>> We're going to pick
up where we left off.
Hopefully you watched the
podcast lecture on Tuesday.
I want to pick up
what we discussed
on Tuesday looking
at protein function.
Specifically we're going to be
talking about how enzymes work.
How do enzymes catalyze key
reactions in the cell and so on.
We're going to start by
talking about some measures
of enzyme activity and then
we'll talk about regulation
of enzymes and then we'll
get into the mechanisms
and then we'll close with
mutagenesis and engineering.
So, what we talked
about on Tuesday is
that proteins have a wide
range of rolls, structural,
binding and catalytic roles.
An example of structural we
saw, for example, collagen
and we saw how collagen
gets organized
into these complex assemblies
that make it possible
to have extremely strong
bones and things like that.
We also talked a little bit
about titan, a muscle protein
that makes muscles capable
of being pulled and stretched
without breaking,
without snapping.
We also talked about binding.
The example we saw there
was FDBT [phonetic] binding
to FD506 and rapamycin.
Then finally we're
up to the part
where we could start
talking about catalysis.
So a protein function has
at least 3 major
roles inside the cell.
Structure, binding and
catalysis and when we talked
about the repeat proteins, for
example, we saw a great example
of binding and I want to just
very briefly we'll take a look
at that in a moment
but I just want
to emphasize something
based on some questions
that I got during
my office hours.
We talked about how non-covalent
receptor ligand interactions can
be described by dissociation
constants in an
on rate and an off rate.
We're now at the point where
we're going to start talking
about this Michaelis-Menten
constant, Km, which is an analog
to Kd and something
that I touched
on at the very end
of the lecture.
Enzymes work by lowering
the transition state enzyme
of the energy of the reaction.
Doing this is, in essence,
how you catalyze a reaction.
You're lowering the
activation energy necessary
for that reaction to take place.
You're increasing the speed
that the reaction takes place
by doing this and
enzymes do this by binding
to the transition state
and stabilizing it
by having counter ions that
stabilize charge functionalities
in the transition state the
enzyme can lower the transition
state energy and this is
surprisingly effective.
We're going to be seeing
some examples of that today.
So, we're going to
be talking today also
about how this catalysis by
enzymes is coupled to the motion
by the enzyme and I'll show
you some examples of this
and what we're going to see is
that enzymes force
the substrate,
the starting material,
into conformations
that favor formation of
the product and by doing
that that accelerates
these reactions.
Okay, very briefly I just
want to touch on one point
about the repeat proteins.
Something I didn't mention
but probably should have
when I talked about the repeat
proteins this is an example
of a repeat protein.
This is ankyrin repeat.
Notice that it has a series
of helix turn helix turn helix
turn helix turn helix turn helix
turn helix so those repeats,
each one of these loops
and then helix, loop
and then helix,
notice that they're all
stacked on top of each other.
Each one of those is
one example of a repeat
and this ankyrin repeat has a
whole bunch of these lined up.
That's where it gets
the name repeat protein.
Make sense?
Then similarly with
Leucine-rich repeats
in this case it's a helix
strand loop, helix strand loop,
helix strand loop et
cetera, but it's a series
of repeating motif,
repeating structural motif.
In fact, these are
actually individual domains.
They fill modestly well on their
own and they can be shuffled
around to a limited extent.
Okay. Any questions about the
topic of protein structure
and for that matter any
questions about anything
that was covered on Tuesday
in Tuesday's lecture?
All right I think in that case
then we're ready to move on.
We're going to talk
next about catalysis.
Today's discussion is going
to be pretty high level.
I'm going to be telling you
stuff that's absolutely not
in the book and, in fact,
actually it's only found really
in the frontiers of the
literature in chemical biology.
So don't hesitate to interrupt
if at any point I
start to lose you.
It's better if you interrupt me
early on than if I go further
down the road and then
you're totally lost
because the truth is this
lecture will be the only time
that you're going to be able
to find this material
I'll be discussing
and I think it's
very, very important.
It's actually the very
frontiers of chemical biology.
Okay so I want to talk to
you today about catalysis
and last time I showed you that
non-covalent binding consists
of ligands hopping on to some
binding site in a receptor
and we describe this
non-covalent interaction using
an equilibrium constant
especially equilibrium constant
called a dissociation constant
that quantifies the ratio
between unbound up here to bound
receptor ligand interactions.
So this is pretty
straightforward stuff.
The only difference with
catalysis is that we're going
to have this similar
receptor ligand interaction
but upon binding to the ligand
the enzyme, in this case instead
of the receptor the ligand
is going to be transformed,
converted into some new product.
So it's a similar
sort of process.
So if we understand
non-covalence interactions,
then we could also understand
catalysis by enzymes.
Okay, I'm skipping
ahead, skipping, skipping,
we talked about this already.
All right.
This is, I believe this is
where we left off, right?
Is this right?
So where we left off,
again, is this idea
that receptor ligand
interactions are governed
by some binding.
In the case of enzymes,
the substrate binds
and then some catalysis
takes place
to convert the substrate
into a product.
S stands for substrate
or starting material
and that gives us, P,
which is the product,
and then the product
has to dissociate.
I think I've talked
about this right?
This was covered, right?
I did cover this.
Okay, good.
So I think I'm actually
over here.
Okay, good.
So here's a typical reaction
scheme I already talked
about on the previous thing.
So, the formation of this
enzyme substrate product,
this Michaelis-Menten complex,
this is sometimes called the
Michaelis-Menten complex,
this complex here of
the activated enzyme
down to the substrate in a way
to catalyze the reaction
has an equilibrium constant
and in the same way that
the Kd was proportional
to the rate constant for the
dissociation the K off to K on.
In the same way,
the Michaelis-Menten equilibrium
constant is equal to the sum
of the off rate and
the K cat [phonetic].
So that's like getting to the
ES and either going backwards
or forwards; that's the rates
going in either direction
for destruction of the complex
versus forming the complex
meaning the rate constant
for K on.
This Km resembles the Kd
for non-covalent
binding interactions
and so it's useful for us.
The strength of the
Km tells us something
about how avidly the enzyme is
going to grab onto the substrate
and how quickly it's going
to form this ES complex.
So this cam is actually
a useful number.
It tells us something about
the conditions inside the cell
because the enzyme has to evolve
up to a sufficient affinity
for a substrate so it can grab
onto the substrate
inside the cell.
If the affinity is too low,
then the enzyme substrate
complex will never form
and the enzyme will never
catalyze a reaction.
On the other hand, if
it evolves to the point
where it's super duper high,
maybe that's not so useful
for the cell because maybe the
enzyme then will be a little
too active.
So these evolve up to the
maximum ability that's necessary
for the cell, it's required
for the conditions found
inside the cell and oftentimes,
for example, we can
engineer enzymes
to have very different
Kms simply by tinkering
with their active sites and I'll
talk about that more in a moment
when we talk about
protein engineering.
So if we look at a, if we look
at a dose response diagram,
this is similar to the dose
response diagrams that I showed
on a previous slide on Tuesday.
Instead on the Y axis we have
our initial reaction velocity
and the concentration
of substrate the point
of inflection here is
going to be roughly the Km,
this equilibrium
constant up here.
