LECTURE #2
Anyway, so let's start.
Pasteur thought up the concept, because
it was France and the issue was wine.
He was also involved in
immunization and the idea of vaccines, and we'll go into it.
the
issue
of
fermentation.
making
wine
was
basically
And the question was:
see if yeast could make alcohol.
making
But,
alcohol,
or
He had an in vivo assay to
And what we mean by an in vivo
assay is the following:
In terms of biochemistry, the definition
of
abstract
function
is
not
an
concept.
The
function is that there is an assay involved.
enzyme is the definition of its function.
definition
of
So an assay for an
And to try to get the
idea across: If I want to assay for polymerase, for example, I
assay for something that makes a polymer.
So, what I'm looking
for is something that will take small monomers and make them into
a polymer.
By definition, this reaction from monomer to polymer
is a polymerase...
the definition of the enzyme.
But, the key is, this isn't the assay.
The assay is something like this.
This is what's happening.
This comes into the second
Nobel Prize we're going to talk about today.
It's a filter; it's
a funnel, attached to a vacuum bottle, or an aspirator.
And what
you have here is a filter, and this is a Buchner funnel.
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And
you've run into the Buchner funnel.
paid for it.
In organic chemistry, you
You may have broken a couple.
But the beauty of the
Buchner funnel, and Buchner himself, is that he converted this
fermentation assay from an in vivo assay to an in vitro assay.
In
vivo meaning "in life, living", in vitro meaning, "in glass", or
as an extract.
And, the idea of biochemistry is to take a living, functional
process, a biological, biochemical process, keep it somewhat at
room temperature or at body temperature, and mimic that process in
some fashion and break it down to all its components.
Let's get back to the polymerase assay.
the concept of an assay.
I want you to understand
In this case, if I have a glass fiber
filter and I label one of these monomers, let's say with P-32,
then the monomer will go through the filter and not stick on the
filter because it's small.
This is small, and the polymer is
large.
If it becomes polymerized, then it will stick on the
filter,
and
you'll
take
that
filter,
and
it
radioactive P-32 charge on it and you can count it.
is really what sticks on the filter.
of polymerase.
will
have
a
So, the assay
That's the only definition
If I use the NTPs (the NTPs are triphosphates of
dioxytriphosphate; we'll go into it later), then I'm assaying for
DNA
polymerase.
We'll
repeat
this.
If
I
use
ribose
riboses, then I'm assaying for RNA, for RNA polymerase.
NTPs,
If I use
amino acids, what do I make here?... What's the polymer version of
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amino acids?... Protein.
proteins though.
There is no one enzyme that makes
There's no amino acid polymerase.
be a complex system.
It happens to
It's called a self-resynthesizing system.
So, for amino acids, it is not just a polymerase; it's a more
complex
system,
and
it's
called
re(protein)synthesizing system".
"self
resynthesizing,
self-
That's how they broke the code,
BUT, even though these are three different enzymes and three
different systems and even structures, the assay is the same.
just changing the monomer and the label.
acids, I might label <<<latridium<<<<.
amino acids.
There's no P-32 in amino
I might label one of the
But, the assay is what sticks on the filter.
How would I assay for something that cleaves a polymer?
there's one thing with polymers and making polymers.
in biological systems is not very complicated.
make
a
I'm
polymer,
what
are
you
removing
When you polymerize, you're removing water.
The reaction
If you're going to
from
Bonds?... Who said, "water"?... Very good.
Now,
the
monomers?...
It's exactly that.
Just think about it.
We're 80% water, so most of the biochemistry is going to be
involved in water metabolism and salt balance.
So, if you don't
know, three out of four times, the answer is going to have to do
something
with
polymerization.
do?
water.
So,
you're
removing
water
for
If you want to cleave it, what are you going to
If you're going to cleave a polymer down to monomers, you're
going to chew it up with enzymes, either digestive enzymes or
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restriction enzymes or enzymes that allow you to sequence it,
you're going to ADD water.
So, that's very simple.
much more to learn in terms of polymerization.
specific mechanism.
There's not
The rest of it is
That's the concept.
If I want to assay for cleavage instead of polymerization, or
breakdown or degradation, how do I assay for it?
label the polymer first.
Well, I would
I would make the polymer and label it.
It would be just the opposite.
The polymer would be, let's say,
100 lb thick on the filter, and if I cleave it, then I lose the
count.
So, instead of gaining counts with polymerization, it'll
be the loss of counts.
But, I would be assaying for cleavage.
So
all biochemical reactions are reversible.
So, the Buchner funnel is very important.
which you assay for polymerization.
