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PROFESSOR: Today what I want
to do within the lexicon

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is tell you about nature's
most spectacularly beautiful

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00:00:28,880 --> 00:00:30,770
cofactors.

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00:00:30,770 --> 00:00:36,390
And these are formed from
vitamin B-12, which you

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00:00:36,390 --> 00:00:38,570
find in your vitamin bottle.

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00:00:38,570 --> 00:00:39,070
OK.

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00:00:39,070 --> 00:00:41,920
So what is the structure
of vitamin B-12, and why

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do I say they are
spectacularly beautiful?

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00:00:44,870 --> 00:00:46,370
So it's very hard
to see, but if you

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00:00:46,370 --> 00:00:47,870
look at the structure
of this, where

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00:00:47,870 --> 00:00:51,060
have you seen a molecule
this complicated

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00:00:51,060 --> 00:00:54,440
with five membered
rings, each of which

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has a nitrogen in this?

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00:00:55,770 --> 00:00:58,510
You've seen this when
you studied hemoglobin,

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00:00:58,510 --> 00:01:02,720
and you think about heme
and proto protoporphyrin IX.

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00:01:02,720 --> 00:01:05,050
If you look at the
biosynthetic pathway of heme,

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00:01:05,050 --> 00:01:09,530
a branchpoint of that pathway
is to make this ring, which

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00:01:09,530 --> 00:01:13,250
is found in adenosylcobalamin
and methylcobalamin, which

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00:01:13,250 --> 00:01:15,470
is what we're going to
be focusing on today.

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00:01:15,470 --> 00:01:19,390
And this ring is
called the corrin ring.

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00:01:19,390 --> 00:01:21,630
So what I want to
do is introduce

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00:01:21,630 --> 00:01:23,690
you a little bit
to this corrin ring

30
00:01:23,690 --> 00:01:27,670
and what's unusual about it
compared to protoporphyrin IX

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00:01:27,670 --> 00:01:30,810
that you've seen before.

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00:01:30,810 --> 00:01:35,130
So the vitamin, as in the case
of all vitamins that we've

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00:01:35,130 --> 00:01:37,500
talked about over the
course of the semester,

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00:01:37,500 --> 00:01:40,200
is not the actual
cofactor used in

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00:01:40,200 --> 00:01:42,190
the enzymatic transformation.

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00:01:42,190 --> 00:01:46,450
The vitamin, which in this case
would have this group replaced

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00:01:46,450 --> 00:01:50,400
with cyanide, is vitamin B-12.

38
00:01:50,400 --> 00:01:54,570
The actual cofactors
that bind to the enzyme

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00:01:54,570 --> 00:01:58,710
have that cyanide replaced
with either a methyl

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00:01:58,710 --> 00:02:01,680
group-- and that's
called methylcobalamin--

41
00:02:01,680 --> 00:02:06,280
or they have it replaced
with 5-prime-deoxyadenosine,

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00:02:06,280 --> 00:02:09,690
and that's called
adenosylcobalamin.

43
00:02:09,690 --> 00:02:13,400
And so the rest of the
molecule is exactly the same.

44
00:02:13,400 --> 00:02:17,230
The only thing that's
distinct is the axial ligands.

45
00:02:17,230 --> 00:02:21,470
So you remember from your
transition metal chemistry

46
00:02:21,470 --> 00:02:24,490
you had when you were
freshman that here you

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00:02:24,490 --> 00:02:29,831
have a cobalt III, and it's
coordinated to four nitrogens.

48
00:02:29,831 --> 00:02:30,330
OK?

49
00:02:30,330 --> 00:02:32,450
So this would be the
equatorial plane,

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00:02:32,450 --> 00:02:35,540
and we're going to draw it
like this, subsequently,

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00:02:35,540 --> 00:02:38,870
because you can see how
complicated this molecule is.

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00:02:38,870 --> 00:02:43,232
And the middle sits
right in this plane,

53
00:02:43,232 --> 00:02:44,690
but you can see
that you don't have

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00:02:44,690 --> 00:02:48,280
complete saturation of
these pyrrole- type rings,

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00:02:48,280 --> 00:02:50,540
so there's some
pucker in contrast

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00:02:50,540 --> 00:02:53,340
with hemes which are very flat.

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00:02:53,340 --> 00:02:56,180
And then these are called
equatorial ligands,

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00:02:56,180 --> 00:02:59,200
and then you have axial ligands.

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00:02:59,200 --> 00:03:01,610
And so there are
a number of things

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00:03:01,610 --> 00:03:05,660
that again I wanted to point
out that are unusual about B-12.

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One is the fact that the
corrin ring again is much more

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reduced than a pyrrole ring.

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00:03:11,960 --> 00:03:13,410
And so it's puckered.

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00:03:13,410 --> 00:03:18,870
And if you look at the visible
spectrum, or you use your eye

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00:03:18,870 --> 00:03:22,570
and use your eyeball method,
you see that cyanocobalamin

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is bright purple.

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00:03:24,110 --> 00:03:27,860
If you replace this R
group with a methyl group,

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this color turns out to
be sort of yellow-orange.

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00:03:31,120 --> 00:03:35,020
And if you replace this
with 5-prime-deoxyadenosine,

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it turns out to be pink.

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00:03:36,330 --> 00:03:40,010
So that's why I say that this
is nature's most spectacularly

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beautiful cofactor.

