Introduction to Chemical Biology 128. Lecture 12. Protein Functions.
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Introduction to Chemical Biology 128. Lecture 12. Protein Functions.


>>Welcome back. This week we get to
talk about protein — function which is the follow
onto last week’s discussion of protein structure, and I
would say last week was all about pretty molecules. We saw beautiful
structures, architectures that were fascinating, but I didn’t tell you what
makes them so special. I didn’t tell you why is it that
those structures allow proteins to do unique things, and proteins really are
the superheroes of biology. These are the molecules that
make it possible to change around whole solutions, and they
make transformations possible that otherwise would
be kinetically and thermodynamically
inaccessible to the cell. So they’re doing reactions. They’re catalyzing
reactions that otherwise — without these, biology would
not be capable of taking place. Okay, so specifically this
week, we’re going to be talking about some pharmacology. We’re going to be looking
at dose dependent response. We’re going to look at
non-covalent binding, and then, by analogy to non-covalent
binding, we’re going to make the
leap to catalytic binding, and we’re going to try to
understand how enzymes work. I want to talk about
how we’re going to measure enzyme activity. We’ll talk about how
they’re regulated. We’ll talk about they’re
mechanisms, and then we’ll talk about mutagenesis engineering. So these topics in here are
going to give you the foundation that you need to
make predictions about how enzymes work, and
the overarching goal is for you at the end of this series, at
the end of these two lectures, for you to be able to look
at some reaction, and then, maybe not design
the perfect enzyme, but make some predictions about
how that enzyme might work. Okay, now let me give
you an example of that, and this is an enzyme that
we won’t be talking about, but it’s one that I
want you to think about. This is actually a quote from
the Iliad, and the quote is, “As the juice of the fig tree
curdles, milk and thickens it in a moment through it
— though it is liquid, even so instantly did
Paeeon cure fierce Mars.” This fascinates me. So in this reaction,
you can take a — a branch off a fig tree, break
it open, and actually this kind of milky liquid flows
out, and you can drop that in a big bucket of milk, and what will happen is
exactly what’s described here in the Iliad. Now, our goal is to
understand how an enzyme like this might work. If I tell you that the
milk is getting a — is getting solidified
by a certain reaction, then you might be able to
predict what the enzyme is, the enzyme mechanism is that
makes that reaction possible. Okay, so that’s our goal. A little bit of a
mystery to set things up. Let me talk about some
announcements first, and then we’ll get into
the meat of the discussion. So this week, please
read chapter 7, work the odd problems as usual. Our midterm is going to
be a week from Tuesday. It will cover through chapter
— actually it’s going to cover through chapter 6,
and not 7, my bad, and it will be comprehensive
in the sense that there might
be some concepts from the first three chapters,
but it will focus largely on the more recent material. Okay, so when you’re studying,
what I’d like you to focus on are ideas and problems that
are in the assigned homework, such as these off
problems in every chapter. I’d like you to focus on the
problems that are discussed in discussion, and then I’ll
also post a sample midterm which will give you an idea
of the types of problems that I’m expecting you to know. Okay, so hopefully you’re
already starting to study for this and that will
be coming up pretty soon. Also coming up, abstracts
for the proposal, the final proposal report,
are due this Thursday at 11 A.M. We’ve already talked
a little bit about the format of the abstract, but I’ve
also released on the website, more details about the
proposal assignment, and I’d like to take
a moment just to review those with you now. So very briefly, let’s
take a quick look. Okay, and on the website, let
me go back, here’s the website of course, here’s the
proposal assignment. I have a very detailed
description of the chemical biology proposal
that you’re going to be writing. In brief, what it tells you is
that you need a simple idea. Okay, so in this first
paragraph it says, you know, “Don’t come up with something
that’s the next [inaudible] Hannah project.” Don’t, you know, tell me, “If I
get a billion dollars I could, you know, do something
like, you know, solve toenail fungus
or something.” Okay? Tell me something
that I can do for, say, one hundred thousand
dollars or even less, ten thousand dollars, let’s say. Those are the kinds of proposals
that attract attention. Clever ideas, things that
people haven’t thought of, that shows brilliance,
that shows creativity. The sort of the big
ideas, you know, sequencing one thousand
genomes, there are people who are doing that, and they’re
creative in their own rights, but that’s not necessarily
what this class is about. So simple, creative
ideas that interests me. Okay, and let me talk to
you about some ground rules. You must choose a topic that
will improve human health and well-being broadly. Okay, so there’s lots
of ways to do this. They can be things like improving the energy
situation on this planet. If you have a new way of
generating energy using enzymes, I would love to see it, and
that would improve human well-being broadly. So, the focus though,
of your proposal, must be squarely qualified
as chemical biology. If your proposal does not hit
the topic of chemical biology, I will know in the abstract
and I’ll give it back to you, and tell you to change
it largely. Okay, it’s very important that it fits the definition
of chemical biology. Your proposal must
have a hypothesis, or a very good reason
not to be driven by to testing a specific
hypothesis, and then after that, you need to think
about back up plans, creative variations,
and further insight. So good proposals are a
little bit like an onion. At the heart you have
some clever idea. You have something that if
you could do this clever idea, it’s going to change the
world, but equally importantly, you have a lot of little back
up plans and contingency ideas. So, if the main idea doesn’t
work out, you have a bunch of back up ideas that
are waiting in the wings that are going to
rescue the whole thing, and turn it around,
and make you famous. Okay, so that’s the ultimate
goal of a good proposal, and along those lines you
really should be having more than one idea. Okay, so a proposal
has one great idea and then there’s a bunch
of other little ideas that are kind of supporting it. Do not propose experiments
that require human subjects, or samples obtained from humans. This is important. I know many of you want to go
to, I don’t know, dermatology, or you know, medical school and
become dermatologists later, and I’ve gotten proposals about picking scabs
and things like that. Those do not interest me. That is not what
this class is about. You know that’s not what
this class is about. So I will not accept any
proposals that require you to collect samples from humans. Your proposal must include
control experiments, we’ve discussed these,
positive and negative controls, we’ve discussed that, and
then the next part is coming up with ideas. How do you do this? The first thing you need to do
is come up with a clever idea. Here is one very
simple formulaic way to be brilliantly creative
for the rest of your career. Okay, if you learn this formula,
you can be incredibly creative. Okay, and I’ll be honest,
this has always worked for me. All I do, is I take
a new technique and then I simply apply
this new technique to a problem that’s
already existing. Okay and you too can do this. We’ve been talking all
quarter about new techniques. You have new ways of
screening [inaudible]. We talked about RNA
aptamers for example. We talked about speech display. We’re going to talk today about
measuring enzyme activities. There are all kinds of neat, new
techniques that you could use. You simply apply
those new techniques to an existing problem
and boom, you’re creative. That’s all it takes. That’s all you have to do to
be creative, is you take — you scan down the list of all the new techniques
we’ve presented in this class, you scan down the list of
problems, you take column A and you take column B, you
put them together and again, boom, you’re creative. That’s all it will