For that matter this point
of inflection also tells us
where 50% of the maximum rate
of the enzyme is going to be.
At the very highest
concentrations
of substrate the enzyme is
going to be running flat out.
So that's like as fast as
the enzyme can possibly go.
It'll be equivalent of giving
the sprinter maximum oxygen,
maximum glucose, everything
he or she needs to do
to run as fast as possible.
So that's up here under
maximum velocity conditions.
Notice that this asymptotically
approaches this Vmax value way
up here.
So, anything where it's
an excess concentration
of substrate that's
called Vmax conditions
and typically enzyme
reactions that are run
in the laboratory are run
under those conditions.
We always have an extreme
access of substrate typically.
Okay, let's take
a look at some Km.
They range widely.
There's a wide range of
possible Kms for enzymes
and here are some
numbers over here.
Now like the Kd a lower Km
value means tighter binding.
Totally analogous to the
dissociation constant.
In fact, it has a very
similar connotation.
So what this tells
us, for example,
is that this enzyme here,
cytochrome P450 binds
benzopyrine
with very high affinity.
This should come to
no surprise to us.
Benzopyrine is this big,
flat hydrophobic molecule
and hydrophobic molecules in
general aren't so soluble.
So the cytochrome P450 in your
liver is going to be grabbing
on to the benzopyrines that
you inhaled on your over here
when you got behind that stupid
shuttle bus that was, you know,
dinking along at
too slow a speed.
So you get behind the exhaust
pipe or that shuttle bus
and you start inhaling unburned
benzopyrines, cytochrome P450
in your livers right now
as we're speaking grabbing
on with great affinity
for these benzopyrines.
On the other hand, there are
some enzymes that don't have
to grab on all that well to
their substrates like aconitase.
This is a key enzyme in
metabolism of glucose
and its substrate, citrate,
is found at high
enough concentrations
that the enzyme doesn't
really have to evolve
to a very high affinity.
So this gives us sort
of a crude measurement
of what the concentration of
the substrate is in the cell.
So what we know is that
there's probably not a lot
of benzopyrine present
but there's probably
tons of citrate present.
Hence, the need for
lower affinity.
Now let's also take a
look at some K cats.
So this is the rate constant
for the decomposition
of the Michaelis-Menten
complex, the ES complex,
that is now being broken
down to form enzyme product.
Make sense?
So in this case again,
there's a wide range of K cats
and this tells us something
about how hard the
reaction is to catalyze.
Harder reactions in
general have lower K cats
but it can also tell
us something
about the evolution
of the enzyme.
Enzymes in general evolve
up to the required function
and really don't go past that.
There's really no, there's
no evolutionary drive,
there's no selection mechanism
that drives the enzymes
to be perfect unless
they need to be perfect
for some particularly
crucial function for the cell.
So, here's one example
of a really crucial
function for the cell.
The enzyme, catalase, breaks
down hydrogen peroxide
into oxygen and water.
This is a crucial reaction.
Hydrogen peroxide creates a
substantial burden on cells.
This is a strong oxidant
and oxidants run around
and wreck havoc on
cellular machinery and so
for this reason cells have
evolved pretty sophisticated
mechanisms to very quickly break
down such oxidation products
and catalase has a K
cat of 100 million.
So this is a really, really
fast catalytic reaction
that takes place.
A slower reaction
would be a protease.
Proteases, of course,
hydrolyze amide bonds.
I believe we've seen
these before.
Their K cats are
much lower likely
because this reaction is
a little more challenging
and a little less favorable
thermodynamically and also
for that matter it's not as
critical perhaps for the cell.
Okays so far?
We're good?
So these are the
numbers that are going
to underlie our discussion
as we start talking
about the properties of enzymes.
These are the same numbers
that you learned about in BIO99
or 98, whatever biochemistry
class you take here at UC Irvine
or elsewhere, these numbers
are kind of the vocabulary
that my biochemistry
friends talk
about when they talk
about enzymes.
Now the truth is as a chemical
biologist I don't get too worked
up about these numbers.
I'm more interested in
understanding the atoms
and bonds basis for
how the enzymes work.
So I guess the best place to
start would be let's start
with the perfect enzyme.
What would be the enzyme
that really can crank,
that could maximize its
ability to turn over reaction
and then we'll look
at some specifics
at the level of [inaudible]
bonds.
So the very perfect enzyme you
might imagine every time it
forms this Michaelis-Menten
complex, the ES complex,
then it goes immediately
to K cat.
So it forms and then boom
it's over to the K cat
and it just immediately
gets converted,
converts the substrate
to the product.
That happens instantly.
On the other hand, the
perfect enzyme is not going
to have any off rate over here.
This off rate represents
lost opportunities.
This is the wasteland
of could have,
should have, maybe should have.
This is the chance the enzyme
missed so instead of going
to product the enzyme goes
backwards and so this off rate
over here is miserable and
inefficient for an enzyme.
So the perfect enzyme is not
going to have an off rate
and so the perfect enzyme you
can basically imagine K off
being 0 and if we have that then
we can imagine rearranging our
Km equation, shown a
couple of slides ago,
such that K on equals
the ratio of K cat to Km.
Again, notice that these little
ks are indicating rate constant
and the big Km is for
equilibrium constant.
So, the very best
enzyme will have an
on rate that's diffusion
controlled.
In other words, it's
limited by the amount of time
that the substrate
rowdy [phonetic]
and motion style
eventually bounces this way
to the active site.
That should be the slowest step
for an enzyme that's perfect
and we've talked about
this before but that rate
of diffusion has a
speed limit of 10
to the 9th per molar per second.
Can't go any faster than
that; that's a physical law.
It's like the speed of light;
you cannot exceed that.
Just because it takes a
little while to bounce
around through all that water
and other stuff that's
present in the cell.
Make sense?
We'll take a look in
a moment at an example
of an enzyme that's far
from perfect and we'll start
to understand what its
sources of imperfection are.
So before we do let me just give
you a little table that I really
like that shows us and
helps us organize enzymes.
This shows us the
rankings of enzymes
in your proteome [phonetic].
So this is a listing from
most common to least common.
It's like a greatest hits of
the 7 categories of enzymes
that are found in
the human proteome.
The most common enzymes
by far are the hydrolases.
These are the enzymes that
introduce water as a way
of breaking a bond
and we're going
to see a couple of
examples of this.
We'll see examples
of glycosylases and
proteases today.
We've already seen
examples of nucleases.
That was stuff like
RNase, right?
Remember when we
talked about RNase
and it was inhibited
by DIPSY [phonetic].
This is a similar sort of thing.
Can someone help this guy out?
[ Pause ]
Thank you.
Okay. Transferases next most
common; second position.
These are examples of enzymes
that transfer functionality
from one spot to another and
we're going to look in detail
at an example of a protein
kinase today and then later
in the class we'll look
at a glycosyltransferase.
Oxcyto reductases [phonetic].
This is like the
enzyme cytochrome P450
that takes the benzopyrines,
introduces an epoxide
and oxidizes the benzopyrines.
You all remember this, right?
