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JOHN ESSIGMANN: We're
still on storyboard 7.

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00:00:22,960 --> 00:00:25,570
We're on panel C.
Panel C is where

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00:00:25,570 --> 00:00:28,030
I introduced the TCA cycle.

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00:00:28,030 --> 00:00:29,830
As I mentioned
earlier, respiration

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00:00:29,830 --> 00:00:32,090
consists of three stages.

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00:00:32,090 --> 00:00:34,810
The first is the pyruvate
dehydrogenase reaction,

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00:00:34,810 --> 00:00:38,950
which we just covered, taking
pyruvate to acetyl-CoA.

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00:00:38,950 --> 00:00:43,210
The second is the TCA cycle,
or tricarboxylic acid cycle

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00:00:43,210 --> 00:00:45,970
taking that acetyl-CoA
and basically

17
00:00:45,970 --> 00:00:49,090
oxidizing it in
order to generate CO2

18
00:00:49,090 --> 00:00:51,550
but also to produce more
reducing equivalents

19
00:00:51,550 --> 00:00:54,920
in the form of mobile
electron carriers.

20
00:00:54,920 --> 00:00:56,680
And then the third
step of respiration

21
00:00:56,680 --> 00:00:58,720
is taking those
reducing equivalents

22
00:00:58,720 --> 00:01:01,100
to the mitochondrial
inner membrane, where

23
00:01:01,100 --> 00:01:03,970
the molecules containing
those reducing equivalents

24
00:01:03,970 --> 00:01:05,470
are oxidized.

25
00:01:05,470 --> 00:01:08,890
And then, the electrons
from that oxidation reaction

26
00:01:08,890 --> 00:01:12,730
are used to power proton pumps
that ultimately will generate

27
00:01:12,730 --> 00:01:16,450
the proton gradient that can
be used for generation of ATP

28
00:01:16,450 --> 00:01:19,990
or for generation of
movement or for other things.

29
00:01:19,990 --> 00:01:22,630
The tricarboxylic acid
cycle, which is sometimes

30
00:01:22,630 --> 00:01:25,630
called the Krebs
cycle, takes acetyl-CoA

31
00:01:25,630 --> 00:01:27,750
from several different sources.

32
00:01:27,750 --> 00:01:30,640
One of those sources is
glycolysis to pyruvate

33
00:01:30,640 --> 00:01:34,030
and pyruvate to acetyl-CoA,
as we've just seen.

34
00:01:34,030 --> 00:01:35,950
And the second
source of acetyl-CoA

35
00:01:35,950 --> 00:01:37,960
is from fatty acid oxidation.

36
00:01:37,960 --> 00:01:40,720
We'll come to that
pathway somewhat later.

37
00:01:40,720 --> 00:01:43,660
Again, looking at
panel C, the TCA cycle

38
00:01:43,660 --> 00:01:46,190
starts with the reaction
of acetyl coenzyme

39
00:01:46,190 --> 00:01:49,300
A, a 2-carbon compound
with oxaloacetate,

40
00:01:49,300 --> 00:01:53,950
a 4-carbon compound, to form
the 6-carbon product citrate.

41
00:01:53,950 --> 00:01:57,450
Citrate will lose 2
carbons as carbon dioxide,

42
00:01:57,450 --> 00:02:00,160
and in the process, there'll
be a series of oxidation steps

43
00:02:00,160 --> 00:02:04,120
that generate three
NADHes one FADH2,

44
00:02:04,120 --> 00:02:08,110
and one either GTP or
ATP by substrate-level

45
00:02:08,110 --> 00:02:09,430
phosphorylation.

46
00:02:09,430 --> 00:02:11,620
In terms of the banking
system of the cell,

47
00:02:11,620 --> 00:02:14,290
the NADHes that are generated
in the mitochondrion

48
00:02:14,290 --> 00:02:17,430
are exchangeable for
about three ATPs.

49
00:02:17,430 --> 00:02:21,070
FADH2 is exchangeable
for about two ATPs.

50
00:02:21,070 --> 00:02:22,690
So if you look up
the total number

51
00:02:22,690 --> 00:02:25,990
of nucleotide triphosphate,
or NTP, equivalents

52
00:02:25,990 --> 00:02:28,390
that can be produced
in the TCA cycle,

53
00:02:28,390 --> 00:02:32,260
you'll get about 12 ATPs
for each 2-carbon unit

54
00:02:32,260 --> 00:02:35,020
of acetyl-CoA that's oxidized.