take for you to come up with a creative new idea, but
it is essential for this idea to be creative, for it to
be novel, and if it is not, I will return it
to you ungraded. Okay, I’m fiercely
defensive about creativity. This must be creative. Okay, now the other
thing is, after you come up with this idea, you
need to verify that, in fact, it’s original. This idea can’t be so
outlandish as to be impossible, but on the other hand, if
it’s already been done, it doesn’t count as creative,
even if it’s brilliant. What I usually do
next, after I come up with a creative idea is
simply type it into PubMed, simply type it into Google, and see what else has
been done in that area. If it turns out that someone
else has done this idea that it thought was
brilliant, yahtzee, yahtzee. I think that’s fantastic. That tells me that my idea
was brilliant enough that’s someone’s willing to vest
their own money in it. It doesn’t bother me at all,
if I’m coming up with ideas that other people are
willing to vest in and do, in fact if anything, that tells
me that I’m on the right track, and that should also tell you
that you’re on the right track. So don’t panic, if it turns
out that other people have come up with the idea before,
that’s perfectly acceptable, and it’s actually kind of
normal, and it’s a good sign. It means you’re on the right — you’re going in the
right direction. Okay, so — okay, so
we talked a little bit about different ideas, let
me scroll down a little. Okay, so — here, after you
finish screening your RICO about your idea, the
real work begins. You have to dig into
the literature, learn the field just a little
bit, and know something about the area that you propose. Okay, so that’s really the
real work of this proposal. If you spend all your
time trying to think of an original idea,
you’re wasting time. Okay, ninety-five percent
of the effort comes after you have the idea. So only give yourself a
limited amount of time to think of a new idea, and then after
that, start doing research and get that idea into shape. Don’t spend a lot of time just
cycling through ideas and, you know, kicking
yourself, like, “Oh, it’s not the world’s
greatest idea.” Fine, you don’t need the
world’s greatest idea for this assignment. What you need is an ability to argue successfully
for that idea. That’s what I’m grading you on. Okay, so along those
lines, you know — okay, so yeah, so anyway,
focus on that sort of thing. Ninety-five percent of the work
comes after you have the idea. Okay, we’ve talked a
little bit about the format of the abstract,
that’s listed here. Here’s some stuff about the
format of the assignment. The assignment is going to be around five pages,
not more than ten. I don’t want to read it
if it’s longer than ten. No one wants to read it
if it’s longer than ten. Somewhere in there, there
should be lots of figures. This is important. The level of detail should
be sufficient for someone in this class to understand
what you’re proposing. You should be able to hand it to
a random stranger in the class, just turn to the person on
your right, and hand them — hand him or her the proposal, and then that person should get
some idea of what’s going on, and should be able to judge it. That’s the level of detail. You don’t need to tell me
about every experiment. You don’t have to tell
me where you’re going to buy the materials, and stuff
like that, and most of all, you don’t have to do
the actual experiment. Okay, good news. You’re Visa card is safe
because I’ll tell you, those experiments are
expensive, and then finally, if you’d like to have your
graded proposal returned to you with comments of plenty, then
you have to give me, in advance, attached to the assignment, a copy or a self-addressed
stamped envelope. If there’s no self-addressed
stamped envelope, then I’m going to assume that you don’t want
any written comments back on your proposal, and
that’s quite all right. I just don’t want to spend time
writing comments if it turns out you’re not going
to pick it up, but if you want comments back,
I will take the time to comment on your proposal, the TA’s
will take time to grade it and comment on it as well, and
so you’ll get back something that has some feedback to you,
but I know not everyone wants that so it’s totally up to you. Last thought, it’s important,
as usual, that you turn it in through the turnitin.com
website. This will be scanning
for plagiarism. Bad news, I’ve already
picked up plagiarism on the last assignment which
is disappointing to me. It happens every year though. It drives me nuts. We already talked about it. I’m not going to spend
more time going over it. Okay, now is the time I
usually ask you for questions. If you have questions,
don’t hesitate to shoot me an email,
ask the TA’s. Miriam and Krithica
will know a huge amount about this assignment. They can help you. Okay, let’s move on. So next I want to talk to
you about office hours. I have office hours this week, even though I’m not
here on Tuesday. I will have office hours at the
usual times, and usual places, Thursday immediately after
lecture, and then Wednesday 2:15 to 3:15, in my office. Miriam has her office
hour on Friday, and Krithica has her
office hour on Tuesday, usual times, usual places. Okay, that’s it for the
announcements let’s get on with our regularly
scheduled program. I want to talk to
you about enzymes. Enzymes are truly remarkable. They are attractive to
celebrities like these two, and they’re also attractive
to chemical biologists, and I would say they’re
attractive for the same reasons. They’re attractive because
they do transformations that would otherwise
be inaccessible. Enzymes make possible the things that would otherwise
be impossible. That would otherwise
just take too long, or require too much energy,
and we’ll talk a little bit about how they do that, and I’m
just, you know, so entertained when I find, you know,
celebrity endorsement on my favorite topic. Here in the New York
Times last year, actually on the same day I was
delivering this lecture last year, February 22, 2012, enzymes
tried to grab the spotlight, and there are tons of enzymes
that are found in papaya, they’re called papain,
and they’re notable for digesting proteins. They’re notable for
digesting skin for example. They’re actually used
as treatments for people who are undergoing therapy after
bad burns, after bad, you know, high temperature
burns, and it’s a way of digesting away
necrotic tissue, and these guys have clearly
some good facial structure here so maybe they’re onto something. Okay, let’s talk next
about how this topic ties in with what I told you
about on — last Thursday. Last Thursday, I was showing
you how simple rules can dictate protein structure,
and in a moment, we’re going to then be
applying these same rules to understand enzymes. Protein structure leads
to protein function. So the shapes that the
proteins were assuming on Thursday are what
allow the proteins, the enzymes in this case, to actually acquire their unique
function, and what I found so beguiling about this idea
of conformational analysis is that the rules are so simple. We’re talking about something
as simple as just eclipse versus staggered ethane, or
ghosh versus anti butane. This fascinates me. This will keep me running to
work for a really long time because that’s such simple idea. These are such foundational
concepts in modern chemical biology that
I can explain to my grandmother. These are things that
totally make sense, right. It makes sense to you that you
would want to avoid electrons which are all — electrons
banging into each other, right. Electrons hate banging
into each other. It makes sense that
things should try to spread the furthest apart,
and if we think about things in that way, then such complex
structures as the proteins that we’re about to look
at, their structures start to make sense as well. We talked about how
as a consequence of these simple rules, some
amino acids can be found in specific types of secondary
structure, and there are tables of this, and you can
even make predictions about the secondary structure of
proteins based on nothing more than the amino acid
sequence, and surprisingly, these predictions turn
out to be pretty good. They’re about eighty
percent accurate, or so, and from there people
have been attempting to predict protein structure
for a long time, and quite a bit of progress has been made in
recent years towards that goal. We also discussed disulfides
which provides spot wells that hold together independent
regions of protein structure that otherwise would, sort of,