I showed you the benzopyrine a
couple of slides ago but earlier
in this quarter when I was
talking about cigarette smoking,
I showed you how the
benzopyrine that looks
like this rather innocuous
flat structure gets converted
into an epoxide and then slips
into the pie stack of your DNA
and alcolates the DNA.
So these are actually
very common enzymes,
these oxcyto reductases
because they're important
for removing toxins.
Dehydrogenases are another
one that's very common
and perhaps we'll get a
chance to see this one today.
Then finally we get
down to the ligases.
These are enzymes that spot well
together, two functional groups
such as attaching ubiquitin
or DNA to something.
Disomerases are used
to convert substrate
into some related
isomeric product.
These are things
like epimerases.
The synthetases we've seen
before we talked aminoacyl
tRNA synthetase.
This was that gargantuan complex
that read out the anticodon
and the various modifications
of the tRNA to make sure
that the correct amino
acid was being attached
to the tRNA during
aminoacyl tRNA synthesis.
So the final one, the lyases,
are doing things
like decarboxylation.
These are actually
breaking carbon-carbon bonds
in dramatic fashion.
So these are aldelases that are
doing aldol reaction, et cetera.
They're either breaking or
making carbon-carbon bonds.
So, I feel like we've
seen many examples
of these different enzymes this
quarter so now I can go through
and just talk about the ones
that are really important
that we haven't seen yet
such as the kinases over here
and I believe that's
where I'm going to start.
Yes, in fact it is.
So it turns out that kinases
have a common fold that consists
of a lower domain down here
and then a larger lobe up here.
The active site is indicated
where these Van der
Waal spheres are.
This is ATP so kinases take ATP
and transfer the gamma phosphate
of ATP to some sort of
hydroxide recipient.
That's generically
what they're doing.
When we talk about the
gamma phosphate, ATP,
adenosine triphosphate, ATP has
3 phosphate groups called alpha,
beta and gamma.
The third one in the
row is called gamma
and so that's the
phosphate group
that the kinases are
going to transfer.
So, again, notice that these
have a conserved dual lobe
structure even though these have
widely disparate activities this
is everything from a receptor
tyrosine kinase over here
to protein kinase over here.
These two have very
different activities.
They phosphorylate
different targets and
yet on the other
hand they all evolved
to have very similar structures.
Now, this class of enzymes like
all enzymes can be inhibited
by substrates, pseudo
substrates,
that mimic the real substrate.
So here's a structure of ATP but
in place of one of the oxygens
in ATP highlighted in
blue we have a nitrogen.
This phosphoramide
inhibits the kinase.
So if you feed this
phosphoramide to a kinase,
to [inaudible] the kinases I
showed on the previous slide,
it's going to be
game over for them.
They're not going to be able
to work because they're going
to bind to this ATP analog
and they're going to put it
in a sloppy old bear hug
but this gamma phosphate
is missing the oxygen
and missing the oxygen
is the same
as saying it's totally inert.
So this is going to basically
be locked in the active site
in the brace of the
active site yet unable
to transfer the phosphate group
and so the net effect is to shut
down the enzyme and this
is a very effective way
of killing enzymes.
You basically know
something about the mechanism,
you make a tiny little
modification of the substrate
and boom it's game
over for the enzyme.
Okay, so you could do this
also with sulfur or nitrogen
so I have shown you the
nitrogen but you could do
that as well and, again, it
sticks in the active site
and inhibits the enzyme.
This approach also works
if you mimic the product
and a large number of
enzyme inhibitors pursue one
of those two approaches
either mimicking the substrate
as shown here or
mimicking the product.
Either approach works great.
So, let's zoom in.
I showed you the structure, the
bi-lobe structure of kinase.
Let's zoom in and take a
look at its active site.
In the active site, there are,
here's the structure of ATP.
There are a series of
conserved magnesium ions,
these balls over
here, that are bound
to the phosphate
groups of the ATP.
The numbers here indicate
an angstrom the distances.
These numbers are pretty low.
If you recall that a
carbon-carbon bond is somewhere
on the order of like
1.5 angstroms
or so these are pretty
close in numbers, right?
This magnesium is
getting awfully close
to this oxygen over here.
These are cozy, cozy
molecules and atoms;
they like being this close.
They like being this close
because they have
complementary charges.
Right? The magnesium
has a plus 2 charge,
the oxygens of these phosphate
groups have negative charges.
So they're attracted by salt
bridges or Coulomb interactions
that we saw earlier
in the quarter.
So, over here there
is the other substrate
for this enzyme reaction
that has a hydroxyl.
I'm showing it to you with
the hydroxyl deprotonated
and after phosphoryl this oxygen
of the substrate has now picked
up the phosphate
group and notice
that the magneskims
[phonetic] here are helping
to stabilize that
phosphate group.
That they're lowering the
energy of binding by forming
that same sort of Coulombic
soft bridge that we saw earlier.
Okay, now if we zoom
in and take a look
at the arrow pushing mechanism
for this enzyme active site,
what we find is that, and not
depicted on this previous slide,
somewhere out here there's
a carboxylate [inaudible]
from aspartic acid.
The carboxylate of the
aspartic acid deprotonates,
the proton of the hydroxy of
a serine for the substrate
and that sets us up
with an alkoxide.
Alkoxide being a
superb nucleophile,
it's negatively charged, can
attack the gamma phosphate
of ATP and, again, the
magnesiums get in on the action.
They're over here
participating fully
and stabilizing this negative
charge on the phosphate group;
that's crucial, right?
You can imagine this reaction
not going in the absence
of those magnesiums because 1
negative charge is not going
to want to approach a negatively
charged phosphate group.
The negatively charged
alkoxide over here is going
to be stymied in its attack.
It's going to get to repelled
by this phosphate group
so the magnesiums are
shielding the phosphate group
and protecting it and preventing
it from getting, from looking
like a negative charge
and so that tees
up this reaction very neatly and
then finally there's a collapse
of this trigonal
bipyrmidal intermediate
and just very briefly
the structure
around this phosphate looks
like a trigonal bipyramid.
Not so, anyway that's
interesting,
and then there's collapse
of this trigonal
bipyrmidal intermediate
to give us our final product.
Okay, so to summarize
the most important aspect
of this is the notion that
the magnesium ions are playing
several roles to make
this reaction possible.
First, they're coordinating
and stabilizing the transferred
phosphate group as a Lewis acid.
So that helps accelerate the
reaction that's a Lewis acid.
Turns out that kinase activity
in the cell is very tightly
regulated and the reasons
for this are perhaps not
clear if you don't know much
about signal transception
[phonetic] and I'm just going
to very briefly cover
it today and then
in a future lecture we'll learn
quite a bit more about it.
In the cell, there's
a series of pathways
that transfer information and
these pathways are controlled
by a transfer of
phosphate groups
to key residues in proteins.
So kinases play a
really key role
in kicking off various
processes in the cell.
So there's cascades of kinases
where 1 kinase phosphorylates
the next kinase
which phosphorylates the next
kinase and so on and so forth.
Turns out that this process
is very tightly regulated
because you don't want
your cells going wild.
You don't want them to be doing
uncontrolled cell division,
for example, and so for this
reason the cell very tightly
regulates kinase activity and
I want to show you a couple
of vignettes about this tight
regulation because it's crucial
to our understanding
of how kinases work.