55
00:02:35,020 --> 00:02:36,760
As a detailed point,
I want to mention

56
00:02:36,760 --> 00:02:38,860
that the two carbons
that entered the TCA

57
00:02:38,860 --> 00:02:42,100
cycle as acetyl-CoA are
not exactly the same two

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00:02:42,100 --> 00:02:45,190
carbons that come out
as CO2 in that cycle.

59
00:02:45,190 --> 00:02:48,160
The carbon dioxides from
the input acetyl-CoA

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00:02:48,160 --> 00:02:51,940
will emerge in later
turns of the TCA cycle.

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00:02:51,940 --> 00:02:54,610
Now we're going to look
at panel D. In panel D,

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we start looking at the
details of the TCA cycle.

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00:02:57,760 --> 00:03:00,550
JoAnne explained why
nature uses thioesters.

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The sulfur allows
enolization stabilization

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00:03:03,520 --> 00:03:06,430
of a carbanion at carbon
2, the carbon that's

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00:03:06,430 --> 00:03:09,630
distal to the coenzyme
A functionality.

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00:03:09,630 --> 00:03:12,400
The carbanion is then able
to attack the number 2

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00:03:12,400 --> 00:03:14,950
carbon, carbonyl,
of oxaloacetate

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00:03:14,950 --> 00:03:18,550
in the reaction catalyzed
by citrate synthase.

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00:03:18,550 --> 00:03:21,940
An intermediate is formed,
citroyl coenzyme A,

71
00:03:21,940 --> 00:03:25,060
which loses its coenzyme
A moiety by hydrolysis

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00:03:25,060 --> 00:03:28,300
in a very thermodynamically
irreversible step,

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00:03:28,300 --> 00:03:30,850
resulting in the
product citrate.

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00:03:30,850 --> 00:03:33,040
This step is at the
top of the pathway,

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00:03:33,040 --> 00:03:36,940
and as is usually the case,
this highly exothermic step

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00:03:36,940 --> 00:03:41,200
makes the pathway,
overall, irreversible.

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00:03:41,200 --> 00:03:43,630
Chemically, citrate
synthase does

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00:03:43,630 --> 00:03:46,810
a mixed aldol-Claisen
ester condensation.

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00:03:46,810 --> 00:03:49,240
The product of the citrate
synthase reaction, citrate,

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00:03:49,240 --> 00:03:52,240
is a tertiary alcohol,
and tertiary alcohols

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00:03:52,240 --> 00:03:54,670
are relatively
difficult to oxidize.

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00:03:54,670 --> 00:03:57,160
The next enzyme in the
pathway, aconitase,

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00:03:57,160 --> 00:04:00,280
which is shown in
storyboard 8, panel A,

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00:04:00,280 --> 00:04:03,580
removes a water molecule and
then adds a different water

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00:04:03,580 --> 00:04:06,850
molecule back to rearrange
the hydroxyl group,

86
00:04:06,850 --> 00:04:08,800
making a secondary
alcohol, which

87
00:04:08,800 --> 00:04:11,200
is much easier to oxidize.

88
00:04:11,200 --> 00:04:15,010
Looking again at panel A,
the hydroxyl group of high

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00:04:15,010 --> 00:04:17,380
isocitrate is
oxidized to a ketone,

90
00:04:17,380 --> 00:04:21,990
with the transfer of hydride
to NAD+ to make NADH.

91
00:04:21,990 --> 00:04:25,190
This reaction is catalyzed
by the enzyme isocitrate

92
00:04:25,190 --> 00:04:27,910
dehydrogenase, ICDH.

93
00:04:27,910 --> 00:04:29,470
The intermediate
in this reaction

94
00:04:29,470 --> 00:04:33,310
is oxalosuccinate, which
is a beta-keto acid.

95
00:04:33,310 --> 00:04:35,340
As we know from what
JoAnne taught us,

96
00:04:35,340 --> 00:04:40,190
beta-keto acids are prone to
spontaneous decarboxylation.

