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JOANNE STUBBE: Hi.
Today, what we're going to do is focus on yet another cofactor
that you get out of your vitamin bottle.
And today we're going to be looking at vitamin B6.
And vitamin B6 is the cofactor that you use whenever you
want to metabolize amino acids.
Where do you get amino acids?
We all eat proteins.
The proteins get degraded to amino acids.
And we can use the energy released.
And trap that energy to do bio synthesis.
And we use amino acids, convert them into central metabolism,
and use them to make fats, or sugars, depending
on what the environment is telling us we need to do.
So let me introduce you to this cofactor,
the vitamin itself, as you have seen now, over and over again,
is not the actual cofactor.
It's Pyridoxine.
And so that's what you eat out of the vitamin bottle.
And as in the case of all vitamins, inside
the cell it has to be converted into the active form
of the cofactor.
And the cofactor we're going to be talking about,
the active form is Pyridoxal phosphate, which is called PLP.
And the structure of pyridoxal-- there two main structures
of the pyridoxal phosphate cofactor, one of them
is involved in 80% of all the chemistry.
And the one we're going to talk about today,
uses both forms of the cofactor.
That fits into central metabolism.
I'll show you how that fits in a minute.
So pyridoxal phosphate has this structure.
You have a pyridine ring, the pKa.
The pyridine ring is 6 5, so it may or may not be protonated.
And the key part of the cofactor-- one
of the key parts of the cofactor, is this aldehyde.
So pyridoxal means that this is an aldehyde.
The second form of the cofactor is pyridoxamine.
So this aldehyde is somehow-- and we
will look at how this happens-- is
converted from this aldehyde, into an amino group.
And this is called PMP, or pyridoxamine phosphate.
Now the other thing I wanted to show you,
before we move on to look at more
details of pyridoxal phosphate, is-- while this
is the form of the vitamin.
You almost never see this form inside the cell.
It is always bound in the active site of the enzyme.
And it binds in the active site of the enzyme--
so here's your enzyme-- through the epsilon
amino group of a lysine.
So you never really, when you purify the protein,
you never isolate the aldehyde.
So this is the aldehyde.
And I won't draw out all the rest of the structure,
but what you have is an amino group,
that's attached to a lysine, in the active site of your enzyme.
And what we're going to do is convert this ketone
into an imine.
So this is chemistry you see over and over and over again.
You've seen it already with carbonyl chemistry,
with carbon-carbon bond forming reactions,
with peptide bond hydrolysis, that we've already
talked about.
But what you do is, you're going to convert this aldehyde
into an imine.
You need to go through a tetrahedral intermediate.
And the carbonyl is polarized, delta plus, delta minus.
You spend a lot of time practicing
this kind of chemistry to form a tetrahedral intermediate.
And I'm going to have a proton transfer.
So one of the things that's tricky about this chemistry
is that, if you count the number of protons
they move around a lot in the active site.
And the fact is, that you want to protonate something
to make it into a better leaving group,
you want to deprotonate something
to make it function more like a nucleophile,
or as a base, and nature has figured out
how to orchestrate all the residues in the active site
to do this.
In most cases, we don't understand all of the details.
And so I'm not going to focus on proton transfer,
but you're going to see many proton transfers
within the active site.
So this gives us a tetrahedral intermediate, or transition
stage.
You've seen that over and over again.
And now what will happen is that you want
to lose a molecule of water.
So again, you need to pronate that, and what you form then
is a new carbon doubly bonded to a nitrogen,
rather than doubly bonded to the oxygen,
that's covalently bound to the enzyme.
And so, this is called an imine.
And because it's an imine of an aldehyde,
it's called an aldimine.
So whenever you isolate your enzyme, whenever
you isolate your enzyme, the pyridoxal
is always covalently bound.
OK, so, this bond is chemically easy to hydrolyze,
but it's always covalently bound.
OK.
So what I want to do now, is make a few generalizations
about pyridoxal, but the first thing is, that in all cases,
whenever you metabolize-- I want to metabolize an amino acid.