flop apart, and we also talked about how readily exchangeable
these disulfides were to allow formation of new
disulfides and exchange of one disulfide for another. The next topic we
discussed was this hierarchy of protein structures
from primary structure, to secondary structure, to
domains, to tertiary structure, to assemblies, etc., and so this
helps us organize our thinking as these assemble onto
complex architectures. So we’re going to be
making reference to this as we start talking about
enzymes, and then we ended with the concept that a
relatively small number of protein domains are
found very commonly in the human proteome. Now the truth is, I didn’t
finish the discussion of this so I need to pick up, just too
very briefly, with just a little but more about protein
structure, and then again, we’re
onto enzymes. So enzymes or — last
thoughts on protein structure, this is an example of an
all beta sheet called an immunoglobulin domain. These often times assemble into
long strings, and these are used in proteins like titin. Titin is found in muscles. Have you ever wondered
why muscles are so strong? You know, you’ve probably seen,
for example on the Olympics, you’ve seen Olympic
weight lifters. You’ve seen them
flexing these steel bars. Right, they’re lifting
this stuff up, and the steel is
flexing, and the titin in their muscle is hanging on. Well what happens is
there are long strings of these immunoglobulin
domains that are wound up like beads on a string. So you have IG domain, that’s
another word for immunoglobulin. So you have IG, IG, IG, IG,
and as you pull on the ends, one of these can unfold without
snapping the whole muscle fiber. So the titin actually is
holding things together. So individual domain can unfold without breaking the
entire protein shape, and that gives muscle, and
specifically gives titin found in muscle, some remarkable
properties as a material. Okay, so on the left, this
is the protein that’s found in muscles, and I’m only showing
you one immunoglobulin domain. Notice that the ends
are 180 degrees apart. This then sets up these
long strings of titin that can extend, you know,
up to the roof up here, then down through
the floor down here. You can have many, many numbers,
large numbers of these lined up. On the right, here’s an example
of an enzyme, along the lines of the kind of things I want
to talk to you about today. This is a really
remarkable enzyme. This is one of those enzymes
that make it possible for you to live on this planet. This is an enzyme called
superoxide dismutase, and its active site is actually
not where you expect it to be. I think when you look at
these immunoglobulin domains, these are beta sandwiches,
your expectation is that the inside is this,
kind of, deep cave, and that’s not the case. Instead, this inside is
chalk full of [inaudible]. This is full of stuff. It’s only actually
on the outside where the real action
is taking place. Outside, over here somewhere,
is where the active site of this superoxide dismutase
is found, and incidentally, it’s mutations in this
enzyme that are responsible for diseases like Lou Gehrig’s
disease which is, you know, a terrible disease, and so
mutations to these kinds of proteins, these
enzymes, have very, very serious medical
consequences. Okay, last thought on
immunoglobulin domains, they are also — they’re
named after the antibodies from which they’re found
in, and this is again, one of these truly
[inaudible] domains. Each one of these lobes over here is an immunoglobulin
domain, and notice how these are
just all strung together into an antibody. It should come as no
surprise to those of you who were attending last week’s
lecture, that the business end of antibodies which are
professional binding proteins, are found at the loops. Okay, so these antibodies are
designed for binding to things. That’s their role in life. They’re professional binding
proteins, and in order to get that kind of binding,
the loops, out here, are exactly where you can
pick up that kind of binding. Those loops are flexible. Remember we talked
about the low level of hydrogen bonds
to the backbone? We talked about how the loops
can accommodate many different shapes and sizes. That’s what equips
antibodies with the ability to recognize foreign attackers. Right, you cannot set up
in advance an antibody against everything
on the planet, these have to be just ready
to pick up random things that they — that you might
encounter when you visit, I don’t know, the taco wagon out
here, or something like that. SO you have to be ready
for that kind of thing, not knowing in advance what
the shape is going to be. So having these flexible
and molecular recognition, versatile domain with
these loops over here to accommodate diverse binding
partners, turns out to be key to understanding their activity. All right, another beta sheet
protein, a good friend of mine, what I’ve published many papers
either using or studying, is a protein called
streptavidin, and good news, we’re going to be
talking about this again in about fifteen
slides from now, so it gives me great
pleasure to introduce you to the wonderful streptavidin. Streptavidin is charged with
binding to biotin, and again, this is an all beta
sheet protein, there’s a little
tiny alpha helix, but it’s largely all beta sheet, and it forms these
wonderful little beta barrels. At one end of the beta barrel, this small molecule called
biotin sits, and we’ll look at the structure of biotin in
greater detail in a moment. This is an example
of a small molecule. The molecular weight of
biotin is 254 grams per mole, it’s tiny, tiny, tiny,
and biotin is trapped by the quaternary
structure of streptavidin, and this is an important
concept. If we look here, at say, the
secondary structure, oh sorry, the tertiary structure
of biotin — streptavidin, you’re expectation
is that biotin is not going to be very firmly held
in this beta barrel. Right, I mean look
at all the space, you can imagine the
biotin just floating away, and coming out of
the streptavidin without very much trouble. So tertiary structure
doesn’t begin to hint at the extraordinary abilities
of this molecule to grab on to biotin, and
here’s what I mean — okay, so now this is
the quaternary structure of streptavidin, and
streptavidin consists of four, it consists of a homotetramer
of four streptavidins that are non-covalently joined
together to trap biotin, and notice there are
four biotins bound here. Now, what’s happening
is the alpha helix from a neighboring
streptavidin is sticking down over the top of biotin. I’ve highlighted that for
you in black over here, and you can see it’s actually
forming a trap door to slam down over the top of the biotin, and prevent the biotin
from floating away. That turns out to be
key to its activity. So streptavidin evolved
to bind biotin with astoundingly high
affinity, and we’ll talk about the exact number
in a moment, but it is really extraordinarily
high, but it evolved to bind this cofactor
called biotin as a way of killing any bacteria
that happened to be present. So, for example, a related
protein called avidin is found in egg whites. Okay, so egg whites have a
high concentration of avidin, and that means that
if any bacteria try to colonize the egg, you know,
I’m talking about hen eggs here, if any of those bacteria try
to get in there and go to town and eat all the juicy richness
of a wonderful egg white, they’re going to die because
they’re biotin will get sucked up by the streptavidin, and
then trapped almost permanently, and that turns out to have fatal
consequences for the bacteria. Turns out, that is
also can have, fairly fatal consequences
for humans. If you live on a diet of
nothing but egg whites, your biotin will also get
pulled out of your body, and this is kind of
an astonishing fact because it turns out it doesn’t
take a lot of biotin for you as a human to survive. Biotin is an essential cofactor
in the synthesis of lipids, and — but however, there are
eccentric people out there who constantly do experiments
like this, and when they show up in hospitals after eating a
diet of nothing but egg whites, the physicians tend
to be totally baffled because they’re not used to
seeing such bizarre symptoms, and so — there was a case, I’ll
send it around, I think this is in New England Journal of
Medicine, this English guy, who showed up and was living
on a diet of nothing but tea and egg whites, and
he had all this, like, bleeding out of his gums and
his pores, and anyway, he was, you know, falling
apart basically and the astonishing thing is it