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00:03:42,580 --> 00:03:45,840
So the other thing is you
have two axial ligands.

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I just introduced you to
these guys on the top face.

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00:03:49,010 --> 00:03:51,120
But what you also see
on the bottom face--

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00:03:51,120 --> 00:03:54,900
the bottom axial ligand--
is this unusual structure,

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which is called
dimethylbenzimidazole.

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00:03:58,620 --> 00:04:03,130
And it's attached to a
ribose, but the unusual thing

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00:04:03,130 --> 00:04:08,590
is in most nucleosides that
you've encountered like ATP,

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00:04:08,590 --> 00:04:12,010
this configuration is
in the beta position--

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00:04:12,010 --> 00:04:13,490
it's on the top
face of the sugar--

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00:04:13,490 --> 00:04:15,080
and here it's in
the alpha position.

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00:04:15,080 --> 00:04:17,320
So that's distinct.

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00:04:17,320 --> 00:04:19,430
And the other thing
that's distinct

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is the decorations around
the side chain compared

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00:04:23,190 --> 00:04:26,750
to what you see in porphyrins.

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00:04:26,750 --> 00:04:29,985
So this is the cofactor we're
going to be talking about,

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00:04:29,985 --> 00:04:34,720
and one of the questions
that you're interested in

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00:04:34,720 --> 00:04:37,820
is where do we find thes
cofactors in metabolism.

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00:04:37,820 --> 00:04:43,050
In a mammalian metabolism, the
appearance of these cofactors

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00:04:43,050 --> 00:04:44,630
is quite limited.

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00:04:44,630 --> 00:04:50,740
So the only place you see it in
metabolism in mammalian systems

93
00:04:50,740 --> 00:04:53,950
is you see
methylcobalamin-- OK, where

94
00:04:53,950 --> 00:04:58,120
this is a methyl group-- in
formation of the amino acid

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00:04:58,120 --> 00:04:59,040
methionine.

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00:04:59,040 --> 00:05:00,829
And so the methyl
group from methionine--

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00:05:00,829 --> 00:05:02,370
I'll show you briefly
at the very end

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00:05:02,370 --> 00:05:06,840
of this little presentation--
comes from this methyl group.

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00:05:06,840 --> 00:05:12,910
You use adenosylcobalamin in
odd chain fatty acid metabolism.

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00:05:12,910 --> 00:05:17,560
So you have fatty acids that are
either an even or an odd chain.

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00:05:17,560 --> 00:05:19,400
When you break the
odd chain ones down,

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00:05:19,400 --> 00:05:21,110
you get proprionyl CoA.

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00:05:21,110 --> 00:05:23,870
The proprionyl CoA,
through a series of steps,

104
00:05:23,870 --> 00:05:27,770
is converted into
malonyl CoA, which

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00:05:27,770 --> 00:05:30,690
then gets converted
to succinyl COA,

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00:05:30,690 --> 00:05:33,530
which feeds into the TCA cycle.

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00:05:33,530 --> 00:05:38,750
And in that pathway, you
use an enzyme-- a mutase--

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00:05:38,750 --> 00:05:40,710
that uses a adenosylcobalamin.

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00:05:40,710 --> 00:05:43,300
We'll talk briefly
about the chemistry

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00:05:43,300 --> 00:05:47,800
of a adenosylcobalamin;
also methylcobalamin.

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00:05:47,800 --> 00:05:48,300
OK.

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00:05:48,300 --> 00:05:50,580
So there were a
few things that I

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00:05:50,580 --> 00:05:54,680
wanted to say about-- some
generalizations that I wanted

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00:05:54,680 --> 00:05:59,740
to make about these cofactors.

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00:05:59,740 --> 00:06:08,470
And at the first one is
that again, the corrin ring

116
00:06:08,470 --> 00:06:12,310
is much more reduced
than the pyrrole ring

117
00:06:12,310 --> 00:06:18,210
that you see in protoporphyrin
IX, which you've

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00:06:18,210 --> 00:06:20,900
seen in hemoglobin before.

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00:06:20,900 --> 00:06:23,190
The second thing
is that you have

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00:06:23,190 --> 00:06:26,860
this unusual
dimethylbenzimidazole axial

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00:06:26,860 --> 00:06:31,290
ligand, which you see nowhere
else in cofactor chemistry.

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00:06:31,290 --> 00:06:34,700
It's only found in this
particular cofactor.

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00:06:34,700 --> 00:06:37,450
The second thing, which I
think is the most amazing,

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00:06:37,450 --> 00:06:41,890
is that what you see
if you look back here

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00:06:41,890 --> 00:06:44,740
is that you have-- if
this is a methyl group,

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00:06:44,740 --> 00:06:47,850
or this is this
5-prime-deoxyadenosine--

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00:06:47,850 --> 00:06:50,600
you have a carbon cobalt bond.

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00:06:50,600 --> 00:06:53,500
Well, this was
discovered in the 1950s,

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00:06:53,500 --> 00:06:56,910
and the first structure
was solved of this molecule

130
00:06:56,910 --> 00:07:00,320
by Dorothy Crowfoot
Hodgkin in 1964,

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00:07:00,320 --> 00:07:02,510
and she won the Nobel
Prize for this work.