It is the method by
What they use now is, instead
of funnels, (but they still use it for the enzyme assay), if you
want to look at the characteristics of the polymer that you're
making, either with RNA polymerase or DNA polymerase, you'll run
the acrylamide gels and you look at bands, but basically, you're
looking at the pieces you make either after you chew them up or
after
you
synthesize
them,
because
Yes?...You will use this kind of assay:
the
acrylamide
gel...
Glass fiber on a funnel.
If the DNA sticks to the glass fiber, then it's a polymer,
because the monomer will go through the glass fiber filter.
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It's
a glass fiber filter.
funnel.
But, the filter sits on this Buchner
That assay, this kind of assay, allowed him to start
analyzing complex fermentation mixes to see what was involved.
So, once you have an assay going, you can say, "I can start
purifying this and ask if this reaction keeps going and try to add
components or remove components and to see how pure I can get this
extract in order to purify something that will run the polymerase
assay".
In reality, you need very little protein.
Sometimes you can assay
to do polymerization, because you're starting with a monomer and
you're
making
very
large
end-product
product.
Very
little
polymerase, very little detectable polymerase will still give you
a functional assay.
That means you can detect its activity even
though you can't detect it chemically, not enough to stain, for
example, on a gel.
So what you've got to know is what the Buchner funnel really was
for, and it's quite clever.
So, it ends up being used to purify
and analyze all the DNA polymerases, the RNA polymerases and the
self-reprotein synthesizing system, which broke the genetic code.
Same assay.
What you do also is get the polymer to stick on the
glass fiber.
You have to precipitate it with acids and to use
trichloroacetic acid.
This leaves holes in your clothes, so if
you see biochemists with smocks or anything with holes in them,
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they were probably doing a polymerase assay.
The key was, once you got an enzyme with a function, could you
make enough of it to structurally characterize, not the product or
the substrate, but to characterize the actual enzyme itself?
The
first
the
question
was,
the
activity was the enzyme.
thing
that
was
responsible
for
They knew it was a catalytic function.
Now a catalyst, in a functional sense, is anything that speeds up
a reaction but doesn't take part in the reaction.
define catalytic activity in terms of structure.
But, we will
Functionally, a
catalyst is anything that speeds up a reaction that would have
occurred
consumed.
anyway,
without
The
question
taking
part
is,
"How
in
it,
does
without
this
being
happen?".
Structurally, (and I won't give you the punchline before we get to
it) most biochemical catalysts turned out to be proteins, so that
was one first step.
There are catalysts that are RNA, RNA acting
as a catalyst, and these are called ribozymes.
That was involved
at the MRC, and the discovery of one of these ribozymes and the
assay was quite simple. It was that degradation assay I was
telling you about.
I was doing 100 millicurie preps of P-32 to
get a small messenger RNA, and somewhere at the top of the gel was
a bigger band with a precursor of a t-RNA (an RNA that's involved
in translation, but it comes as a larger piece) and it has to get
processed down to a smaller piece.
And the precursor tyrosine t-
RNA for... is tyrosine, PSU+. I won't go into it.
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Anyway, it was a band on a gel.
hot.
It was very radioactive and very
I would cut out this band on a gel, and this guy would come
up from downstairs, by the name of Sid Altman, and he would ask me
for this band and I would cut it out and give him this band.
And
this was that hot, from 150 millicuries, that if you kept the band
out of the gel in a glass, Sorvall tube, for three months in the
freezer, the tube, the glass tube turned brown.
dose we were getting was very high.
So, the radiation
And, 75 roentgens to the eye
is blindness, cataracts.
So, the early guys in nucleic acid took a heavy dose.
Sanger took
a radiation dose...
So, Sid Altman would take this and he would
assay it this way...
Remember I said it was a large polymer, and
he would add extracts, and the extracts were not polymerases.
extracts were enzymes that would <<<<tape garbled.
called RNases, anything that cut RNAs.
The
So they were
The extract he did, he
called RNase P for precursor, because this was a precursor and it
would concert into a smaller t-RNA.
is
about
125
nucleotides.
nucleotides
and
this
So this was about... A t-RNA
was
about
200,
maybe
250
And, there was a conversion between this and this,
and this enzyme was involved in this conversion.
And all he would
do was assay to see if the band that he started with when he added
some of his RNase P would give him a smaller band.
If it cut the
bigger band to give him a smaller band, two small bands, then he
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knew he had RNase P.
And it turns out, RNase P was not a protein.
It turns out RNase P was an RNA also.
So, here you had an RNA
cleaving another RNA with a protein component.