Okay. So, here's one example.
This is an example from the
enzyme protein kinase A,
cyclic A and P regulated kinase
and the way this works is
there's actually a regulatory
subunit here in blue
that binds to the kinase
and actually has
an inhibitory loop
that blocks access
to the active site.
So does everyone see how this
dark blue thing is binding here
and then it has this long finger
that fits into the active site
and blocks the kinase from
binding to any substrates.
This shuts down the
kinase and the ability
to shut off the kinase
is crucially important.
If you don't have this,
the kinase will be running
around rampant wrecking
havoc turning on stuff,
shedding off stuff causing
death and destruction
and general mayhem and I do
mean death and destruction.
These kinases are
that important.
Now, when levels of a reporter
molecule called cyclic AMP
[phonetic] reach a
certain concentration,
this cyclic AMP binds to
the regulatory subunit
and causes the regulatory
subunit to dissociate
from the catalytic subunit
of protein kinase A.
So these 2 molecules get forced
apart as the blue one flips
into a new confirmation
upon binding
to cyclic AMP the
thing changes its shape
and it no longer has affinity
for protein kinase
A. This is good news
for protein kinase A.
It frees it up to go off
and do the mission
that its wanted to do
for its entire life, which
is to run around the cell
and phosphorylate
anything that moves;
nearly anything that moves.
It actually has a
little bit of specificity
but for the most part
protein kinase A likes
to phosphorylate lots of
different binding partners.
This is a pretty
promiscuous molecule.
Now here's the thing another
way of regulating enzymes is
to phosphorylate them.
So this is one way we have some
regulatory protein that binds,
a second way is to
phosphorylate residues
that are near the active site.
So, for example, this
non-hydrolyzable analog of ATP,
which has the nitrogen in place
of oxygen this is
the molecule I showed
in a previous slide this tells
us where the active site is
but over here are 2 residues
that can be phosphorylated
to flip on this map kinase, this
P38 gamma map kinase and so one
of these is a tyrosine
and the other is a serine
and so this kinase waits around
until it gets phosphorylated
and at that point
it goes into gear.
So this is like an on/off
switch for the kinase.
In the absence of this,
the enzyme doesn't have
the right confirmation.
It doesn't have the right
confirmation it can't be
a kinase.
So the phosphorylation of
the kinase puts it in gear,
turns it on and sets it going.
Make sense?
Any questions about
what you've seen so far?
Okay. That's the basics.
I want to talk to you about
the really neat stuff,
the latest results in
thinking about how kinases work
and thinking about their
motions and, again, this is kind
of an abrupt departure from sort
of standard material presented
in biochemistry classes
and it really represents the
frontier in chemical biology.
Many of the next
experiments I'll be talking
about we're done
actually with Miriam.
She's one of the
leaders in this area.
So, the thing is I want to talk
to you about how enzymes work
at a mechanistic level and how
they actually work dynamically,
how do they move when
they do these reactions.
So I should tell you
that enzymes have great
motions associated
with their activities.
This is unlike the case
of conventional catalysis
by organometallic complexes that
you learned about back in Chem51
or if you learned
about it in Chem125.
In those cases, the
organometallic catalysts binds
and perhaps it plays
some Lewis acidic role,
but we don't think
about its motion.
We don't think about it
having some, you know,
movement associated
with it, some dynamics.
Enzymes it turns out for the
most part almost all have very
wild and very quick
motions associated with them
and a frontier in
chemical biology is
to understand how those
motions impact catalysis.
How do those motions
allow enzymes
to be effective catalysts?
So, it turns out that if
you get a big, you know,
round bottom flask full of
enzymes, you'll never be able
to see those individual motions
and the reason is they
tend to get blurred out.
So, if we look at a large
number of molecules,
we'll never see the individuals
in motion because all
of the enzymes in that flask
are going to be running along
at different speeds and
everything gets blurred out.
So instead in order to
see individual motions,
we have to look at
single molecules.
To understand this a little bit
better let me offer an analogy.
Let's imagine that I convinced
the Orange Company Marathon
folks to reroute the
marathon so instead of being
on Sunday morning,
instead I convinced them
to run it here Thursday at 10:10
AM and I convinced them to start
at that end of the classroom
and send all of the runners
through the classroom from
that door to this door.
So now everyone is running
through here all 10,000 runners.
You can imagine what you're
going to see is just a blur
of pumping arms and legs.
Everyone is going to be
trying to get in and out
of this classroom as
quickly as possible.
It's going to be
total pandemonium.
That's the situation when we
look at an ensemble of enzymes.
Its total pandemonium.
It's a blur of arms and legs.
We don't see anything;
everything gets averaged out.
By see I mean using
tools like spectroscopy,
using tools that you're familiar
with from your other classes.
So over the last 15 years
there's been a revolution
in this area of chemical
biology or biophysics
where scientists have started
to look at individual molecules
in isolation from all of their
other friends and neighbors.
So now instead of having
the entire marathon coming
through the classroom
let's imagine
that I convince each
runner to come running
through one at a time.
So they're going
to start over here
and then come running
through here.
What you will see because
each runner is isolated
from all the other runners is
you'll be able to see their arms
and legs moving, right?
Because now there's no
blurring out effect.
Furthermore if you look
closely, you'll be able
to see some runners
moving faster than others.
Maybe one running has
a different stride
than her neighbor.
She comes running through
and I don't know maybe she
extends her leg a little longer
than the runner behind her,
but if we convince them
to be isolated from each
other, then we can really start
to get information
about how they move
and that's a situation
we find ourselves
in when we start looking
at enzymes and so this area
of single molecules allows us
to look at confirmational steps
at intermediates and the
kinetics and dynamics
that underlie enzyme
function and this, again,
is a major frontier and
it's a really exciting area
to be involved in research.
Okay, so everyone
with me so far?
Everyone understands
this idea of looking
at single molecules right?
Okay, good.
I want to talk to you next
about how we're going ought
observe our single molecules.
There are a number of
different fluoresce techniques
for looking at single molecules.
Patch clamping you
may have heard
of is a 40-year old
proven technology
that works really
well for looking
at individual receptors;
that works fine.
In the last 5 years or so,
groups here at UC Irvine
have been at the forefront
for inventing a sort of tiny
little microphone that allows us
to listen in to enzymes as they
run and it has some advantages
over those other techniques.
So that's what I want to
talk to you about today.
So, this is a collaboration
between my laboratory
and Phil Collins in the
Department of Physics here
at UC Irvine and he's pioneered
ways of building circuits
that are based on
carbon nanotubes
and this is an example
of a carbon nanotube.
This is basically a carbon
graphite graphene layer that's
folded up into a cylinder.
It looks sort of
like chicken wire.
These wires though are
amazingly conductive.
Carbon nanotubes are
really remarkable material,
they have remarkable
mechanical properties,
they have remarkable properties
for deducting electricity
and for conducting heat.
In terms of conducting
electricity,
all the electricity is going to
be flowing through the outside
of the wire through
these bonds out here
and the electricity
is not flowing
through the middle of the wire.
So this is unlike, for example,
the copper wires that are used
in wiring the walls;
wiring the electrical outlet
over there in the walls.