Pyridoxal phosphate requiring enzyme
is going to be the key player.
OK.
So here's an amino acid.
OK, here's the alpha position, the beta position,
and the gamma position.
Well what's so amazing-- I remember first hearing
about this-- is that you can do chemistry
at all these positions.
And the way nature figures out how
to do this is by orchestrating the active site,
with acid based catalyst sitting around in the right place,
to allow you to do the chemical transformation
that this protein has evolved to do.
So let me to show you what the alpha position,
so this is the alpha position, you can cleave this bond.
I'll show you how this happens.
You can do all this chemistry with a few simple chemical
transformations, takes practice, but once you sort of get
what these transformations are, it's
amazing what you can get this cofactor to help you do.
And I'll explain that in a minute.
So you get cleavage of the carbon carbon bond
that's loss of CO2 that's a decarboxylation reaction.
We don't talk about that in 507, but that kind of reaction
generates all of our neurotransmitters.
You're going to cleave this carbon-hydrogen bond.
Remember, amino acids are in the S configuration--
but for example in cell wall, in bacteria,
they can be either the S or the R configuration,
so you can cleave that bond, and put the proton back
on the other face, that's a racemization reaction.
You can cleave this carbon-carbon bond
between the alpha and the beta position,
that's a reaction-- remember, we talked about carbon-carbon bond
formation-- this is the reverse aldol reaction.
And the one where we're going to focus on today, which
is the one that fits best into central metabolism,
is what happens to this carbon-nitrogen bond.
So we have an amino group of our amino acid
is going to get converted into a ketone group.
So, this group, is going to get converted into this group.
And to do that, we're going to use the imine of pyrodoxial
phosphate that we have in the active site of the enzyme.
So this is sort of like a carbonyl
and that's going to get converted
into the amine, the pyridoxamine.
So these are the two forms of the cofactor.
So what are we going to be focusing on, is
how this reaction, actually, happens, and this
is the most complicated of all the pyridoxal prostate
dependent reactions.
So that I'm going to come back to this in a minute,
but what I want to do is make a few generalizations
about where you're going to see pyridoxal phosphate chemistry
in primary metabolism.
So what we're going to see is that the TCA cycle,
tricarboxylic acid cycle, or the Krebs cycle,
plays a central role.
It's found in the mitochondria.
You're going to see this over and over again,
over the course of the semester.
Things feed in and out of the cycle.
A cycle means it goes around and around,
and if you remove something from the cycle
and don't put anything back into the cycle, the cycle stops.
And you're in serious trouble.
So one way-- one thing-- one way to feed in and out
of this cycle is through amino acids.
So this reaction, which we're going
to call the transemination, or a transamination,
is metabolism of amino acid, we'll
see into an alpha keto acid.
So if you look at the TCA cycle and we
look at this reaction, what you'll see
is you have this compound, called alpha ketoglutarate.
OK, so alpha ketogluterate and that
going to interconvert with the amino acid
and so this ketone is going to be converted
into an amino group, and so we're
having the amino group converted into a ketone group.
And pyridoxial is going to be converted into pyridoxamine.
So we're going to have PLP converted into PMP.
I'm going to show you how that works.
So, if you feed in glutamate from your diet,
it-- by this pyridoxal phosphate dependent reaction--
can feed into the Krebs cycle.
If you want to make amino acids, on the other hand,
you can suck some of the alpha keto acid
out, and convert it into amino acids.
And you have to have a way of controlling whether you
feed in, or you, actually, remove your metabolites
from the cycle.
If you come up here and look at oxaloacetic and aspartic acid--
So oxaloacetic acid is also an alpha keto acid, OK.
And the amino acid-- can you see?
I'm probably too close to the edge-- OK,
and so here we have the amino acid.
So here we have amino acid alpha keto acid, amino acid alpha
keto acid.
If you go further up and go pyruvate feeds
into the TCA cycle-- pyruvate comes
from the glycolysis pathway, which
again is breakdown of sugars, pyruvate is an alpha keto acid.
