbohydrates.
And some of these complex
structures are kind
of familiar to us.
This top one is cellulose.
Cellulose is nothing
more than glucose.
You know the sweet stuff
that tastes so good?
Yeah it's glucose except it's
joined by beta glycosidic bonds.
OK. So the stuff for this
table, that glucose stuff.
That's actually-- it's
actually-- is this totally nuts.
The cellulose in what
is taste this glucose.
The problem is of course and
the reason why cellulose doesn't
taste so good to us
is that we have no way
of hydrolyzing these
beta linkages of glucose.
Instead, we're very adept at
hydrolyzing the alpha linkages
of starch, OK, shown here.
Starch forms this
[inaudible] that kind of wined
around each other
as a consequence
of having this beta linkage, OK?
So it's a beta glycosidic bond.
Difference between alpha and
beta could not be bigger, right?
One hand, starchy things tastes
good, that's the potato chips,
cellulose things,
not so good, right?
You start, you know,
chewing on a two by four,
let me know how good
that taste for you.
OK. So, these are ubiquitous
forms of carbohydrates
that are found in nature.
In fact, the majority of biomass
found in our planet is stored
and cellulose or
in starchy forms.
And so, this is totally
ubiquitous.
Another very ubiquitous bio--
polysaccharide and I'm
calling this polysaccharides
because they're just really
long chains of glycans.
Another very ubiquitous
polysaccharide is chitin.
So chitin is the outer
exoskeletons of insects,
of shrimp or crustaceans, right?
And I don't know about
you, but when I ate shrimp.
I'm one of those people that
usually peels off the peels
or I spit them out but I
don't like chewing on them.
I don't like eating them.
I do have friends that
for whatever reason,
they eat the shrimp whole
with the peels and everything.
The truth is, they don't
get any nutritious benefit
out of those peels, OK?
Because chitin is
indigestible to us.
It's actually the Aza
nitrogen analogue of starch.
It actually has the
same data linkages.
But it has this N-Acetyl
functionality attach--
that replaces a hydroxyl
of glucose.
And so, all those
exoskeleton of arthropods,
it's not digestible to humans.
We do not have functional
chitinases in our stomachs.
This is really too bad because
actually this would be a great
source of energy.
And it's very likely that
humans are human ancestors.
The-- not, you know,
homo sapiens humans
but the way distant
ancestors to us.
Actually, probably we're
capable of digesting these sorts
of shell exoskeleton things.
And we could see
evidence of this.
When we look in the
human genome,
we can see non-functional
chitinases
that are still carried along
which again suggested diet
that our ancestors ate.
That was very rich in bugs.
So it's very likely we're eating
all kinds of buggy things.
And maybe we had a functional
chitinase that would allow us
to digest the chitin and
get energy out of it.
All right.
More-- these are-- now
I'm switching gears now.
I want to talk next
about oligosaccharides.
Oligosaccharides,
we're going to define
as being much more
determinant link
of that more determinant
structure
where polysaccharides are
a little less determinant.
Here's the first example
I'm going to show you.
This is the oligosaccharide
that's found
in your knee joint, OK?
This is the oligosaccharide
that lubricates these joints
and makes a possible
top bone on bone stuff
without grinding a part of
the bones after 30 years, OK?
So hyaluronan is synthesized
as a continuous extrusion
on the cell surface of the cells
that are found near this joint.
It synthesized by chondrocytes
and it forms this weird
gel-like cushion, OK?
So in this case what's
happening is we're starting
with the UDP precursor.
And much as like what I
showed with the GDP precursor
on the previous slide.
The UDP is just a
superb living group.
And so, UDP steps out the door
in a new glycosidic bond forms.
And this basically gets extruded
by the contra sites straight off
into this joint region and this
gives us a nice hyaluronan gel
that cushions to joints.
What is it about this
that cushion joints?
OK. Well I don't think it will
come and said, he surprise you
to find out that this stuff
is very water soluble,
abundant opportunities
for hydrogen bonding,
hydrogen bonding here,
hydrogen bonding down here.
The carboxylate functionality
is very nice as well,
lots in hydrogen and bonding
that carbohydrate functionality
also pushes the strands apart
from each other.
This makes a nice cushion.
It makes a nice little
watery layer that soaks
up the water and is very stable.
All right.
Last thought of the day,
glycosylated proteins.
So I've been showing
you carbohydrates
that are kind of free floating.
What we find though,
when we look carefully
at cells is we find
the shrubbery
of glycosylated proteins
all over the place
on the outer surface of cells.
And when we come back next
time, we'll be talking about all
of the abilities that
this endows cells with.
So why don't we stop here.
Look forward to talking to
you when I get back from Rio.
[ Silence ]
------------------------------731e8b51c460--