would take just a few micrograms a day of biotin for him
to be totally healthy, but there’s such a high level
of avidin and its homologue against streptavidin
present in egg whites that is was actually
leeching all of the biotin out of his body. Okay, pretty extraordinary
biochemistry, pretty extraordinary
molecular recognition. It absolutely fascinates me to understand how
this works better. It’s something I’ve spent a
lot of time thinking about. Okay, yet another example of a very common
protein fold that’s found in the human proteome,
the WD proteins consist of these beautiful,
propeller like assemblies where there’s actually seven
of these little triangles. They look kind of like
slices of a pizza that are — that come together to form
these large assemblies. These act as scaffolds to organize big machines
found inside your cells. So this, each one of these
spaces over here might bind to a different protein,
and bring it together like it’s an assembly line for putting together really
complex things inside your cell. Absolutely fascinating stuff. You know, something else
that’s truly bizarre, what’s up with the
seven fold symmetry? You know, I think I’m ready
for four fold symmetry, but we humans don’t like to
think in seven fold symmetry. So I’ll just leave
that as something for you to puzzle over. Also, another very common
— and I’m switching gears, we’re going down the chart of the most common protein
structures found in humans. Collagen is a very
common protein structure. It’s actually an unusual
three stranded coil. It differs from the alpha
helical coils that we saw, the twist is different, and
it’s a little hard to see, but there’s actually
three different colors. There’s a green color, a
purple, and a blue color, so there’s three
different colors here. There’s these three strands that
are winding around each other, and this makes a very strong
framework for collagen. Collagen is another one of these
ubiquitous structural proteins found in the body. Notably, this protein requires a
post translational modification introducing a hydroxide
into prolene residues, and that has the
effect of setting up one particular preferred
structure, a particular twist in the collagen triple helix. Without this hydroxide,
the protein is unable to assume that confirmation. All right, let’s switch gears,
I want to talk about GPCR’s, this is the class of proteins
that makes it possible for you to see me, it makes it
possible for you to smell, for you to taste,
pretty much any of your senses are
totally dependent on this class of protein. So I think we should
take a moment and be grateful for
their existence. How do these things work? Okay, so this is a really — how is it that you’re
going to sense a photon? How can you actually, you know,
respond to light which is, you know, you can’t
bind light right? So how do these things work? All right, so in short,
these sensing proteins, again they’re called G
protein-coupled receptors, or GPCR’s, are all alpha
helical, and notice that, you know, there’s these — they
each have seven alpha helices. These seven alpha helices
transit plasma membranes in the cell, so going through
here is the plasma membrane. So there’s an alcide,
an exterior, and there’s an interior
facing the cytoplasm, exterior is facing the
extracello [inaudible], and these change confirmation
upon binding to things. In the case of a
photon, the GPCR responds by having an isomerization of
a carbon-carbon double bond. So as the photon hits it
isomerizes this carbon-carbon double bond, flips it
from one configuration to another configuration,
from trans to sis and back, and in doing so that
rearranges the conformation of these residues down
here that are found on the inside of the cell. Okay, last thought. Notice that this is a coiled
coil, and if you look carefully at this, it is a
left-handed coiled coil. Almost all of the coiled coils
found in nature are left-handed. Notice that your
hand can trace this out in a left-handed manner,
it doesn’t want to trace it out if it’s right-handed. Okay, so these are very
common because alpha helices in general are hydrophobic
secondary structure, they fit nicely into membranes, and this property
makes them very useful for sensing what’s
happening outside the cell and communicating it to what’s
— to the inside of the cell. Next one, here’s another
example of an alpha — sorry, I’m going to
talk very briefly about alpha beta proteins. This is an enzyme that we’ll
talk more about very shortly. This is a barrel-like protein. Here’s the barrel in the center. This is the active site. So you can mix and match
alpha helices and beta sheets. I’m showing you this
because I didn’t want to give you the impression that
secondary structures never mix, in fact, there very
commonly mixed together. Repeat proteins. So repeat proteins are
another very common assembly of proteins. These are — this is an example
of an anchoring repeat protein. Notice that is has two sides,
the concave side has the loops, and wouldn’t you know it, the
loops turn out to be the key to this binding activity. This is a protein that used
— it’s very versatile. It’s used in many
different contexts, and what happens is
these loops are mutated to give a particular binding
property that’s then useful for the cell. It’s corollary — is
a leucine rich repeat which now has loops
on the convex side. Okay, so the anchoring repeats
had loops on the concave side, this one has loops on the convex
side, and these loops then, can grab on to the
binding partners. Okay, analogous to what we
saw with the antibodies. Okay, so this is a very — these two are very versatile
structures that can be evolved through organisms pretty
readily, and that gives — that puts organisms with
new binding activities which in turn can
be used to respond to environmental
changes etcetera. All right, last structure that
I want to talk to you about, peptide binding domains. This is an example
of the SH2 domain. This tiny little domain
shown here in yellow, binds phosphotyrosine proteins, so proteins that have been
post-translationally modified using a class of enzymes called
kinases, which we’ll talk about in a moment, bind
to this SH2 domain, and fit into very deep pockets. Okay, so there’s s
deep pocket down here. So these evolve basically
to have specificity, to bind to a particular
sequence. So they’re not binding to every
phosphotyrosine, they’re picking out specific binding partners. Another peptide binding domain
of note are SH3 domains. These bind polyproline helices. This is the I plus 3
helix, the 3/10 helix that we saw during
Thursday’s lecture, and this binding pocket
is a very shallow one. It almost looks like
the peptide is resting on the top of this SH3 domain. Okay, it’s like a butterfly,
kind of a lighting on the top of the SH3 domain, and notice
how delicately folded it is into this three-fold
symmetry helix. Right, so you can actually,
if we look down one axis of this helix, you can see how
it’s forming this polyproline I plus 3 helix. Okay, it’s hard for
me to talk about this without having a few favorites, and I know as a chemical
biologist I shouldn’t have favorite molecules, but I do. This is one that I spent
years of my life thinking about in my wasted youth. I was interested in these
MHC receptors for reasons that are too bizarre to explain
right now, but surprising to say, these are the receptors
that your immune system relies on to let it know when
the red coats are coming. This is — these are the
receptors that raise the alarm when foreign invaders are trying
to take over your physiology, and the way this works, is a
small percentage of peptides that are synthesized by
the cell, are digested and then displayed out on the
surface of the protein just like little flags, and the idea
is that if a virus has taken over the cell, little flags
of virus will appear outside on the surface of the cell, and the immune system
then knows, “Oh no. That cell has been
infected with viruses. I better kill this cell. I better mount a
strong response.” This is a very effective way
of alerting the immune system. Okay here’s some
old friends as well. Notice down here. Do you recognize that domain? Does that look familiar? Yes! That is the same domain
we saw a few slides ago. This is the beta sandwich
immunoglobulin domain, and here it is making a cameo
in a slightly different, but equally important role. Here it is actually
lofting the peptide out, off the surface of the cell. Down here’s the surface of
the cell, here’s the scaffold of the immunoglobulin
domain, kind of holding it above the surface of the cell,