So this is a CH3, so this is the simplest,
and this can get interconverted into alanine.
So what you see is this same theme-- amino group ketone,
amino group ketone.
I'm being sloppy, here.
Most amino groups are protonated,
because the pKa, you should remember, is around 9.
So they're, mostly, protonated in solution.
I should protonate this over here, as well.
And so you have a ketone group and amino group.
So this is called anaplerotic pathways
where things can feed in and feed out of central metabolism.
And this is the only time, during the course of 507,
that you're introduced to amino acids,
and how they're metabolized.
So what I want to do now is then,
briefly, show you how this transformation, actually,
works.
OK.
So I'm going to give you some general rules.
So the transformation I just showed you--
an amino acid into an alpha keto acid-- looks to be complicated.
And, I told you, the cofactor changes its structure
from an imine into an amino group.
And the question is, how does that happen?
So I want to show you a bunch of simple, basic rules
that allow you to think about all pyridoxal phosphate
requiring chemistry.
So I'm going to write down the rules,
and then I'm going to show you how
it works on the reactions we were just
looking at this these transeminations,
or transamination reactions.
So how do we think about the mechanism of these PLP enzymes?
So the first thing is-- the first step
is-- so you're going to start out with an imine bound
to the active site of your pyridoxal phosphate
and an amino acid.
I'll abbreviate it aa.
And the first thing you do is, you're going to,
remove this imine and form a new imine with the amino acid.
So that's called a transimination reaction, OK.
So what we're going to do is form a new imine.
And so the imine from the amino acid,
and I'll show you how this happen,
is going to switch with this one.
And so what you're left with, in the active site,
is the amino group of the lysine.
OK.
So this lysine-- nature has figured out
how to minimize the numbers of acid and base
groups constrained in the region, the active site, where
all the chemistry happens.
And she uses this lysine, which initially is holding covalently
the cofactor, in the active site.
She then uses this lysine to do general acid, general base
catalysis.
And she uses it over and over and over again.
Now every pyridoxal enzyme is distinct
and has additional groups in the active site.
But we know a lot about this chemistry.
We even know what the groups are,
but we're going to look and talk about generalizations.
So the first thing, we have to do
is, we need to free up our general acid base
catalyst, lysine, and we need to covalently bind
the amino acid, which is a substrate, into pyridoxal.
So that's the first step.
And I'm going to come back to this in a minute.
The second step in all of these reactions
is-- all amino acids have an alpha hydrogen,
that alpha hydrogen has a very high pKa.
It's very hard for a normal amino acid side chain,
in the active site, to remove that proton,
because it's not acidic enough.
So what I'm going to show you is pyridoxal
increases the acidity of that alpha hydrogen,
making it easier to do the chemistry.
So the second step in almost all pyridoxal reactions,
is removal of the alpha hydrogen.
And we can do that, because PLP makes the hydrogen more acidic.
And I'll show you why that's true in a minute.
And then, the third thing is, once you remove that hydrogen,
then you get a look at the chemistry you want to catalyze.
And we're going to be talking about this transimination
transamination reactions.
But remember I told-- you can do all this chemistry at alpha,
beta, gamma.
So you need to assess what the substrate is,
and what the product is, and then, within the active site,
you're going to have to do a lot of manipulation
with acid and base catalysts to get you
into the final stage, where you can release the product you
want to release.
So the last step in this reaction--
in all pyridoxal reactions-- so we do some chemistry in here.
And I'll show you what the chemistry is
with transamination reactions.
The word transimination reactions-- now
I keep saying transamination and transimination.
That's because most textbooks call it transamination,
because they think about pyridoxal as the aldehyde.
But, in reality, pyridoxal is always in the imine,
covalently bound.
And so that's why it's that transimination, rather than
a transamination.
So the last step then, is, hydrolysis.
And I'll show you how that happens, or transimination
to reform this structure.
So we get ourselves back to where we started.
So there's three simple steps, and in the middle,
depending on what the reaction is, you have
to do additional manipulations.