This property makes the outside
of the wire superbly sensitive
to tiny little protobations
[phonetic] on its surface
and that's what we're
going to do.
So, here's the way we do this.
Students in the Collins
Laboratory
and my laboratory start
with silicon wafers
that are about this big.
We go to the engineering
building across campus.
The students put on bunny suits
and we build using
photolithography circuits
that look like this.
There are these contact pads to
which we attach wires and then
down here you get down to these
inner digitated electrodes
that do not touch each other.
So this is an open
circuit, but somewhere
out here we sprinkle an
iron malignum catalyst
that catalyzes growth of one
of these carbon nanotubes,
a single-walled carbon
nanotube across the wires
to complete the circuit
and I false colored
it in red over here.
Okay so now what we do
next is we turn this wire
into the world's
tiniest microphone.
We turn it into a device called
the field effects transistor.
It's not so important
how that works.
What matters is it's
more or less the same
as the microphone
found in my cell phone.
Same principle.
Next we're going to glue
individual proteins directly
onto the microphone and
listen as they flap around.
So runners, if we had
runners running through here,
you'd expect to hear the
pounding of their feet, right?
You'd expect to be able
to interpret the noise
of their feet to
tell us something
about their stride whether
or not they're accelerating,
whether or not they're slowing
down, et cetera, whether
or not they have funny
heel strike, et cetera.
Right? Make sense?
So we're going to do the
same thing but with proteins.
Now I know that you're
probably thinking proteins don't
have noise.
I have proteins all over my body
and I'm not hearing
anything right now.
The truth is the noise
is very, very tiny.
It is so tiny that
it's very hard to hear,
but moving charges do make noise
and I'll give you
one example of this.
If you're at the beach
with a bonfire down here
at little Corona
Beach State Park
and you have this
big bonfire going,
you know how the whip
kind of whips the flames
and the flames make this
kind of neat whooshing noise?
That sound of the flames
moving is due to plasma
in the flames that's
charged ions in the flames
that are moving around.
So charged functionalities
make noise as they get pushed
around and, in fact,
actually there's a speaker,
a loud speaker, these are
really expensive stereo speakers
on the order of like $20,000
a pair, they better sound good
at that price, but it's actually
based upon having a plasma
that's moved around by
a little magnetic coil.
So you can actually hear
charged ions moving around
and that's what we're going to
do when we glue in the protein.
So let me show you what it looks
like when we have
protein glued in.
This is the schematic
diagram over here,
we have the carbon nanotube,
here's a protein glued in,
this protein is streptravidin,
familiar, right,
to everybody in this classroom?
We have streptavidin conjugated
to a tiny little dot of gold
and that's shown here.
So the little dots here
that's the gold attached
to streptavidin and then the
horizontal lines are the wires,
the carbon nanotubes, and the
vertical are the electrodes
over here and you can
see we're getting 1,
1, 1, 1, 1 attachment.
So the breakthrough that
Phil and I came up with
with our coworkers, our
friends the graduate students,
was that we developed
a way of attaching one
and only one attachment each
time to the carbon nanotube.
This means then that we're
isolating the enzymes away
from all their buddies, which
means then we can start looking
at confirmations
and intermediates.
Okay, this is a long
introduction.
We're going to get back to
the kinases in a moment.
Before I do let me
just set the stage.
Here's the experiment again.
We have the electrodes, we
have the carbon nanotubes.
It turns out that, of course,
you can't run your cell phone
in water so you can't run one of
these tiny microphones in water.
I think I did this
experiment last week.
I dropped my phone
in a bucket of water,
actually it was my cat's
water bowl, and I pulled it
out quickly enough
but it definitely did
cause some damage.
So electronics and
water don't mix.
I don't think it surprises
anyone in the classroom.
So what we have to do
is, but all biology,
of course, takes place in water.
So this creates a dichotomy
and to solve this what
we do is we cover up all
of the electronics with a layer
of polymethyl methacrylate,
this is shown here in gray,
and then we blast the little
tiny window using something
called an electron
beam that just opens
up a little tiny region of
the carbon nanotube and that's
where we're going to
do the experiment.
Now all of the images
I have been showing
up to now are electron
micrographs using
electron microscopy.
We're now going to
get really small
as we start imaging individual
molecules of proteins.
This is so small that you can't
really see them very readily
using electron microscopy
except if you use that trick
that I showed on a previous
slide where you cut things
with gold; that was
a streptavidin thing.
So now we see these things
we now are going to have
to use atomic force microscopy
where we're now getting
down to really tiny resolutions
of 1 nanometer or so.
So here's what it looks like.
This is 1 enzyme attached
to the carbon nanotube
and this is an AFM image
atomic force microscopy image,
just showing the
windowed region,
just showing the carbon
nanotube that's exposed.
This little blob right here is
actually the enzyme attached.
It has the right dimensions for
that 1 enzyme and now we turn
on the microphone and it's
lights, camera, action, okay?
At that point then we're
ready to listen in.
One more image.
This is before and
then that's after
and where it's circled you
can see very clearly the
enzyme attached.
Everyone still with me?
Questions so far?
All right.
Yes?
[ Inaudible response ]
Oh, thanks, these other
blobs that are [inaudible].
Those are other little enzymes
that we can't get rid of.
It's actually enormously hard
to do these sort of images.
It just turns out the
proteins are kind of sticky,
there might even be some salt
crystals somewhere out here.
So there's always
some garbagy stuff
that we've been totally
unable to get rid
of despite a lot of work.
It took a lot of work to get
images that are this clear.
[ Inaudible response ]
Okay, that's a good question.
That's a really good
question actually.
Thanks for not being on the
study section that asked that.
So the question is would
an enzyme in isolation
from itself behave differently
than an enzyme that's next
to its neighbors in the same
way that a crowded field
of runners is going to run
differently than a solo runner.
We don't know.
I would like to think it's
going to run the same,
but it is a legitimate caveat
and I thank you for that.
I will have to think
about that some more.
Thank you.
>> So why do we study that?
[inaudible].
>> Yeah, so, enzymes
frequently aren't really,
so it's true enzymes are
in really crowded conditions
inside the cell, but it's not
like there is like
a thousand enzymes
that are all doing the same
thing crowded together.
It's more like there's a
couple of enzymes that are kind
of jammed in with hundreds of
other molecules inside the cell,
you know, so they're not
all doing the same thing.
So we can recreate
that sort of thing.
We can recreate the crowded
conditions inside the cell
and compare.
We haven't gotten
to the experiment
but I'd love to do it.
Okay so let's get
back to kinases.
So, again, this is protein
kinase A. It still has the 2
lobes that I showed earlier.
Here's the big lobe down here
and here's the smaller
lobe up here.
Somewhere close to this
upper lobe Miriam together
with Lisa Moody, a
former graduate student
in the laboratory,
engineered a single cysteine.
The cysteine, of course,
has a sulfur functionality,
a thiol [phonetic]
functionality and that allows us
to attach site specifically
the enzyme to the nanotube,
to this microphone down
here through a pyrine.
So a pyrine is making
yet another cameo
in today's lecture.