making it a little easier for the flag to be seen
by passing T cells, especially cytotoxic T
cells that take an interest in these things, and
then can go into action and kill the cell if necessary. All right, one last
example of this. The last example is a
slightly different variant of these MHC receptors. In this case, this is
one called class two. The one on the previous
slide was called class one. The details, not so important, but this guy actually
displays peptides not that are being synthesized
by the cell necessarily, but peptides that are
being engulfed by the cell, so that the cell is
kind of randomly taking up extracellular
material, and so, again this gives the immune
system a different look. So the MHC class
one tells about — reports on what’s
happening inside the cell, the class two reports on what’s
happening outside the cell, and notice that the structure
of this peptide when viewed down the same axis that we
looked at earlier form SH3, it again starts to
bear some hallmarks that we’ve seen before. Notice that is also has
that, sort of, three-fold — it kind of looks like a triangle
type of geometry, and yes, this is also assuming a
polyprolene type helix, analogous to the
I plus 3 helices that we saw earlier
in this class. Okay, so hopefully
some of the concepts that we saw earlier are
finally coming into play, and what I want you
to do is I want you to first have an aesthetic
appreciation for these things. These are beautiful. Okay, I like to think
of this one as like a hotdog
in a hotdog bun. I mean look at this thing,
it’s so juicy I could eat it! But equally importantly, it has these immunoglobulin
domains again that have the wonderful function of lifting it off the cell
surface, at the same time, it’s presenting lots of
surface area out here so the cell can recognize
whether or not this flag belongs
to self or non-self. It can even interrogate this one
receptor and determine whether or not the original cells are
from self versus non-self, and this is really
one of the challenges for organ transplantation is to
deal with this class of proteins where every one [inaudible]
different humans have different MHC receptors, and this is one
way that the immune system keys in on organs that
have been transplanted and knows to kill them. Okay, now obviously I can talk
about this particular topic for hours, but we don’t have
hours so we’ll have to move on. All right, I want to finish our
discussion of protein structure by talking about
higher order assemblies, and I guess our best example
of this is a structure that I introduced to
you earlier, collagen. Collagen, again,
plays this key role of structurally strengthening
bones, of joints, of doing all kinds
of important things. This is, yet again, one of those
proteins that makes it possible for the weight lifter to hoist,
you know, do the dead lift over his head, and
have the bar flex. I’ve always wanted to do that,
but I don’t think it’s going to happen, but just to see
the bar flexing is, you know, a thing of beauty right? How is that possible? Okay, so obviously these
things are really strong, collagen is remarkably
strong, it also is assembled into an ordered structure where that ordered
structure supports each of its constituent fibers, and
I guess the best example of this that you’d be familiar with
is something like a rope. Right, individual strands of
the rope, of a fibery rope, not so strong, but when
you wind them together, and they’re all supporting
each other, and then suddenly it
gets really strong that you can anchor an
aircraft carrier to a dock or something like that. All right, so here’s
the way collagen works. You have to control its assembly
in a way so that it doesn’t get, you know, kind of
prematurely winds itself up, and then get all tangled. Okay, so what happens is
the triple helix is formed with some caps on the
ends, and these caps on the end remain
for quite a while. So the end cap pieces are
then brought together, and then the whole assembly
with the caps still in place, is secreted outside the cell
and where these red arrows are, these caps are then snipped off
using proteases, the scissors of the cell, and that gives
you a formation of fibrils. Okay, so without that
this does not happen. Okay, without that you get this
kind of sticky tangled up mess, but what ends up happening
instead is a very detailed oriented assembly
where each step in the process is carefully
controlled, and that’s essential to making structures
that are really strong. Okay, now I want to move on. I want to talk to you
next about enzymes. We’ve seen protein structure,
enough about protein structure. Let’s talk about what
they’re good for. Okay, obviously they’re good
for strength and structure. I want to talk about
cytolysis next. Okay, so in order to
talk about cytolysis, I have to introduce you to
some measurements of strength of binding, of [inaudible]
efficiency, and so the first thing
I’m going to have to do is to find a few equilibrium
constants for you. The first of these is used
to describe the strength of a non-covalent
receptor ligand interaction where the receptor
is indicated in R and the ligand is this little
sphere indicated in L. Now if you have a bunch of receptors
on the surface of the cell that want to bind to ligand,
the ligand is going to hop on, it’s going to hop off,
it’s going to hop back on, it’s going to hop back off. So we need some way of
describing the occupancy. How many of receptors are
actually bound to the ligand? How many of the ligand
are free in solution? So the way chemists do this is
using equilibrium constants. These equilibrium
constants are kind of special so they get a special name, but
they’re more or less the KEQ that you learned
back in Chem one. So here’s the way this works, we can describe some receptor
ligand interaction that’s formed as having a dissociation
constant in which the receptor and the ligand dissociate
from each other, and the dissociation constant
KD is equal to, you know, concentration of receptor
times concentration of the ligand divided
by the concentration of the complete receptor
ligand interaction. The inverse of the dissociation
constant is the association constant, abbreviated
KA, but again, these are just fancy
equilibrium constants; however, they tell us quite a bit about
the strength of an interaction, and I’m going to be
referring to them. One thing that you need to
know is that a lower KD, a lower dissociation constant,
means a stronger interaction and we’re going to
stick with KD’s. Okay, everyone out in
the pharmaceutical world, in the biochemical
world, discusses things in terms largely of KD’s,
KA’s, you basically — every time I hear
someone give me a KA, I mentally take the inverse
of that number and then think about it in terms of KD. It’s just a convention,
okay, but what matters is that a lower KD means
a stronger interaction. That means more of the
ligand is bound here. Right, so you have more
of the complex form, bigger number down
here, lower KD. Let’s take a look
at some of these. Okay? I’ve been talking to
you a little bit earlier about organ transplant
and rejection. So after people receive
a transplanted liver, they’re given a class of drugs
called immunosuppressant’s that suppress the immune
system, and we know quite a bit about receptor ligand
interactions through classic studies done
by Stuart Triburn and others that looked at how these
immunosuppressant’s work. Here’s the — here are two
examples of immunosuppressant’s, on the left is a small
molecule called FK506, and on the right is a small
molecule called rapamycin. They both work by
targeting a binding protein that the Schreiber
laboratory named FKBP for FK506 binding protein,
and here it is neatly fit into this receptor
which is FKBP. So the ligand is the
immunosuppressant drug, the receptor is the FKBP, and notice that this is finding
a really deep binding pocket to bind to. Let’s zoom in. Okay, let’s take a closer look. So imagine now that we can zoom in just looking at
the green ligand. What we would see is
something like this where in blue this is the region of the small molecule
that’s bound by FKBP. Okay, so again, on
the left is FK506, on the right is rapamycin. Notice some similarities here. Okay, notice that in blue, these largely have
the same structures. Okay? That’s not a coincidence. That same structure helps
orient the molecule, and make it so that this half of the molecule can very
readily bind to FKBP. Notice the part that’s
not shaded. These two are wildly divergent. Okay, the molecule on the left, the part that’s unshaded