But all pyridoxal enzymes go through these three,
general steps.
The first step, I told you, in all these reactions
is transimination.
OK.
So here's our Schiff-base, and it's your pyridoxal.
It's covalently bound to the lysine in the active site.
Now one thing that students often find confusing
is the protonation state of this imine.
And that's because it's right around neutral pH, pH 7.
So depending on what's in the active site,
it could be protonated, or not protonated,
if it's protonated of course it enhances reactivity
for nucleophilic attack.
So you want it to be protonated.
So the active site is going to manipulate itself
to put in the protonated state.
So here you have an amino acid, and here we
have a protonated imine, and so this is the nucleophile,
and it can attack the carbon of the imine
to form a tetrahedral adduct.
So that's what we're doing here.
So, this, is going to attack, this,
to form this tetrahedral adduct.
And, you've seen again, the tetrahedral
chemistry over again, when I showed you
how you formed this imine in the first place.
So tetrahedral chemistry, tetrahedral intermediates,
transition states, which collapse to form back
imines, or carbonyls, happens over and over, and over again
in pyridoxal chemistry.
So now what happens is we have this tetrahedral intermediate,
or transition state-- I have it in parentheses, because it's
a high energy intermediate, it doesn't sit around,
and let us look at it.
Most of the time you can never see it.
It's very high on a reaction coordinate.
This, collapses then, and when it collapses,
what do you generate?
You generate the lysine in the active site.
So now we have generated a residue
in the active site that can function
as a general acid, or general base,
catalysis through the rest of the chemical transformations.
And what've we done, is we've converted this imine
with lysine, now to an imine of the amino acid.
So that's what transimination is, one imine to another imine.
The imine that's covalently bound
to the protein through pyridoxal, to an amino acid
imine.
OK, so, that's the first step.
The second step is that ultimately,
in almost all pyridoxal reactions, you
want to remove this alpha hydrogen.
And that alpha hydrogen, again, is extremely non-acidic
but by complexing the amino acid to pyridoxal--
this is what the function of the cofactor
is-- you are enhancing the acidity of this alpha hydrogen.
You're making it easier for a group in the active site,
a general base catalyst, like lysine,
can now pull off this proton to generate this intermediate.
Now why is this hydrogen more acidic?
Well, if you look at the structure,
you can draw all kinds of resonance structures
which shows that this carbanion is more stabilized,
because it's attached to the pyridoxal cofactor.
So if you look at this, you can draw this resonance structure,
which is shown here, and you can draw 20 other resonance
structures.
OK, so, the key here is you can remove the alpha hydrogen,
because you're able to delocalize
these unpaired electrons on this carbon over the entire system.
So let me show you what that looks like.
So if I draw-- this is called Dunathan's hypothesis-- so
here's our pyridine ring, here's our imine,
and here's our amino acid.
Here's the carboxal-- here's the side chain in our group
and here's a carboxylate, OK.
So the idea is, you have like a benzene ring-- if you haven't
seen pyridine rings-- but you have a pi cloud that
delocalizes-- where these electrons are completely
delocalized over the aromatic ring-- but here,
you also have a pi cloud and these things are close
enough so you can delocalize.
Now what you're going to do-- and the way nature decides
what chemistry happens is since we want to cleave, in this case
the carbon-hydrogen bond, she places
that carbon-hydrogen bond-- by complexing the carboxylate
and complexing whatever the R group is--
she places that carbon-hydrogen bond
perpendicular to this plane of the pi aldimine system.
So what you're doing then is the lysine, that we just liberated
through doing the transimination reaction,
you now generate an empty P orbital
with unpaired electrons in it, generate the carbanion
and now this system can completely delocalize.
So, again, this pyridine ring is planar.
So you can, completely, delocalize the electrons
over this whole system.
This is a pi cloud.
And now this is already set up so that it can delocalize over
this whole system.
So what you've done then is because of the ability
to stabilize this carbanion you're
making that hydrogen more acidic.