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No chemist had ever seen a
carbon cobalt blonde bond,

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00:07:06,840 --> 00:07:10,520
and thought in fact biochemists
were crazy that they even

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00:07:10,520 --> 00:07:12,750
proposed such a structure.

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00:07:12,750 --> 00:07:14,310
So this structure--
and we'll see

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00:07:14,310 --> 00:07:16,890
that this is where all
the chemistry happens--

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00:07:16,890 --> 00:07:18,830
is completely unique.

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00:07:18,830 --> 00:07:22,230
And people spent 25
years figuring out

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00:07:22,230 --> 00:07:24,540
how this cofactor
actually worked

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00:07:24,540 --> 00:07:27,820
to do the transformations that
I'll very briefly introduce you

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00:07:27,820 --> 00:07:28,320
to.

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00:07:28,320 --> 00:07:33,460
So this is the first
example of a carbon cobalt--

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00:07:33,460 --> 00:07:36,630
and will see that cobalt is
in the plus 3 oxidation state

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00:07:36,630 --> 00:07:45,400
bond-- so it's the first
organo-metallic cofactor.

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00:07:45,400 --> 00:07:48,690
And what people also found
by studying this molecule

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00:07:48,690 --> 00:07:52,850
is that the cobalt can actually
exist in three oxidation

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00:07:52,850 --> 00:07:53,690
states.

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00:07:53,690 --> 00:07:58,520
It can exist in the
cobalt I state, where

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00:07:58,520 --> 00:08:01,810
in the d z squared orbital--
if you don't remember what a d

150
00:08:01,810 --> 00:08:04,070
z squared orbital is, you
need to go back and look

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00:08:04,070 --> 00:08:08,480
at your freshman chemistry--
you have two electrons.

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00:08:08,480 --> 00:08:14,820
And it turns out that cobalt
I is a super nucleophile.

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00:08:14,820 --> 00:08:18,940
And we'll see that that plays
a key role in the chemistry.

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00:08:18,940 --> 00:08:21,270
And so again, this
is the cobalt. The d.

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00:08:21,270 --> 00:08:25,600
We're only looking at one of
the orbitals of the cobalt.

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00:08:25,600 --> 00:08:33,130
On the other hand-- and
this is found in methyl--

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00:08:33,130 --> 00:08:35,580
this is going to be
used in methylcobalamin.

158
00:08:35,580 --> 00:08:40,340
So when you have a methyl
as the axial ligand,

159
00:08:40,340 --> 00:08:43,760
you're going to use cobalt in
this oxidation state, which

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00:08:43,760 --> 00:08:45,880
is the plus one state.

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00:08:45,880 --> 00:08:48,860
For almost all other
B-12 dependent reactions,

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00:08:48,860 --> 00:08:54,780
you have cobalt II, which
has-- you've lost an electron,

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00:08:54,780 --> 00:08:58,630
so you have only one
electron by itself.

164
00:08:58,630 --> 00:09:00,570
And its d z squared orbital.

165
00:09:00,570 --> 00:09:03,090
And this really
dictates the chemistry.

166
00:09:03,090 --> 00:09:08,410
And so this is found in
adenosylcobalamin chemistry.

167
00:09:08,410 --> 00:09:13,320
And then in the resting the
state you have cobalt III.

168
00:09:13,320 --> 00:09:17,120
And cobalt III has none
of the electrons in the d

169
00:09:17,120 --> 00:09:18,990
z squared orbital.

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00:09:18,990 --> 00:09:23,950
And this is basically the
resting state-- its most stable

171
00:09:23,950 --> 00:09:26,260
state-- where you
find this cofactor.

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00:09:26,260 --> 00:09:29,590
So in cyanocobalamin,
which is vitamin B-12,

173
00:09:29,590 --> 00:09:33,671
the cobalt is in the
plus 3 oxidation state.

174
00:09:33,671 --> 00:09:34,170
OK.

175
00:09:34,170 --> 00:09:36,530
So we're going to see
very briefly-- we're

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00:09:36,530 --> 00:09:41,290
not spending much time on
this-- is the cobalt I state.

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00:09:41,290 --> 00:09:42,650
It's a super good nucleophile.

178
00:09:42,650 --> 00:09:44,900
It affects the chemistry
of the cobalt II state.

179
00:09:44,900 --> 00:09:47,240
It has one unpaired electron.

180
00:09:47,240 --> 00:09:49,380
You haven't been
introduced to chemistry

181
00:09:49,380 --> 00:09:51,000
with one unpaired
electron, which

182
00:09:51,000 --> 00:09:53,250
is radical chemistry-- that
most people don't spend

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00:09:53,250 --> 00:09:56,770
a lot of time talking about
adenosylcobalamin because it's

184
00:09:56,770 --> 00:09:59,090
radicals, and they don't
learn much about radicals

185
00:09:59,090 --> 00:10:01,360
in introductory
organic chemistry.

186
00:10:01,360 --> 00:10:05,780
But I'll show you briefly
how the enzymes work

187
00:10:05,780 --> 00:10:08,850
that use this cobalt II state.

188
00:10:08,850 --> 00:10:14,500
The other thing I wanted
to mention was the colors.

189
00:10:14,500 --> 00:10:18,640
And so if you want to understand
how these cofactors work,

190
00:10:18,640 --> 00:10:20,100
the different
number of electrons

191
00:10:20,100 --> 00:10:23,060
govern what colors
you can end up seeing.