So, this ribozyme,
RNase P, by Sid Altman, was the discovery of the first set of
enzymes that were not proteins.
That's how common and part of the dogma it was that protein should
be
enzymes,
and
it
turns
out
that
RNA
could
be
an
enzyme.
Anything can be an enzyme if it has certain characteristics.
The
characteristic is that is speeds up the reaction and doesn't take
part in it.
It has another characteristic structurally, which
we'll go into later.
Anyway, Sid Altman won a Nobel Prize for this.
down on that website.
You can track him
So, again, this degradation cleavage,
thinking you have another enzyme to cleave RNA...turns out not to
be protein, but RNA.
Those were called ribozymes.
Now, Sid
Aldman, when he was working, had a pinhole in his test tube, and
he was doing the same purification that I was, and he contaminated
himself
completely
with
50
millicuries
of
enormous radiation in the whole laboratory.
P-32,
which
caused
I had some training
in radiation physics, and Fred Sanger was the radiation officer,
but
he
didn't
know
anything
about
it.
I
had
this
>>>>>trolley<,<<<<< with lucite that I would use to do my preps,
and I used to do it at night.
Now with P-32, it has a decay of
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two weeks.
You have to remember that.
So, once you make your
product, you have to work day and night because in two weeks you
have half of it left.
left.
In four weeks, you have a quarter of it
So, once you start your prep, you work day and night and
you try to work at night all night until you're walking around
with a >>>>trolley... and it's very radioactive, so you don't want
people around.
It's very hard.
If you have a little Pasteur pipette and you squeeze it too fast,
the spray will contaminate the whole room.
the war stories.
I'm just telling you
Anyway, Sid Altman contaminates himself, and
you'll remember this, and what happened... He had to go to the
bathroom to clean himself up and as he's leaving, Sanger and I
said, "Do you want anything?
Is there anything we can get you, do
you
said
need
anything?".
millicuries of P-32."
He
yes,
That's dedication.
"Order
another
50
Anyway... and there he
was, contaminated, and he wanted more P-32.
Nobel Prize.
me
That's why he got a
So, that's the dedication you need to persevere.
He ended up being at Yale.
Now he's a professor there.
So, that's the concept of ribozymes, enzymes and degradation, and
the assay.
So, that was the assay for the enzyme.
Now, structurally, what we're going to have to do is define what a
catalyst is.
It's going to be a little complicated because what
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will
happen
is
structurally,
we'll
end
up
defining
proteins.
Protein structure will be in crystallographic structure.
First
sequence, a la Sanger, and then crystallographic structure, a la
Max Perutz and more complex structure, Nigel <<<<ongland and Aaron
Klug.
The question of structural definition of a catalyst will
also depend on what binding is and what the nature of binding is
and what the characteristic of what is bound between the enzyme
and the substrate and what the shape of the substrate is when the
enzyme binds it.
antibodies.
It turns out, you can mimic an enzyme with
And the antibody mimicking of enzymes is... the
question is whether you can get the right shape of a substrate
that the antibody will be mimicking what's called "the transition
state".
If the antibody mimics the binding to a transition state
(and we'll go over this in more detail) then what you have is an
antibody that has catalytic activity.
So, the definition of a
catalyst, in terms of a protein, will NOT be whether the enzyme
binds the substrate but the question is whether the enzyme binds
an intermediate state called "the transition state".
that
ANYTHING
catalyst.
that
binds
a
transition
That's the definition.
state
It turns out
will
act
as
a
So, the binding of a transition
state creates the catalytic activity.
Then the question is, "What is a transition state?".
shape.
The
first
person
to
start
crystallizing
That has a
enzymes
to
determine that they were proteins was a guy named Sumner, who also
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ended up at Rockefeller... Northrop, both of them... Sumner and
Northrop crystallized enzymes involved in the degradation of other
proteins, while Sumner just in urease; that's a functional in the
breakdown of urea.
Now,
once
you
crystallize
evidence for it's purity.
view.
But,
if
you
get
a
substrate
or
a
protein,
that's
That's from a biochemical point of
a
crystal,
you
can
diffraction from the physicist's point of view.
also
do
x-ray
So, the purity of
a substrate is involved in actually crystallizing something.
And,
it's ability to be used to analyze it for its structure requires
that it be a crystal.
So, there were two schools that started to
work on these enzymes.
Once you had a crystal.
At the same time,
Wendell Stanley, when the Rockefeller Institute was in New Jersey,
showed that tobacco mosaic virus could be crystallized.