So if you wanted to say, for example, cleave-- remember,
I told you at the very beginning,
you might be able to decarboxylate-- that enzyme,
would place that CO2 perpendicular
to the plane of the pi aldimine system
and use the same strategy to stabilize the resulting
carbanion intermediate.
We're not going to talk about that chemistry in 507.
So what you've done then-- what the beauty of pyridoxal
is that she's increased this acidity,
and allows you a great deal of flexibility.
Because once you generate that carbanion-- those of you who've
had 513-- you'll, immediately, recognize if you have a leaving
group on a carbon adjacent to this carbon,
you can do an elimination reaction.
So it sets up a whole series of transformations.
For today, were only focusing on the transimination reaction.
How do we convert this to an alpha keto
acid, and the pyridoxal to pyridoxamine?
That's the question we're asking.
So we do this chemistry.
And now what we need to do, we can use this resonance
structure, we want to ask the question, what is the product?
Well, we want to get to an alpha keto acid.
OK, and if you look at this structure,
this molecule is an imine of an alpha keto acid.
So this is, exactly, the state we want to be in,
but then we have all of this-- we
have this reactive intermediate here.
So what we want to do is protonate some place here
to generate this state.
So that the last step in all pyridoxal reactions
is hydrolysis.
And now we're set up with the hydrolysis reaction
to generate the alpha keto acid.
So, we then want to ask the question,
where can we protonate?
And, so, again, we have this lysine
which we now have used to pull off the alpha
hydrogen. It's now protonated.
So now instead of being a general based catalyst,
here, it's functioning as a general acid catalyst.
And so now what can happen is you can pick up
a proton from this lysine, it's supplying it with that proton,
to generate this structure and regenerate
lysine that can function now as a general base catalyst.
So it's toggeling between general acid and general base
catalysis.
And now remember, what we want to get in the end
is this pyridoxamine, and we want this alpha keto acid,
and now we're set up to rapidly go to an alpha keto acid.
Where have you seen chemistry like this before?
You've seen it in the aldolase reaction
that we talked about with carbon-carbon bond forming
reactions.
So the last step in all PLP dependent transaminations
is hydrolysis.
So here we have the lysine acting as a general base
to activate water for a nucleophilic attack
on this imine, which is activated to have water add,
and now you form again, your tetrahedral transition state.
I have all of these unstable species in brackets.
We really-- sometimes we see them, sometimes we don't.
But you have to work hard to see them.
And now this simply, the tetrahedral adduct,
collapses to form pyridoxamine and forms the alpha keto acid.
So that's where we wanted to get.
But now what happens, is we're in the form of the cofactor--
instead of being in the aldehyde form we're in the imine form.
So what we want to do now is reverse
this reaction using a different alpha keto acid,
and we will generate a different amino acid.
So now what can happen-- so in their pyridoxamine form,
we then can bind a different alpha keto acid--
remember, in the first slide, I showed
you three different alpha keto acids--
oxaloacetic acid, alpha-ketoglutarate,
pyruvate-- you can reverse this whole process.
And, in the end, what you end up with is a different amino acid
and you regenerate your imine of pyridoxal.
So out of all-- the only pyridoxal phosphate
requiring enzyme that goes from the aldehyde,
or imine, to the pyridoxamine are
these transamination reactions.
And, so, this is the most complicated.
Normally, at the end of your reaction
you wind up in this state.
And this probably seems extremely confusing to most
of you, but after you solve three, or four, problems
where you have to look at the actual transformations,
and think about this tetrahedral chemistry, imines,
and amino groups, and alpha keto groups, you will, I think,
be able to actually easily see how
ingenious nature has been to actually design
pyridoxal phosphate.
And, I think, the most amazing thing, of course,
is that pyridoxal phosphate without any enzyme--
I told you can catalyze many, many reactions you
do all the reactions with the en-- it does it
spontaneously, at room temperature, at pH 7.
What the enzyme does, is only allows one of these reactions
by having everything positioned exactly the right way
in the active site.
So this is one of the cofactors, that I
thought was amazingly cool when I
was in graduate school, that made
me want to become a biochemist.