As you might expect, the pyrine
is going to pie-pie stack
onto the carbon nanotube because
it's so hydrophobic it's looking
for a nanotube to stick
on to and it turns
out the enzyme is very
firmly held in place.
This is like a very special
kind of molecular glue
that sticks these 2
molecules together but notice
that it's being held in a
non-covalent interaction
and in practice this thing is
held in place for 10-12 hours,
we don't see it coming off.
It's really stuck in
there very firmly.
Again, here's another AFM image
and that little blob attached
to the carbon nanotube
is our enzyme.
Yeah, question over here.
Sergio?
[ Inaudible response ]
Yeah, so we do AFM
this technique
of atomic force microscopy
before we get started
with the experiment just to make
sure that we have 1 attachment
and we use a special
technique called in liquid AFM.
If we see 2 attached, we wash
it away and then start over.
If we see 0 attached, we
start over at the attachment.
Question over here?
>> So when you have 1 and you
use AFM I'm sure you have 1.
>> Yeah.
>> For example the second
one is attached afterwards
when you start [inaudible].
Afterwards we don't
add any more enzyme.
So we have like purified buffer
that has no enzyme around.
So, and I know what
you're thinking.
You're wondering what
about this blob over here?
What if it decides to get
up and wander over here?
It turns out these blobs
are pretty firmly stuck
down on the surface.
Another thing is if
another one attached,
we'd hear that one running
alongside it in the same way
that 2 runners would
make different sound
than 1 runner, right?
You'd expect to hear a different
rhythm do we could detect that.
We don't see it.
Other questions?
These are great questions
you guys.
Yeah over here?
[ Inaudible response ]
So, we only add 1 enzyme so we
know that we have PK around,
yeah, and then the
AFM image confirms
that we have 1 that's attached.
All right.
Any questions in the back?
Anthony?
[ Inaudible response ]
Okay, I'm getting to that.
Okay. Let me show you.
Okay. [Laughter]
Anthony is impatient.
Okay. So you guys have exchanged
songs by Sound Cloud, right?
Raise your hand if you don't
know what Sound Cloud is?
Okay, this is great.
You guys are totally savvy.
So when you do Sound Cloud, you
know how like there's this post
that comes with it
and it tells you
about the loudness of the thing?
I'm going to be showing you
images that are like that,
data that's like that
where we're going to
be watching noise.
That's shown here where
this is time on the X axis
and on the Y axis this
is the current flowing
through the nanotube.
So that's going to be our noise.
Enzyme by itself flutters
around a little bit
but for the most part
it's totally quiet.
So if you don't feed the
runner, you know, some oxygen
or glucose, you know,
some bars or whatever,
the runner doesn't
start running.
Enzymes are like that too.
Unless they get substrate
this enzyme happens
to be totally quiet.
Some enzymes we find kind
of randomly flutter around.
In our technique, we will
not be able to pick that.
Now when we add ATP, we see
a new blimp that appears.
You see this lower
blimp over here?
Each one of these corresponds
to the ATP down state.
So we go from up here where
the enzyme is open to down here
where it's bound and then back.
We can measure how long the
enzyme spins in this bound state
and derive a dissociation
constant, a KD, for this enzyme,
this PKA/ATP interaction.
When we do that, we
find that KD corresponds
to what's measured
ensemble kinetics.
That tells us that actually
we're seeing something very
similar to what's seen in
the more crowded cases.
Okay? Next we wash away
the ATP and then add
in a peptide that's
a peptide substrate.
This has the serine
hydroxide that's going
to be phosphorylated by
the gamma phosphate of ATP
and again we see some
intermediate confirmation
as the enzyme goes
from open to bound.
Again, do you see how there's
like more blimps down here?
The enzyme binds
to its substrate,
it's peptide substrate,
with greater affinity.
It grabs on tighter,
it has a lower KD.
Everyone still with me?
Make sense?
Check this out.
This is the really cool one.
This is the enzyme plus
ATP plus the peptide
and now we see 3 levels.
So this is 1 second over
here, this is now 2/10
of a second I'm zooming in.
What we see is that these 3
levels correspond to ATP bound
and then [inaudible] down which
then gives us a catalytically
committed confirmation.
So when the enzyme starts
working, it goes between open,
intermediate and catalysis.
I'm just going to
call these 1, 2, 3.
This is the waltz of PKA.
So here's the enzyme waltzing
going along 1, 2, 3, 1, 2, 3, 1,
2, 1, 2, 1, 2, 1, 2, 3, 1, 2, 1,
2, 1, 2, 1, 2,3, 1, 2, stuck, 3;
1, 2, 3, 1, 2, 3,
1, 2, 3, 1, 2, 3.
So that's an enzyme in action.
This is what the enzyme looks
like as it goes about
its business.
Now what's hugely
inefficient is the K off.
Do you remember earlier I told
you the perfect enzyme should
have 0 K off?
In this case, we see
the enzyme in real-time
in action being inefficient.
Here it is being inefficient
as it goes 1, 2, 1, 2, 1, 2.
This is it with a K off.
That catalytic inefficiency
is what dooms the enzyme
and makes it waste
opportunities.
The enzyme is trying to make
up its mind, go to product,
back to substrate, product,
substrate, product, substrate,
and that lack of decision
is what makes this enzyme
inefficient as a catalyst.
There are other kinases such as
kinases involved in metabolism
that are far, far more efficient
that this enzyme over here.
If you're looking for something,
could I ask you to wait
until after the class is over?
Oh, no, you're here for the
class; you're just kind of late.
Okay, no problem.
Welcome. All right.
So anyway this is the enzyme in
action and what this shows us is
that the enzyme fluctuates
speed enormously.
This is kind of mind boggling
and I'm just going
to tell it to you.
It turns out that the enzyme
speed fluctuates from second
to second by a factor of 100.
So this is like going
out to the 73 out here,
the freeway out here,
and then you know,
pulling up alongside
a Honda Civic
and then suddenly the Honda
Civic goes from 55 miles an hour
to 5,500 miles an
hour, and then back
down to 50 miles an hour all
in the course of a second.
So these enzymes are widely
changing their speeds.
They're changing speeds
much faster than any runner,
they're changing speeds up
to speeds that are widely
that are almost inconceivable
to us humans
and really that's
the stuff of life.
It's essential that this enzyme
is able to alter its speed
in order for it to be regulated.
Remember earlier I talked
about the regulation
approaching kinase A.
That regulation is going to
control its speed and in doing
so that turns the enzyme
from being a widely
efficient catalyst
to being a catalyst
that's not even worthwhile
that doesn't operate on
a timescale that's useful
for the cell and this
is really the essence
of how catalytic
biology takes place.
Any questions about this?
Yeah?
>> Do you see any
pattern along the on/off
or just random process?
>> Brilliant question.
We spent a lot of time looking
for patterns in our data
and looking for correlation
between one step or another
and we, the short
is pretty random.
There's a small amount of a
memory effect in the sense
that the enzyme if it hits
the intermediate state,
it's more likely to go down to
3 than it is to go back to 1.
So that's actually a
thermodynamic effect
that the enzyme has evolved to
do K cat in preference to K off.
All right.
Let's talk about a
different enzyme.