192
00:10:23,060 --> 00:10:26,360
And again I always use
this is as an example

193
00:10:26,360 --> 00:10:29,550
on MIT'S campus in the spring.

194
00:10:29,550 --> 00:10:35,120
Cobalt II has spectacular orange
color like the orange azaleas,

195
00:10:35,120 --> 00:10:39,630
and cobalt III is
like the pink azaleas.

196
00:10:39,630 --> 00:10:41,580
And so they're really
dramatically different.

197
00:10:41,580 --> 00:10:43,420
And this is why I say
this is nature's most

198
00:10:43,420 --> 00:10:44,710
beautiful cofactor.

199
00:10:44,710 --> 00:10:45,560
OK.

200
00:10:45,560 --> 00:10:52,130
So what I want to do now is
briefly talk about mechanism.

201
00:10:52,130 --> 00:10:54,900
And I'm going to mostly
talk about mechanism

202
00:10:54,900 --> 00:10:59,380
of the cobalt II state, and
how adenosylcobalamin works,

203
00:10:59,380 --> 00:11:02,030
since that's the one
that's most complicated.

204
00:11:02,030 --> 00:11:03,570
So what I'm drawing
here is if we

205
00:11:03,570 --> 00:11:11,070
look at the structure
of adenosylcobalamin,

206
00:11:11,070 --> 00:11:13,900
you see you have a cobalt
with four nitrogen.

207
00:11:13,900 --> 00:11:15,800
These are the four
nitrogens and these

208
00:11:15,800 --> 00:11:16,970
are the two axial ligands.

209
00:11:16,970 --> 00:11:18,550
So this is the
abbreviation I'm going

210
00:11:18,550 --> 00:11:21,650
to be using in all the
subsequent chemical

211
00:11:21,650 --> 00:11:23,740
descriptions of what
I'm talking about, OK?

212
00:11:23,740 --> 00:11:26,290
So I'm not going to draw this
structure out over and over

213
00:11:26,290 --> 00:11:27,140
again.

214
00:11:27,140 --> 00:11:27,650
OK.

215
00:11:27,650 --> 00:11:33,770
So here's our adenosylcobalamin
that we just talked about.

216
00:11:33,770 --> 00:11:35,290
And there's DMB.

217
00:11:35,290 --> 00:11:38,230
It's a dimethylbenzimidazole
axial ligand,

218
00:11:38,230 --> 00:11:40,870
and then you have the
5-prime-deoxyadenosine

219
00:11:40,870 --> 00:11:43,080
in the top face, which is
where all the chemistry is

220
00:11:43,080 --> 00:11:44,060
going to happen.

221
00:11:44,060 --> 00:11:47,390
So the business
end of the molecule

222
00:11:47,390 --> 00:11:49,550
is going to be this
part of the molecule.

223
00:11:49,550 --> 00:11:53,430
And the key to everything
is this carbon cobalt bond.

224
00:11:53,430 --> 00:11:56,240
Now, what's unusual about
a carbon cobalt bond?

225
00:11:56,240 --> 00:11:58,580
It's very weak.

226
00:11:58,580 --> 00:12:02,560
If you go to 40 degrees,
the bond breaks.

227
00:12:02,560 --> 00:12:04,560
Most things-- you can go
to hundreds of degrees,

228
00:12:04,560 --> 00:12:06,000
and the bond is stable.

229
00:12:06,000 --> 00:12:07,620
So it's thermally labile.

230
00:12:07,620 --> 00:12:11,720
And then the other thing that's
unique about this cofactor

231
00:12:11,720 --> 00:12:16,430
is that it's light sensitive.

232
00:12:16,430 --> 00:12:19,860
So if you put adenosylcobalamin
out on the bench top

233
00:12:19,860 --> 00:12:22,260
here, within two
minutes the whole thing

234
00:12:22,260 --> 00:12:25,870
would be destroyed because you
would break this carbon cobalt

235
00:12:25,870 --> 00:12:26,380
bond.

236
00:12:26,380 --> 00:12:26,900
OK?

237
00:12:26,900 --> 00:12:29,040
And that's the key
to the chemistry

238
00:12:29,040 --> 00:12:31,250
of how this cofactor works.

239
00:12:31,250 --> 00:12:31,960
OK?

240
00:12:31,960 --> 00:12:35,290
So I'm going to give
you a generic mechanism,

241
00:12:35,290 --> 00:12:38,660
and then I'm going to
focus on the mechanism

242
00:12:38,660 --> 00:12:40,700
of methylmalonyl-CoA
mutase, which

243
00:12:40,700 --> 00:12:43,220
you find in human metabolism.

244
00:12:43,220 --> 00:12:45,330
Before I get started,
let me just show you

245
00:12:45,330 --> 00:12:48,120
what the reaction is and
show you why it's unusual,

246
00:12:48,120 --> 00:12:50,110
and then we'll go back
to the actual mechanism.

247
00:12:50,110 --> 00:12:50,610
OK.

248
00:12:50,610 --> 00:12:54,710
So what does
methylmalonyl-CoA mutase do?

249
00:12:54,710 --> 00:13:00,050
Again it plays a central
role in odd chain fatty acid

250
00:13:00,050 --> 00:13:02,430
metabolism.

251
00:13:02,430 --> 00:13:03,900
OK, so this is CoA.