looks completely different than the thing on the right. Okay and that’s not too
much of a surprise either because it turns out that these
two molecules effect different pathways in T cells to
suppress the immune system, and these ligands act
as sort of like the meat in the molecular sandwich, and they recruit two
different top layers of bread. This one over here recruits a
different protein than this one over here; however, both of
these molecules bind to FKBP with very high affinity, and
the way we know this is high affinity is we refer to their
KD, the dissociation constant as having a subnanomolar
KD range. That’s really good. That’s really, really
right binding, and it turns out that most of
the pharmaceuticals that are approved tend to have
affinities for their targets in this, kind of,
very low KD range. Why that is will be apparent
to you in a few slides. Okay, so this is one way of describing non-covalent
interactions. I next have to tell
you how we’re going to be describing speeds
of reactions, the kinetics that make reactions possible. Two kinds of reactions that
we’re going to be seeing in this class, kind number one,
are unimolecular reactions. These are reactions where you
have some reactant and it goes through a transformation,
and that’s it. Okay, there’s no other
species that’s implicated in the reaction mechanism. That’s it, it’s just this one
tetrahedral intermediate falling apart, collapse of the
tetrahedral intermediate. The rate of unimolecular
interactions equals some rate constant, little tiny K
times the concentration of the starting material. Okay, this K over here, I’m
emphasizing tiny K for a reason. The little K indicated
rate constants; it’s totally different than
the equilibrium constants I was showing you on the
previous slide. Never the twain shall meet. They’re two totally
different things. It drives me crazy though that
they’re both symbolized by K, and there’s nothing
I can do about that. Okay, we’re stuck with that. It’s old timing nomenclature. All right, the next one. We’re also going to see
bimolecular reactions. These are reactions that have
two reactants that are colliding with each other, and
in that collision, resulting in formation
of a new product. The rates of these bimolecular
reactions are going to be equal to some little tiny K, the rate
constant times the concentration of reactant one, in
this case, hydroxide, times the concentration
of reactant two which we’re calling
Y in this case. Make sense? Okay, I expect that
this has been a review. This is something
you’ve seen before. In biology though, these
rates vary enormously. Okay, check this out. This rate constant, K1, ranges
from 10 to the 13 per second to 10 to the minus 7 per second. That’s 20 orders of magnitude
difference in speeds. Okay, this is, at the wild, you
know, fast end of the scale. These are things that
are a total blur, and at the super slow end of
the scale, these are things of geological times that are so slow they simply don’t
even matter in biology without some sort of
catalyst to speed it up. Okay, so these are the
parameters we’re going to use. Let’s now think about kinetics, first of non-covalent
interactions, and then we’ll talk
about enzymes. Okay, so for a non-covalent
interaction, you can imagine the ligands
hopping off of the receptor. When that happens, there
will be some speed of this that will have a rate
constant of little K off. Similarly, ligands can
hop onto the receptor, and again there’s a rate
constant, little K on. Naturally, if this
is at equilibrium, then you can actually,
you can work out that the KD equals the
ratio of the K off to the K on. Okay? Everyone still with me? Again, this is at equilibrium
where the — let’s see, okay. Everyone still with me? Okay, good. Here’s the thing, the typical
rates of binding are, again, wildly different, and this is a
table from the book, table 6.1. I want you to take a
moment to just gaze at the truly all
inspiring nature of the differences
in speed here. Okay, so let’s just take a
moment to appreciate this. What I’m showing you is a
series of different receptors, and over here a series
of different ligands. At the top, these
are small ligands. These are small molecules like
biotin that we saw earlier, and at the bottom,
these are large ligands. Okay now, check this
out, this is really cool. Notice that the on rates
for all the small ligands, roughly the same, very,
very little difference. They’re you know, they’re all
right in that 10 to the 8 range, and hey guess what, 10 to the 8
is kind of near the speed limit. The speed limit for zooming
through the cell is going to be somewhere around 10 to
the 9 per molar per second. Okay, that 10 to the 9
is a physical constant. You can’t bounce through
water any faster than that, and so these small molecules
are zooming along about as fast as they possibly can to
fit into their receptors, but check this out, off rates,
these off rates vary enormously. They range from say nine over
here, up to 100,000 over here, and down here, in
the big things, huge changes in off
rates as well. Okay, so what this tells
us is if you are trying to design the perfect
pharmaceutical, the perfect therapeutic
to treat, I don’t know, muscular dystrophy or something,
you want to spend a lot of time thinking
about off rates. Off rates are where the
big money is when it comes to therapeutics, when it
comes to pharmaceuticals. They all have roughly
the same on rates, what differs is off rates, and those off rates are how
you can determine whether or not a pharmaceutical
can be given at low dose versus a high dose, but I’m
getting ahead of myself. We haven’t talked
about this yet. Okay, let’s talk about
the large ligands. Large ligands have enormously
variant on rates, you know, there’s too many zeroes here
to count, and then over here, enormously variable off rates. You know, this should make
sense to us just intuitively because large molecules
aren’t going to be able to zip through the cell as
quickly as small molecules. Right, they’re going to
get, you know, side tracked. They’re going to try to
bind to other things. They’re going to, you know,
their diffusion rates are going to be slower, for example. Okay, now let’s put
it all into effect. Let’s put everything we’ve
seen so far into one, you know, summary that tells us
what it is that we care about in terms of
treating patients. Okay, so the way it works
is, what we want to do, is we want to have some
biological response. Okay, if our goal is to
cure patients of, say, toenail fungus, then our
biological response is going to be, you know, what percentage of their toenails are
clear from the fungus? Here’s the way this works. On the Y axis, this is the
percent biological effect that biological effect results from ligands binding
to some receptor. Okay, we’ve seen, for
example, antibacterial products that are binding
to the ribosome. In that case, the ribosome would
be your biological receptor, and the biological effect
would be death of the cell, that would be killing
the bacteria. Okay and so, in general, we see
sigmoidal biological responses when we look at receptor
ligand binding. Okay, when it’s graphed as
log of the concentration of drug along the X axis, and percent biological
response at the top. At the very top, at 100
percent biological response, this will take a very high
concentration of drug. Okay, notice that the numbers
are bigger on this side, and then smaller over here, this
is 10 to the minus 9 over here, 10 to the minus 1 over here,
and concentration of molar. I realized it’s — the
axis is a little confusing. Bear with me, there’s a
reason we did it that way. Okay, so up here at a
really high concentration, 100 percent biological effect. Okay, but maybe at that
concentration you end up with a drug that has to be
given with pills that are like, you know, the size of, you
know, erasers or something, and no one likes to swallow
things that are really big. So we compromise. Instead, what we want is we want to have a 90 percent receptor
occupancy in [inaudible] to see some sort of effect. Okay, that’s our goal. So we’re going to be
measuring biological potency through these dose responses
effect, and the major goal of pharmacology is
to get up here into this 90 percent
receptor occupancy where you get 90 percent
biological effect, greater than 90 percent
biological effect. Now typically the
numbers up here, you know, obviously we’re approaching
an asymptote so things are, you know, can go for a
really long time up here. So instead, we describe