This
suggested that even a living thing like a virus was a chemical in
some fashion, though a complex chemical, and this crystallization
showed
that
characterized
components
virus.
it
was
into
could
be
biochemically
its
components
separated
and
pure.
Then,
of
and
RNA
reaggregated
it
could
protein.
to
get
be
The
viable
But, also, later on, it could be used for, not x-ray
diffraction, but for optical diffraction.
We'll go into that.
Where you use a laser to set up a diffraction pattern because it's
too large for x-rays, and you analyze the diffraction pattern from
the laser diffraction pattern. That was done by Aaron Klug, who
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was also your uncle, who is now Sir Aaron Klug, and he was Crick's
student.
Crick
was
Perutz'
student,
and
Perutz
was
Bragg's
student, and Bragg created x-ray diffraction.
So, once you start getting crystals of things, you get purity and
you can also get structure.
Structure is very important because
you want to define, from the structure, its function.
Northrop
then
crystallized
enzymes
chymotrypsin, trypsyn, also papain.
involved
in
protein
degradation,
that
are
involved,
named
These are enzymes that are
but
they
cleave
at
specific
sequences in the protein, and therefore, allow the development of
protein sequencing.
Protein sequencing allowed, again, the purity
of these enzymes allowed that, and that protein sequencing, by
Sanger, allowed him to do a structural characterization.
dimensional, ???????, of proteins and enzymes.
It's one
So, it was the
start of characterizing these polymers in terms of their sequence.
So, these enzymes were beginning to be characterized in terms of
their sequences to determine why they act as catalysts.
go back to Haldane.
He was very clever.
Again, we
He's the one who said,
"I shall sacrifice my life for one brother or one sister or two
cousins."
That was his definition of a population gene.
said something very profound about enzymes.
He also
What he said was that
enzymes, in principal, interact with their substrate with weak
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bonds and that the interaction is not a covalent interaction...
Well, it could be.
But, that these weak bonds allowed it to
interact and to disengage as rapidly as they interact.
He also
suggested that this interaction is involved with a small area,
that there was a small, active site involved in that interaction.
If you have a large protein structure, let's say an enzyme, and
there's a substrate that's sitting in an area, what he basically
said is that the interaction between the enzyme and the substrate
is going to be with very weak bonds.
These could be hydrogen
bonds, salt linkages and hydrophobic bonds.
More importantly,
that the number of interaction sites between the substrate and the
enzyme are not many.
They are in the range of, let's say, three
to five different contact points.
How one knows that is that if
one mutagenizes an enzyme, one does not inactivate its activity,
except a very small proportion of the time and only in the regions
that are involved with interacting with the substrate.
If you
look at a mutagenesis like a target and the active site is a small
part
of
the
target
(and
this
is
the
whole
target)
then
the
proportion... let's say this is 5% of the target... actually it's
much smaller... let's say 2% (this being 2% and this being 100%),
you've inactivated only 2/100% of the time, or 1/50 of the time.
If this were large, you'd be inactivating it a lot of the time,
maybe 50%.
So, it's suggested that, just on mutation data, that
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the
activity,
or
the
functional
part,
of
the
enzyme
itself,
involved in the catalytic activity, was a small part of the
interaction.
There's another philosophical reason why you don't need more than
three
contact
points.
It
asymmetric in direction.
asymmetry.
is
because
two
contact
points
is
But, three contact points gives you
That means, if I contact here and here, I can't
differentiate this kind of interaction.
But, if I have three
points, this middle one allows me to differentiate direction and
allows me to have asymmetry.
So, the minimum that you need to
have asymmetry would be about three contact points, maybe four or
five.
If there are 20 amino acids, and you have somewhere in the range
of five possible contact points, that means there are about 3.2 x
106 million possible ways you could interact.
but it's not infinite.
That's quite large,
It turns out that antibodies bind in the
range of five amino acids, four to five amino acids.
So, if I'm
binding a substrate with an antibody... So let's take an antigen,
something on the surface of a virus, that contact point, those
contact points are in the range of four to five amino acids.
So,
the total universe of possible bindings of antibodies and antigens
are also very small.
but it's defined.
I mean, it's large in one sense, 3,000,000,
That means what you're talking about in terms
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of possible antigens are about 3,000,000.
The possible number of
antibodies (we'll go into it) is also about 3,000,000.
tells
you
(1)
that
the
binding
site
is
small
and
So that
(2)
it's
combinations of those that give you the interaction.
As I said before (and we'll show it next time) this, if you can
get the right binding to the right substrate, you can convert an
antibody into a catalyst and we'll talk about that next time.
Those are called catalytic antibodies.
END OF LECTURE #2
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