252
00:13:03,900 --> 00:13:05,800
You need to go back and
look at your lexicon

253
00:13:05,800 --> 00:13:09,080
if you can't remember
the structure CoA.

254
00:13:09,080 --> 00:13:10,750
But just remember
it's a thioester.

255
00:13:10,750 --> 00:13:12,231
That's all you need to know.

256
00:13:12,231 --> 00:13:12,730
OK.

257
00:13:12,730 --> 00:13:16,140
So this is the
substrate, and this

258
00:13:16,140 --> 00:13:17,770
is called methylmalonyl-CoA.

259
00:13:23,301 --> 00:13:23,800
OK.

260
00:13:23,800 --> 00:13:29,735
So adenosylcobalamin
catalyzes rearrangements.

261
00:13:32,240 --> 00:13:36,880
And this reaction has
fascinated chemists for 30 years

262
00:13:36,880 --> 00:13:38,860
because there was no
chemical precedent--

263
00:13:38,860 --> 00:13:41,390
just like there was no chemical
precedent for carbon cobalt

264
00:13:41,390 --> 00:13:41,890
bonds.

265
00:13:41,890 --> 00:13:43,460
When biochemists
discovered this,

266
00:13:43,460 --> 00:13:46,220
there was no precedent for the
reaction I'm going to show you.

267
00:13:46,220 --> 00:13:47,430
So what is the reaction?

268
00:13:47,430 --> 00:13:49,510
It's a rearrangement.

269
00:13:49,510 --> 00:13:51,800
And it's a rearrangement
because this hydrogen

270
00:13:51,800 --> 00:13:54,650
moves from this
carbon to this carbon.

271
00:13:54,650 --> 00:13:57,300
And this whole group--
this thioester--

272
00:13:57,300 --> 00:13:59,750
moves from this
carbon to this carbon.

273
00:13:59,750 --> 00:14:01,260
So that's the actual reaction.

274
00:14:01,260 --> 00:14:03,140
It's reversible.

275
00:14:03,140 --> 00:14:09,298
And so what you do is you
generate succinyl CoA.

276
00:14:14,000 --> 00:14:16,450
OK so this hydrogen
is moved over here.

277
00:14:19,050 --> 00:14:21,370
And this is succinyl CoA.

278
00:14:21,370 --> 00:14:24,050
And we haven't
talked about it yet,

279
00:14:24,050 --> 00:14:26,170
but succinyl CoA
plays a central role

280
00:14:26,170 --> 00:14:28,610
in metabolism in the TCA cycle.

281
00:14:28,610 --> 00:14:29,240
OK.

282
00:14:29,240 --> 00:14:31,660
So the question that
we want to focus on now

283
00:14:31,660 --> 00:14:34,000
is how do you catalyze
this weird rearrangement.

284
00:14:34,000 --> 00:14:35,450
What is this cofactor?

285
00:14:35,450 --> 00:14:39,790
This big, huge molecule with
this weak carbon cobalt bond.

286
00:14:39,790 --> 00:14:40,950
What does it do?

287
00:14:40,950 --> 00:14:41,690
OK.

288
00:14:41,690 --> 00:14:44,810
So I'm going to come
back to this in a minute,

289
00:14:44,810 --> 00:14:46,980
but just let me show
you what it does.

290
00:14:46,980 --> 00:14:47,480
OK.

291
00:14:47,480 --> 00:14:50,860
So this is a generic
mechanism, because while I

292
00:14:50,860 --> 00:14:55,220
said there's only one
enzyme in humans that

293
00:14:55,220 --> 00:14:58,310
uses adenosylcobalamin,
if you move into fungi,

294
00:14:58,310 --> 00:15:00,500
or you move into
archaea, or bacteria,

295
00:15:00,500 --> 00:15:04,990
you find there are many B-12
dependent reactions that

296
00:15:04,990 --> 00:15:07,660
also do rearrangements,
but different kinds

297
00:15:07,660 --> 00:15:08,720
of rearrangements.

298
00:15:08,720 --> 00:15:09,450
OK.

299
00:15:09,450 --> 00:15:11,030
So what's the generic mechanism?

300
00:15:11,030 --> 00:15:11,530
OK.

301
00:15:11,530 --> 00:15:14,246
So the generic mechanism
is the following.

302
00:15:14,246 --> 00:15:16,490
So here we have our
adenosylcobalamin.

303
00:15:16,490 --> 00:15:18,280
Here's our substrate.

304
00:15:18,280 --> 00:15:18,780
OK.

305
00:15:18,780 --> 00:15:26,301
And the idea is we need to move
the hydrogen from here to here.

306
00:15:26,301 --> 00:15:26,800
OK?

307
00:15:26,800 --> 00:15:28,980
Does it just jump through space?

308
00:15:28,980 --> 00:15:30,590
And the answer is no.

309
00:15:30,590 --> 00:15:33,020
The cofactor
adenosylcobalamin is

310
00:15:33,020 --> 00:15:35,860
going to remove the
hydrogen, and then it's

311
00:15:35,860 --> 00:15:37,770
going to transfer it.

312
00:15:37,770 --> 00:15:39,880
You're going to generate
a reactive speciea,

313
00:15:39,880 --> 00:15:43,450
and then it's going to transfer
it back to form a new product.