biological potency in terms of an effective concentration
for 50 percent effect. That 50 percent effect takes
place, right here, at the point of reflection for this
sigmoidal dose response curve, and so we can compare
two different drugs just by comparing the EC50 where the
more potent drug will be the one that gets the same
50 percent response, but at a lower concentration. Right, it means then that
the patient can be treated with a lower dosage
to get the same effect and the same benefit. Okay, so you know, I think it’s
worth us taking a moment to talk about this because like
90 percent of the students in this class are going
to be spending the rest of their lives battling
with this sigmoidal curve, and trying to get up here and
sometimes being down here. Okay, let’s talk about how you
measure biological response. Often times, in chem
bio laboratories, this is measured using ELISA, enzyme-linked immunosorbent
assay, and before I can tell you
a little bit about how that assay works, I need to
tell you about some reactions that are catalyzed by enzymes. There are two enzymes that
are kind of the work horses for chem bio laboratories. One of these is called
peroxidase the other one’s called phosphatase. These are reliable enzymes that
will catalyze reaction that lead to turn over of dimolecules. So here are two molecules
up here, this is — these two aromatic molecules. These guys up here are clear. Okay, so if you made a
solution of these guys, it would look more
or less clear, it might look a little
yellow, but more or less clear. However, after the enzymes — these two enzymes
catalyze these reactions, what ends up happening
is you get a dye that has a deep color,
a very strong color. This one forms a dark black, actually more brownish
color, very dark color. This one over here forms
a bright yellow color. So both of these give
us reliable indicators that we can use to follow how
much activity is taking place at a certain dosage. So let me show you
how this works. What we do is we use plates, and I think I’ve talked
about those before. They’re enzyme — they’re called
ELISA plates in colloquium in the lab, and these plates
have 96 wells on them. Okay, so that’s over
here, and each one of these wells can be coated so that the surface is
coated with the receptor. Okay, so here’s the
receptor down here. The problem is, if they’re
surface likes to bind receptors, it will also bind to
a ligand and we don’t like that kind of thing. So what we do instead
is add a blocking agent, typically something like
dried milk, nonfat milk. Not dry, it’s a solubilized,
and so we take nonfat milk and we coat any other place
on the surface of this well that otherwise might start to bind non-specifically
to the ligand. We then add the ligand. If stuff binds, we wash
away the non-binders and then add an antibody
against the ligand. So wherever the ligand is bound, we’re going to get an
antibody stuck to it. This antibody is a
special antibody, unlike the antibodies I showed
earlier, this one happens to be covalently tethered
to the enzyme that I showed on the precious slide. That enzyme is peroxidase,
and so this means when you add the dye, this
peroxidase goes to town and turns over the dye in this
well, and then you can look at a large number of wells
to get a dose response curve. Okay, this works really
well, this is totally robust. You can use this
in your proposals. It works great. Okay, let’s talk next about other receptor
ligand interactions. Okay, so I’m showing you
ELISA’s as an example of catalytic receptor
ligand interactions. I want to get back
to streptavidin. So streptavidin has this
remarkable half-life of 200 days. Recall that this is the protein
that is found in egg whites, and if you live on a
diet of egg whites, eventually you’re leeching all
of the biotin out of your body. This half-life over here, of
200 days, starts to make sense because it starts
to explain why it is that you can actually leech all
of the biotin out of your body. It’s KD happens to
be an astonishing 10 to the minus 15 molar,
or Pico molar, subpicomolar really,
[inaudible] molar. This would be 10 to the minus
15 so sub-pico molar KD. That’s extraordinary because
earlier I showed you rapamycin and FK506, and I said, “That Nano molar
affinity was really high, that’s a really great
binding partner.” In this case, this is one
that’s a million times better, and is really strong. Okay, now chemical biologists
have learned to use this for all kinds of assays. One thing we do pretty often
is attach biotin covalently to small molecules, and use this
kind of like lures for fishing. Okay, so you all know
about fishing right? So the way this works, you
have a line, you throw the line in the water; at the end
of the line is a hook. The fish are smart, okay. The fish are not going
to eat some random hook so instead we’ll put
some sort of lure on the hook that’s then going to
bring the fish up to the hook. Okay and classically I guess
it was worms; I don’t fish with worms, I fish with flies. I also like to fish
with small molecules, and when I do, I
always use biotin. Okay, so here’s the way this
works, so here’s the biotin, it’s now covalently tethered
to some small molecule. This becomes the lure, okay. So this side over here is
going to attract proteins from the cell, and then
this side over here is going to be attached — is
going to act as a handle, so that’s the part the
you grab on to, and hold, and where this really useful is if you have some
new small molecule and you don’t know what
it binds to in the cell. This happens to us all the time. We’ll have some molecule that
we pulled out of, I don’t know, a fruit fly screen, where
we’re looking for molecules that make fruit flies less
drunk or something like that, and we want to know,
“How does that work?” Okay, so the way you would do
this, is you have some linker and then biotin, and then
you basically go fishing and hope for the best. A quick word about the linker. The linker matters a great deal because do you remember earlier
I told you about the trap door, and how the neighboring
streptavidin subunit slips over the one beta
barrel, and traps it? Without this linker, it’s
very hard for these molecules to be strongly held by biotin. The linker allows the trap
door to get closed all the way and get wedged tightly shut. Without this, then some
molecule like this, that has a ring system
nearby, would basically get into the trap door, and
block it and make it harder for it to close all the way. So the linkers actually
matter quite a bit. Okay in practice, here’s
what it looks like. In practice, we have
these columns that we flow cell lysates
over, and what we’re looking for is we’re looking for
molecules that will stick to the column that binding to — not to biotin, but
to the product, the target that’s then
tethered to the biotin. Okay, so this is the
way we go fishing. The handle is over here,
that’s the biotin, it’s grabbed onto by streptavidin, and
then the lure’s hanging out and you send through
junk from the cell, just cells that are
chopped apart, and you hope that stuff sticks. Other molecules that don’t
bind to the target flow through and don’t stick, and
again, this works great. Okay, so everything you know about KD’s are now
being applied. Okay, so now what
I’ve shown you, is I’ve shown you an example of a really strong
receptor ligand interaction. I’ve shown you how to
measure biological effect. Let’s now zoom in
and start talking about catalytic receptors. This is a great example of
a non-catalytic receptor. Enzymes are basically
catalytic receptors. Everything that we have
been discussing in terms of dissociation constants,
in terms of binding, in terms of molecular
architecture, all of that comes into play when we
discuss enzymes. So what do we talk about
when we talk about enzymes? We like to talk about
these in terms of a few simple parameters,
and again, these are parameters that are familiar to us
from our earlier discussion. So earlier I talked about receptor ligand
interactions having K ons and K offs, similarly enzymes
substrate interactions are going to have K ons and K offs. They can either form
the complex, the enzyme substrate complex, sometimes called the
Michaelis Menten complex, or they decide not
to, and the key — the one little twist here is the
fact that the enzyme is going to catalyze transformation of
the substrate to some product. Okay, substrate is a fancy
word for starting material, and what is happening
here is we’re going to form this intermediate
complex, and this intermediate complex is
going to very quickly collapse. If it collapses to the left —