The second enzyme I want to talk
to you about today is lysozyme.
This is an example
of a hydrolase
and remember I'm moving down or
actually I'm still at the top
up here in our most
common enzymes.
This enzyme was discovered
about 100 plus years ago
and it's been intensively
studied.
This is the X-ray crystal
structure of lysozyme and,
in fact, it was the very first
enzyme X-ray crystal structure
ever solved was this enzyme.
The active site up here has
an angstrom hinge motion
and so this enzyme has kind
of a packed down type motion
as it hydrolyzes
the glycosidic bonds
of the polysaccharides bound
on the cell surface
of bacteria cells.
So here's a bacteria cell
wall and an enzyme is going
to cut apart the glycosidic
bonds between each one
of these glycan [inaudible]
found on the cell wall.
So it's going to be chopping
apart the polysaccharide.
In doing this, this will
basically burst apart the cell.
In the absence of this, the
enzyme is basically going
to be chewing apart the
bacteria and this has the effect
of killing the bacteria.
You're breaking the cell
walls, they explode, et cetera.
This enzyme is found in high
concentration in chicken eggs;
that's the hen egg whites
over here, and it's present
to prevent colonization
by bacteria
and you might recall
avidin was isolated also
from chicken egg whites.
So biochemists have been
studying what makes eggs
so special as sterile
vessels for a very long time.
Okay let's take a quick look
at the arrow pushing mechanism
for how this enzyme operates.
In this mechanism,
the enzyme goes
through a covalent intermediate.
Let's start over here.
So, I'll zoom again now to
the polysaccharide region
of the cell wall
of the bacteria.
The enzyme is going to cleave
this bond that's indicated
with an arrow and
this is an example
of a hydrolase meaning
it's going
to introduce water
across this bond.
So the first thing that the
enzyme does is torque this
N-acetylmuramic acid
[inaudible].
So it's going to
torque this carbohydrate
from being a nice
chair confirmation
to being a boat confirmation.
This is a crucial aspect
for what makes enzymes
such effective catalysts.
This enzyme is going
to be catalyzing a reaction a
thousand times more efficiently
than if the reaction just
had to happen by itself.
I actually think it's
like a hundred thousand
times more efficiently.
In order to do this,
the enzyme is going
to be physically
bending the substrate
and by physically bending
the substrate this helps
to accelerate the reaction.
So here it is pushed up
into this boat confirmation.
Notice that the boat
confirmation neatly sets
up an SN2 attack by this
carboxylate of aspartic acid
to attack back side displacement
style, this glycoside.
This is crucial.
So the glycoside gets protonated
by 1 glutamic acid and then
over here a carboxylate
from a nearby aspartic acid
then attacks doing a backside
SN2 displacement.
This protonated glycosidic bond
then is a very effective leading
group because the second
arrow highlighted in red
over here kicks electrons to
a positively charged oxygen
which is all too eager to
accept those electrons.
Okay, this gives us a
covalent intermediate.
This is another common way that
enzymes accelerate reactions.
In this case, we're seeing it
form a covalent intermediate
and then this covalent
intermediate gets hydrolyzed.
So, half of the polysaccharide
floats away,
water comes in gets deprotonated
by the glutamic acid
the glutamate up here
and then this hydrolyzes
the ester bond
between the polysaccharide
and aspartic acid.
So some notable features here.
I'm showing you an example
of acid base catalysis.
The enzyme is simultaneously
acting as both an acid over here
and as a base over here.
In fact, it's even
wilder than that.
Check this out it's the same
functionality this glutamic acid
that acts as both the
acid and the base.
To me that's an elegant
simplicity
that makes enzymes so beguiling.
Right? If we were in the
chemical laboratory trying
to make molecules using
glassware and such,
we'd either dump in a bunch of
acid or dump in a bunch of base
but you wouldn't add
simultaneously both acid
and base because they
would neutralize each other
and enzymes have
evolved to be able
to simultaneously catalyze
things using both acid
and base catalysis.
Furthermore, this
enzyme has evolved
to form a covalent intermediate
and sometimes this is referred
to as a ping-pong mechanism
that eventually gives us back
the hydrolyzed glycosidic bond.
Really, really beautiful.
This is what you learn
in biochemistry classes
and the problem with it is that
it neglects enzyme dynamics,
which are really critical.
As the enzyme moves, it can then
help to torque this conformation
of the ring into this
boat conformation.
In the absence of this movement,
it doesn't make sense
really why it is
that the enzyme is
actually going
to be torquing this substrate
because the substrate binds
in the chair conformation up
here why should it get pushed
into this other conformation
unless the enzyme is doing the
pushing and that, in
fact, is what we see.
So, same idea we're
going to attach an enzyme
to the carbon nanotube and then
listen in as the enzyme works.
When we do that, here's
a paper from the lab
from about a year ago.
What we see is that, again,
the enzyme by itself is
relatively quiet and then
when we add the substrate,
the polysaccharide,
the peptidoglycan that I showed
on an earlier slide
the cross link net,
there's an immediate jump upward
and then there's all
this noise in here.
This is the enzyme
chilling on a substrate
and we get to listen in.
It's just like Sound
Cloud basically.
Okay. Some controls.
I haven't been showing
you controls
but these are everything
in biology.
This is substrate by
itself in red overlaid
and then this is enzyme
by itself again it's
relatively flat and purple
and then what we have enzyme
plus substrate we see this
notion where what we're seeing
here is enzyme open, closed,
open, closed, open, closed,
open, closed, open, closed,
open, closed, open,
closed, this is, you know,
on a third of a second
but we get
to watch the same enzyme
cranking over for a long period
of time and, again,
what we find is
that the enzyme is
highly variable.
It accelerates, it slows down,
it speeds up, it slows down,
it accesses different
conformations.
In fact, it acts as at least 2
dramatically different speeds
and you can actually see
that in the 40 seconds
of data over here.
Do you see how there's
this dense region
and then a less dense
region and a dense region?
The dense region corresponds
to rapid switching.
The enzyme has an
overdrive gear that it goes
into so it flips
gears to second gear
and it starts cranking
along at a much faster speed
and you can see that over here
where the enzyme is going open,
closed, open, closed, open,
closed, open, closed, open,
closed, open, closed, open,
closed like 300 times
per second whereas
over here it's doing open,
closed, open, closed, open,
closed, open, closed, open,
closed like 50 times per second.
So this is dramatic.
It's 6 times faster
and what's crazy is
that the enzyme does
this all day long.
It switches between first gear
and second gear, first gear,
second gear, back and
forth and a big mystery
in the field is what's
up with second gear.
So to address that question
I'm going to skip some stuff.
To address that question
together
with collaborators we
chemically synthesize aversion
of the polysaccharide that
didn't have cross links.
This is like 1 strand of the
net that I showed you earlier.
Same polysaccharide now we're
using chemical synthesis
to access a new substrate
and what we find is
that the enzyme has a
different type of activity.
I do have to show you
some more controls.
I can't get away from
this they are crucial.
These are mutant
enzyme active sites.
Do you remember earlier I
showed you the carboxylates
that are required for
enzymes to operate?
So we mutated those
carboxylate residues
and the enzyme no longer works
and, therefore, it never closes.