314
00:15:43,450 --> 00:15:46,570
So the cofactor-- and this
took people a long time

315
00:15:46,570 --> 00:15:49,200
to figure out because there
was no chemical precedent

316
00:15:49,200 --> 00:15:52,480
for this-- is mediating
the hydrogen transfer.

317
00:15:52,480 --> 00:15:56,370
So that's what I'm going to show
you-- how that actually works.

318
00:15:56,370 --> 00:16:00,150
So here what happens
is the whole key

319
00:16:00,150 --> 00:16:03,210
to the chemistry of
adenosylcobalamin--

320
00:16:03,210 --> 00:16:06,860
which again, most of you
haven't seen something like this

321
00:16:06,860 --> 00:16:13,360
before because you're not
exposed to radical chemistry--

322
00:16:13,360 --> 00:16:18,270
is that you have homolysis
of the carbon cobalt bond.

323
00:16:18,270 --> 00:16:19,250
So what does that mean?

324
00:16:19,250 --> 00:16:21,940
It means one electron
goes to the axial ligand,

325
00:16:21,940 --> 00:16:25,190
and one electron
goes to the cobalt.

326
00:16:25,190 --> 00:16:30,680
So the cobalt III is reduced
from cobalt III to cobalt II,

327
00:16:30,680 --> 00:16:34,040
and what you're left with
is this radical species

328
00:16:34,040 --> 00:16:42,531
of 5-prime-deoxyadenosyl
radical.

329
00:16:42,531 --> 00:16:43,030
OK.

330
00:16:43,030 --> 00:16:45,470
So this is the reactive species.

331
00:16:45,470 --> 00:16:49,230
Because what you're doing
in these transformations is

332
00:16:49,230 --> 00:16:53,660
pulling off an amazingly
non acidic hydrogen.

333
00:16:53,660 --> 00:16:57,670
And normal amino acid side
chains cannot do that kind

334
00:16:57,670 --> 00:16:58,220
of chemistry.

335
00:16:58,220 --> 00:17:01,350
You have to go to these
reactive radical species

336
00:17:01,350 --> 00:17:03,960
to be able to do
this tough chemistry.

337
00:17:03,960 --> 00:17:07,220
So here we generated
a radical species.

338
00:17:07,220 --> 00:17:09,720
It is sitting right
next to the substrate

339
00:17:09,720 --> 00:17:11,450
in the active site
of the enzyme.

340
00:17:11,450 --> 00:17:12,940
And so what does it do?

341
00:17:12,940 --> 00:17:17,170
It removes a hydrogen atom--
a hydrogen with one electron.

342
00:17:17,170 --> 00:17:17,670
OK?

343
00:17:17,670 --> 00:17:20,859
And so again, this is
free radical chemistry

344
00:17:20,859 --> 00:17:24,589
that most of you don't
think about that much.

345
00:17:24,589 --> 00:17:27,380
But what you do is the
hydrogen from the substrate

346
00:17:27,380 --> 00:17:31,260
is now transferred
to this axial ligand

347
00:17:31,260 --> 00:17:33,300
and that stays stuck
in the active site.

348
00:17:33,300 --> 00:17:36,440
So we've seen this
guy move over here.

349
00:17:36,440 --> 00:17:38,700
And now what you've
done is transferred

350
00:17:38,700 --> 00:17:41,440
one radical from the cofactor
into a second radical--

351
00:17:41,440 --> 00:17:42,910
the substrate.

352
00:17:42,910 --> 00:17:43,410
OK.

353
00:17:43,410 --> 00:17:44,450
So that's the key.

354
00:17:44,450 --> 00:17:47,890
Now it's carrying this
hydrogen, and eventually it

355
00:17:47,890 --> 00:17:51,031
wants to put the hydrogen
back on to form the product.

356
00:17:51,031 --> 00:17:51,530
OK?

357
00:17:51,530 --> 00:17:54,200
That's part of the
rearrangement reaction.

358
00:17:54,200 --> 00:17:58,970
So what happens now is--
and this looks like magic.

359
00:17:58,970 --> 00:18:01,790
I'm just showing that a
substrate radical goes

360
00:18:01,790 --> 00:18:03,690
to product radical,
and I will show you

361
00:18:03,690 --> 00:18:06,400
how this works in the case
of methylmalonyl-CoA mutase

362
00:18:06,400 --> 00:18:07,690
in a minute, OK?

363
00:18:07,690 --> 00:18:09,410
So there's some kind
of rearrangement.

364
00:18:09,410 --> 00:18:12,420
Remember we had two things
rearranging the hydrogen,

365
00:18:12,420 --> 00:18:16,730
but we also had a second--
a thioester rearranging.

366
00:18:16,730 --> 00:18:19,200
So we have a
rearrangement reaction,

367
00:18:19,200 --> 00:18:21,960
and this is reversible
in the reaction I'm going

368
00:18:21,960 --> 00:18:24,680
to be talking about today.

369
00:18:24,680 --> 00:18:27,680
And so this is the same is this.

370
00:18:27,680 --> 00:18:29,250
Structurally these are the same.

371
00:18:29,250 --> 00:18:31,640
I've just written
the methyl group.

372
00:18:31,640 --> 00:18:33,990
And so we have a
substrate radical

373
00:18:33,990 --> 00:18:36,320
converting into a
product radical.

374
00:18:36,320 --> 00:18:36,820
OK?

375
00:18:36,820 --> 00:18:39,940
And now what we want to do
is generate the product.