if it collapses to the right, then it is going to then form a
product, and this product will, you hope, quickly dissociate. If it collapses to the left, then the substrate
diffuses away, and this goes through
an off rate. Okay, quick word about
this, in this case, I’m showing that the
enzyme product then has to dissociate from the enzyme. If that doesn’t happen, then the
enzyme is trapped and inhibited in this state, and guess what? That actually happens,
and in fact, it’s one of the reasons
why, often times, this last step can be a rate
determining step for enzymes that you would not expect
this to be a problem for. All right, let’s zoom
in and take a look at the reaction coordinate
diagram for an enzyme catalyzed
reaction. I will tell you in advance, that
this is grossly over simplified. The real reaction coordinate
diagram is complicated and messy, and I’m going to show
you the theoretical idealized version first. Okay, so enzymes
work by stabilizing, and thus lowering the energy
of the transition states. Doing that accelerates
the reaction. Okay, so here’s a typical
reaction, on the Y axis, this is the change in
energy, delta G. Higher on this axis means less stable. Lower on the axis means more
stable, and as you know, higher means it takes
more energy which means then, in
turn, it’s slower. On the X axis, this is
often times called the reaction coordinate. It’s nothing more than
the conversion of going — or the pathway between enzymes
plus substrate going all the way to enzyme plus product
over here. Okay and here it is
going through a couple of different intermediates,
enzyme substrate intermediate, enzyme product intermediate, and
then also a transition state. Okay, so in order for — so let’s talk about the
uncatalyzed reaction first. Uncatalyzed reaction, in blue,
substrate starts off over here, and gets converted to product. That takes a lot of energy. Okay the enzyme in this
case is uninvolved, that’s why it has a plus sign. It’s kind of hanging
around as a spectator. Let’s just imagine
that would happen. So if that happens, there is
this activation energy, here, the difference in energy
between the transition state and the starting
material is very high. We can actually derive the speed
of the reaction from knowing that energy, right, and we
know a bigger energy over here, higher activation energy,
means a slower reaction, and often times these
reactions are too slow to be biologically useful. If we relied upon spontaneous
reactions taking place, you wouldn’t be a
human, you would just — it wouldn’t be possible
for biology to take place. Instead what happens
is, enzymes stabilize, bind to the substrate, lower the
energy of the substrate complex, and in turn, most
importantly, lower then energy of the transition state. The red arrow here is exactly
how much lower the energy is. The bigger that arrow is,
the better the enzyme, right because that’s how much
— how greatly improved it is. Similarly, the enzyme
then binds to the product, and then eventually
dissociates from the product. The product dissociates. Okay, let’s take a
look at an example. The example is first a
unimolecular reaction. I realize this is kind
of a hairy example. Bear with me it’s worth it. So in this case, what’s
nice about this example is, this reaction is a
concerted reaction, meaning it goes smoothly
in one fell swoop. Just one, you know, step
in this reaction mechanism. This is a paracyclic
reaction, and it has — it involves the transformation
of chorismate on the left, to perruthenate on
the right, and again, the enzyme is called
chorismate mutase. By the way, this is a key step
in the shikimic acid pathway which all the plants
on this planet rely on to produce phenylalanine
and the aromatic amino acids. This is one of those things
that we humans could not exist on the planet without. We cannot do this reaction. Plants and microorganisms
are the only organisms that have the necessary enzyme
to catalyze this reaction, and for that matter,
the only organisms that have this shikimic
acid pathway which is why indirectly you must
eat plant material to survive. Okay, so check out this
reaction mechanism. This is a beaut. This is awe inspiring. Okay, so in this reaction
mechanism, electrons are going to bounce down here, bounce,
bounce, and bounce some more, all the way up to here, producing a new carbon-carbon
bond between this carbon over here and this carbon. These guys are going
to join together to produce this new
carbon-carbon bond that has the wedge
coming out towards us. Okay, so in order
for this to happen, this part of the reaction
mechanism has to be sticking out towards us, and this whole
thing is just going to swing over the top, and all six
electrons are going to be flying at once, fist of fury style,
to give you, in a neat swoop, this new carbon-carbon bond
while breaking apart this old carbon-oxygen bond, and breaking
a carbon-carbon double bond to form a new carbon-carbon
double bond. This is really spectacular
stuff. This is the stuff
of just, you know, of graceful electron
choreography in motion. Okay, let’s take a closer look,
and see exactly how this works, and by the way, I think if you
try to do this reaction in lab, you can get it to work,
but it’s going to be slow. It’s going to be a
dog, so check this out. This is the way the
enzymes do it. This is the way they
make this happen. What they do is they grab on to
the handle up here, and force it into some semblance of the
necessary transition state, and in doing so they’re going to lower the activation
energy of this reaction. Okay, so here’s what
it looks like bound, or actually I’ll tell you
in a moment what this is. This is kind of like the
transition state bounds an enzyme, and in practice, the transition state
looks like this. Here, in dashed lines, are the
bonds being made and broken, and in order to get this
reaction to take place, the enzyme has to grab on
to this thing over here, and basically force it over
the top so that this carbon and this carbon are next to
each other to form a new bond. If that doesn’t happen, then
no reaction takes place, and so what the enzyme is
doing is just grabbing on and forcing this thing into
the right configuration to allow the reaction
to take place. Beauty, it’s a true
thing of beauty. Okay, now here’s the way we know
that that’s how that happens. This is classic work
done by my advisor from my undergraduate days at
UC Berkeley, Paul Bartlett, and the Bartlett laboratory
synthesized an inhibitor of this reaction
that looks like this. It kind of mimics
this transition state, and guess what? This mimics the transition
state so well that it sticks in there and just plugs it up. It binds really well
to this enzyme because the enzyme evolved to
bind to this transition state. That’s how it works, and so since enzymes
bind transition states with the highest affinity, this strategy can potentially
yield the best inhibitors. Binding to transition states
is the same thing as saying, “You’re going to
catalyze the reaction.” You’re lowering the energy
that’s required for the reaction to take place, and here we’re
seeing a dramatic example of this. By binding to this
transition state, you’re forcing the
substrate to get its carbons in the right location for
this reaction to take place, and if you don’t do
this, the reaction, the stuff is just swinging
all over the place, and if it’s swinging all over
the place, it’s finding lots of other stuff to
do with its time, and the reaction
never takes place. I have so much more I
want to tell you about. When we come back on Thursday,
I have something really cool that I’ve been saving
up for a while. I want to talk to you about
how it is that enzymes work, not just in terms of
binding transition states. By the way, this is kind of the classical stuff
you learn in biochemistry. It’s not wrong, it’s
actually totally right or else I wouldn’t say it to
you, but I want to talk to you about the other aspect
of enzymes which is enzymes
also have motion. They actually are going to be physically forcing
together these transition states to lower the transition
state energy, and in turn, accelerate reactions, and
to me, that’s really one of the most extraordinary
aspects of enzymatic catalysis. So let’s stop here, when
we pick it up on Thursday, we’ll be talking
about motion as a mode for accelerating
chemical reactions.
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4 thoughts on “Introduction to Chemical Biology 128. Lecture 12. Protein Functions.

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