The other one of these enzyme
mutations traps the covalently
bound form of the substrate
that I showed earlier
in the ping pong mechanism
and the enzyme never can
hydrolyze back off the substrate
and, again, it never
gets back to closed.
Okay, so I get to finally tell
you what the difference is
between first gear
and second gear.
This is a day in the
life of an enzyme.
Here is how it spends its time.
So this is an enzyme cranking
along happily being fed either
the linear substrate or
the cross link substrate.
In the case of getting
the cross link substrate,
it gets to hydrolyze things
about 50% of the time,
but if you feed it the linear
substrate, it goes wild.
It gets hydrolyzed glycosidic
bonds 88% of the time
and so then this over here
is second gear in blue;
that's non-productive
rapid shatter and over here
in the cross link the blue
is much more apparent.
So, in other words, the
cross link substrate,
the substrate found on the
surface of the bacteria cell,
corresponds to second gear.
So what we think is happening
is the enzyme is mowing
across the surface of the
cell chewing contentively bond
after bond after bond after bond
happily hydrolyzing them all.
Then it hits one of
these peptide cross links
and gets stuck and when it
gets stuck its response is
to start chattering away.
It flips gears and it just
starts going 6 times faster
and what we think
is happening is
that it then transits
along the peptide
down to the parallel
polysaccharide.
So in the same way that DNA
is a bi-prime directionality
and 3 prime directionality
polysaccharides have a
directionality as well and
it turns out the surface
of the bacteria cell
is a highway
of parallel polysaccharides.
So the enzyme comes along
hits a cross link, goes down,
zooms along, hits cross
link, goes down, zooms along,
down, across, down, across.
So what the enzyme is doing is
zigzagging across the surface
of the cell as it chews apart
the surface of the bacteria
and in retrospect this totally
makes sense because, again,
the enzyme evolved to
poke holes in bacteria
and by doing a 2
dimensional rip in the surface
of the bacteria this makes
the enzyme much more effective
at killing its bacterial
targets.
Questions?
Yeah?
[ Inaudible response ]
Yeah, well, it's a
non-productive chatter
that we think is
moving along one
of these peptide cross links.
Yeah, Anthony?
[ Inaudible response ]
Until it finds a
new glycosidic bond
and then it goes to town again.
Okay, let's move on.
I want to talk to you
about other enzymes.
We have lots to talk about.
I want to talk to you very
briefly about proteases,
which cleave amide bonds.
We've seen these, we've
seen examples of these.
In blood, there's a whole
cascade of proteases
that are used to respond
to damaged blood vessels
with a series of factors; factor
7A, factor 10A, et cetera,
proteases where one cuts one and
then the next one cuts another
and the next one cuts
another, et cetera.
All the way down to the point
where you get production
of fibrin which then could
crosslink to form to replace
and fix the damage up here.
So this is an important
mechanism for blood clotting
and naturally if you're missing
any one of these proteases
or one of these proteases
happens to be mutated,
you're in big trouble.
Your blood will not clot.
This happens in inbred families
such as the royal families
of Europe and the turn
of the century this is the
Czar Nicholas II, whose wife,
Alexandria, passed on
the gene for hemophilia
to their son Alexei down here.
Again, this is a mutation it's
either factor 9 or factor 8,
which are both X-linked genes.
These are genes found
on the X chromosome
so they're passed
along by the mother.
Okay, apoptosis is
also regulated
by a series of proteases.
Apoptosis, the cell suicide
mechanism that we talked
about earlier in this quarter,
and each protease activates the
next one so you have a protease
up here called a
cast [phonetic] phase
that cleaves the next
cast phase in line
which then cleaves
this, et cetera.
So this guy cleaves this guy,
which cleaves this
guy, et cetera.
Let's take a closer look at
an example of a protease.
The protease I want to show
you is one of my favorites.
It's isolated from one
of my favorite fruits,
papaya and it's perhaps
appropriately called pupae
[phonetic] because it's
isolated from papaya.
It's an example of a
cysteine-based protease.
Furthermore, it's
another example
of a nucleophile-based enzyme
mechanism and I chose this one
because cysteine, of course,
is the preeminent nucleophile
as illustrated earlier today
when we talked about engineering
in a single cysteine on the
surface of the enzyme as a way
of attaching it to a specific
spot on the carbon nanotube.
So here's the mechanism
for how this enzyme works.
In practice, it's actually a
fairly complicated mechanism or,
sorry complex mechanism,
in the sense
that it's a concerted mechanism.
Specifically here's the
nucleophilic file functionality
of the cysteine in active site.
I don't think I pointed it out
here but here's the cysteine;
that's the business end of the
molecule in its active site.
In a concerted mechanism,
the cysteine is simultaneously
deprotonated
to attack nucleophilically
the amide bond
and then this amide bond
carbonyl gets protonated
by an acid residue that hovers
above the carbonyl
of the amide bond.
This happens in one fell
swoop from nucleophile
to protonation all at once.
That is really, that
kind of concerted dance
of catalytic efficiency
is yet another example
of what makes enzymes so special
the fact that everything is kind
of held together at once lowers
the transition state energy.
So now you don't
necessarily have
to stabilize protonated
carbonyl in an active site.
Instead, you wait until
electrons appear up here
on this oxygen before
it gets protonated.
So that lowers the
transition state energy
for this transition state.
There's other ways of
depicting this as well.
I prefer this one,
concerted mechanism.
So, all of these, okay,
I'm going to skip the
serine-based proteases.
They're similar
to the cysteine-based
protease I showed earlier.
I'm going to skip the
zinc proteases as well
but there's quite a few others.
Like the kinases the proteases
because they're involved
in crucial processes
in human physiology
such as blood clotting, which
you would not want to happen,
you know, here and there,
because these evolved
at such crucial processes the
enzymes themselves are tightly
regulated; oftentimes regulated
by some loop in a pro-enzyme
that has to be cleaved.
The terminology pro means
a reaction takes place
that then converts it into
the active functionality
of the molecule.
So, for example, pro-drugs
are precursor drugs
that are then converted
into the active drug
by some enzymatic process and
over here we see a pro-enzyme
that has a loop blocking access
to the active site of the enzyme
that gets cleaved and then that
allows the pro-enzyme piece
to dissociate and
turn on the enzyme.
So, enzymes can be
very readily inhibited.
You can do things like have
transition states analogs.
We've talked about transition
state analogs before.
Here is the transition state
for hydrolysis of an amide bond
and here's a very effective
phosphoramide transition state.
Notice that this also has
the tetrahedral geometry
of this transition state
up here and if you do that,
you can actually very
effectively inhibit this enzyme.
Other types of inhibitors
phosphonase down here,
phosphoamides over here.
These KIs are the dissociation
constant for binding
where it's KD except it's
for inhibiting, binding
and inhibiting the
enzyme and, again,
smaller numbers equals
more potent enzymes
and notice what a
champion phosphoramidate is
with a near picomolar inhibitor.
Okay, now there's a million
other things I could talk
to you about.
I'm going to pick
them up next Thursday.
When we come back, we'll
be finishing off Chapter 6
and going on to Chapter 7.
Midterm will cover
through today's lecture.
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