376
00:18:39,940 --> 00:18:43,520
And so the hydrogen
from this carrier--

377
00:18:43,520 --> 00:18:46,430
your axial ligand-- is now
going to be transferred

378
00:18:46,430 --> 00:18:52,060
by hydrogen atom transfer back
to p dot to form the product.

379
00:18:52,060 --> 00:18:55,800
So again, it's one
electron chemistry,

380
00:18:55,800 --> 00:18:58,540
and doing one
electron chemistry,

381
00:18:58,540 --> 00:19:02,710
the hydrogen is transferred back
to p-- and then what do you do?

382
00:19:02,710 --> 00:19:05,670
You lose one radical, and
you generate another radical.

383
00:19:05,670 --> 00:19:08,350
You regenerate the
radical we started

384
00:19:08,350 --> 00:19:12,970
with-- the 5-prime-deoxyadenosyl
radical on the axial ligand.

385
00:19:12,970 --> 00:19:17,100
Now I have written here
hydrogen in black and in red.

386
00:19:17,100 --> 00:19:18,690
Why is that true?

387
00:19:18,690 --> 00:19:20,870
Because here's a methyl group.

388
00:19:20,870 --> 00:19:23,070
And if you have free
complete freedom of rotation

389
00:19:23,070 --> 00:19:25,360
around that carbon
carbon bond, you

390
00:19:25,360 --> 00:19:28,240
can pull off either-- the methyl
hydrogens become equivalent,

391
00:19:28,240 --> 00:19:31,930
so it can pull off a hydrogen
red or a hydrogen black.

392
00:19:31,930 --> 00:19:32,430
OK?

393
00:19:32,430 --> 00:19:35,210
Can't distinguish in the
active site of the enzyme.

394
00:19:35,210 --> 00:19:38,680
So what you now generate is
the same thing we started with,

395
00:19:38,680 --> 00:19:40,090
except we have a product.

396
00:19:40,090 --> 00:19:42,170
But we have
5-prime-deoxyadenosyl radical

397
00:19:42,170 --> 00:19:43,070
cobalt II.

398
00:19:43,070 --> 00:19:45,560
Here we have
5-prime-deoxyadenosyl radical

399
00:19:45,560 --> 00:19:46,670
cobalt II.

400
00:19:46,670 --> 00:19:50,710
And what you do is you
re-form the carbon cobalt bond

401
00:19:50,710 --> 00:19:52,810
at the end of every
turnover, and now you're

402
00:19:52,810 --> 00:19:55,560
ready to start all over again.

403
00:19:55,560 --> 00:19:57,070
So that's the reaction.

404
00:19:57,070 --> 00:20:00,280
This 5-prime-deoxyadenosyl
radical.

405
00:20:00,280 --> 00:20:06,430
This axial ligand
sitting over here

406
00:20:06,430 --> 00:20:09,770
acts as a hydrogen
atom transferring agent

407
00:20:09,770 --> 00:20:11,920
to remove a hydrogen
from the substrate

408
00:20:11,920 --> 00:20:14,550
and to transfer it
back to the product.

409
00:20:14,550 --> 00:20:15,404
OK?

410
00:20:15,404 --> 00:20:17,070
I'm going to show you
how this works now

411
00:20:17,070 --> 00:20:21,390
in the case of
methylmalonyl-CoA mutase.

412
00:20:21,390 --> 00:20:25,230
So here's our
methylmalonyl-CoA, and we're

413
00:20:25,230 --> 00:20:28,180
going to be converted
into succinyl CoA.

414
00:20:28,180 --> 00:20:32,780
So this guy is migrating, and
this guy is migrating there.

415
00:20:32,780 --> 00:20:34,030
That's the goal.

416
00:20:34,030 --> 00:20:38,290
And so what happens here is
you cleave the carbon cobalt

417
00:20:38,290 --> 00:20:45,070
bond to form cobalt II and
5-prime-deoxyadenosyl radical.

418
00:20:45,070 --> 00:20:49,300
And now this
5-prime-deoxyadenosyl radical

419
00:20:49,300 --> 00:20:54,310
can remove a hydrogen
atom from the substrate.

420
00:20:54,310 --> 00:20:56,280
So it leaves you
with another radical.

421
00:20:56,280 --> 00:20:57,520
This radical goes away.

422
00:20:57,520 --> 00:20:59,490
You generate another radical.

423
00:20:59,490 --> 00:21:02,480
And now this hydrogen
from the substrate

424
00:21:02,480 --> 00:21:05,610
is transferred to our cofactor.

425
00:21:05,610 --> 00:21:08,430
So it's the hydrogen
transferring agent.

426
00:21:08,430 --> 00:21:11,000
So now the question is, how
does this weird rearrangement

427
00:21:11,000 --> 00:21:11,560
reaction end?

428
00:21:11,560 --> 00:21:15,200
How does this CoA migrate
from one place to another?

429
00:21:15,200 --> 00:21:17,050
Again, there was no
chemical precedent

430
00:21:17,050 --> 00:21:18,640
in the literature for this.

431
00:21:18,640 --> 00:21:20,880
And the answer is we still
don't know the answer.

432
00:21:20,880 --> 00:21:23,960
So there are two
mechanistic possibilities.

433
00:21:23,960 --> 00:21:28,130
One is that you go through a
three membered ring-- cycle

434
00:21:28,130 --> 00:21:29,620
propane ring intermediate.

435
00:21:29,620 --> 00:21:31,570
So what you can
picture happening

436
00:21:31,570 --> 00:21:36,400
is that one of the
electrons from the carbonyl

437
00:21:36,400 --> 00:21:40,960
forms a bond with the unpaired
electron on this carbon.

438
00:21:40,960 --> 00:21:43,760
And you generate this
cycle propane intermediate.

439
00:21:43,760 --> 00:21:46,140
So you've gone from this
radical to another radical.

440
00:21:46,140 --> 00:21:47,930
We haven't lost any radicals.

441
00:21:47,930 --> 00:21:52,250
But now what we want to do is
we want this group to migrate.

442
00:21:52,250 --> 00:21:55,410
So now what happens-- this
intermediate can collapse.

443
00:21:55,410 --> 00:21:57,220
It can collapse back
to form starting

444
00:21:57,220 --> 00:22:00,380
material and the
5-prime-deoxyadenosyl radical.

445
00:22:00,380 --> 00:22:03,230
Or you can break this
bond, in which case

446
00:22:03,230 --> 00:22:07,600
you collapse to form the direct
precursor to the product.

447
00:22:07,600 --> 00:22:09,790
So it can break down
in either direction,

448
00:22:09,790 --> 00:22:11,690
and the reaction's reversible.

449
00:22:11,690 --> 00:22:13,870
And if it breaks down
in this direction,

450
00:22:13,870 --> 00:22:15,140
you form a new radical.

451
00:22:15,140 --> 00:22:17,950
This radical is distinct
from this radical.

452
00:22:17,950 --> 00:22:22,970
And now what happens is that
the hydrogen that you removed

453
00:22:22,970 --> 00:22:26,510
from your starting material
can be returned back

454
00:22:26,510 --> 00:22:27,780
to the product.

455
00:22:27,780 --> 00:22:33,660
And what you generate then is
5-prime-deoxyadenosyl radical

456
00:22:33,660 --> 00:22:35,870
and cobalt II.

457
00:22:35,870 --> 00:22:38,820
And now when you transfer
the hydrogen back,

458
00:22:38,820 --> 00:22:40,910
you form your product
like I showed you

459
00:22:40,910 --> 00:22:41,940
in the previous slide.

460
00:22:41,940 --> 00:22:44,830
And now you re-form
the carbon cobalt bond

461
00:22:44,830 --> 00:22:48,520
to re-form the active
form of the cofactor.

462
00:22:48,520 --> 00:22:52,800
So alternatively you can
make this arrangement

463
00:22:52,800 --> 00:22:56,930
happen by another mechanism
which is called a fragmentation

464
00:22:56,930 --> 00:22:57,519
mechanism.

465
00:22:57,519 --> 00:22:59,310
And I won't go through
the details of this,

466
00:22:59,310 --> 00:23:01,518
but you can sit and look at
this for those of you who

467
00:23:01,518 --> 00:23:05,930
are really interested in
sophisticated proposals

468
00:23:05,930 --> 00:23:08,910
for the chemical mechanisms
of the rearrangement.

469
00:23:08,910 --> 00:23:15,710
So adenosylcobalamin chemistry
was unprecedented every step

470
00:23:15,710 --> 00:23:16,650
along the way.

471
00:23:16,650 --> 00:23:21,080
And it's now known to be
widely used, but not so much so

472
00:23:21,080 --> 00:23:21,633
in humans.

473
00:23:21,633 --> 00:23:25,590
But really humans are only
a small part of the world.

474
00:23:25,590 --> 00:23:27,460
We have many, many more
bacteria and archaea

475
00:23:27,460 --> 00:23:28,380
than we have humans.

476
00:23:28,380 --> 00:23:32,160
So this is a pretty
important transformation.

477
00:23:32,160 --> 00:23:35,620
So hopefully now when
you see this again

478
00:23:35,620 --> 00:23:38,140
it won't be completely magic.

479
00:23:38,140 --> 00:23:41,130
But this is a challenging
reaction that most of you

480
00:23:41,130 --> 00:23:42,550
haven't been exposed to before.

481
00:23:42,550 --> 00:23:44,580
But hopefully some
of you get excited

482
00:23:44,580 --> 00:23:47,860
about radical mediated
transformations.

483
00:23:47,860 --> 00:23:51,660
Just let me close by saying--
from bioinformatic studies

484
00:23:51,660 --> 00:23:53,790
in the last five
years or so, we now

485
00:23:53,790 --> 00:23:57,050
know there are over
50,000 reactions that

486
00:23:57,050 --> 00:23:59,720
use free radical chemistry,
yet we don't talk

487
00:23:59,720 --> 00:24:02,600
about radical chemistry in 507.

488
00:24:02,600 --> 00:24:04,960
So hopefully some of you
will get interested enough

489
00:24:04,960 --> 00:24:08,390
in biochemistry to come
in and start figuring out

490
00:24:08,390 --> 00:24:12,010
how all these radical
dependent reaction occur,

491
00:24:12,010 --> 00:24:15,680
not in primary metabolism, but
in many secondary metabolic

492
00:24:15,680 --> 00:24:16,631
pathways.

493
00:24:16,631 --> 00:24:17,130
OK.

494
00:24:17,130 --> 00:24:18,900
Thank you.