97
00:04:40,190 --> 00:04:43,000
So the second half of the
isocitrate dehydrogenase

98
00:04:43,000 --> 00:04:46,090
reaction involves the
loss of carbon dioxide

99
00:04:46,090 --> 00:04:49,810
in what's usually considered
to be an irreversible step.

100
00:04:49,810 --> 00:04:51,760
I do want to point
out, however, that

101
00:04:51,760 --> 00:04:54,790
under certain circumstances,
you can re-add the carbon

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00:04:54,790 --> 00:04:57,160
dioxide in order to
make the reaction go

103
00:04:57,160 --> 00:04:59,200
in the other direction.

104
00:04:59,200 --> 00:05:03,850
After the ICDH reactions, which
generated NADH and resulted

105
00:05:03,850 --> 00:05:06,970
in loss of CO2, the product
is alpha-ketoglutarate,

106
00:05:06,970 --> 00:05:09,640
a 5-carbon keto acid.

107
00:05:09,640 --> 00:05:12,190
Take a look at the structure
of alpha-ketoglutarate

108
00:05:12,190 --> 00:05:16,150
in the upper right-hand
portion of storyboard 8,

109
00:05:16,150 --> 00:05:19,990
panel A. If you hold your
finger over the top 2

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00:05:19,990 --> 00:05:22,190
carbons of
alpha-ketoglutarate, you'll

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00:05:22,190 --> 00:05:24,980
notice that the
residue is pyruvate.

112
00:05:24,980 --> 00:05:28,280
So pyruvate plus an
acetyl functionality

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00:05:28,280 --> 00:05:31,580
equals, effectively,
alpha-ketoglutarate.

114
00:05:31,580 --> 00:05:33,830
Now take a look back
at the mechanism

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00:05:33,830 --> 00:05:39,200
by which pyruvate is oxidized
by pyruvate dehydrogenase.

116
00:05:39,200 --> 00:05:42,290
It's going to be a very similar
mechanism for the oxidation

117
00:05:42,290 --> 00:05:44,090
of alpha-ketoglutarate.

118
00:05:44,090 --> 00:05:46,520
The product of the
alpha-ketoglutarate

119
00:05:46,520 --> 00:05:49,280
dehydrogenase reaction
is succinyl-CoA.

120
00:05:49,280 --> 00:05:51,740
Again, if you look at the
structure of this molecule,

121
00:05:51,740 --> 00:05:56,420
succinyl-CoA, it's actually an
acetyl-CoA with an acetyl group

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00:05:56,420 --> 00:05:57,970
put onto one end.

123
00:05:57,970 --> 00:06:01,370
Now let's go back and look
at the alpha-ketoglutarate

124
00:06:01,370 --> 00:06:03,290
from a different perspective.

125
00:06:03,290 --> 00:06:07,010
I want to make a point here
in that alpha-ketoglutarate

126
00:06:07,010 --> 00:06:09,280
is an alpha-keto
acid, and if we were

127
00:06:09,280 --> 00:06:11,840
to replace its keto group
with an amino group,

128
00:06:11,840 --> 00:06:13,460
you'd convert this
keto acid into

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00:06:13,460 --> 00:06:16,130
the amino acid glutamic acid.

130
00:06:16,130 --> 00:06:19,670
As with most enzymatic reactions
involving such nitrogen

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00:06:19,670 --> 00:06:24,440
functionalities, a pyridoxal
pyridoxamine phosphate cofactor

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00:06:24,440 --> 00:06:27,950
will be needed to interconvert
the alpha-ketoglutarate and

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00:06:27,950 --> 00:06:29,810
glutamic acid.

134
00:06:29,810 --> 00:06:33,590
So glutamic acid can serve as
a source of alpha-ketoglutarate

135
00:06:33,590 --> 00:06:36,890
if the cell is starved for
TCA cycle intermediates.

136
00:06:36,890 --> 00:06:38,960
Alternatively,
alpha-ketoglutarate

137
00:06:38,960 --> 00:06:41,570
can be a source
for glutamic acid

138
00:06:41,570 --> 00:06:45,560
when a cell may need amino
acids for protein biosynthesis.

139
00:06:45,560 --> 00:06:48,280
Now, let's turn
to panel B, where

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00:06:48,280 --> 00:06:52,370
we'll start with succinyl
coenzyme A. Looking

141
00:06:52,370 --> 00:06:54,980
at the molecule of
succinyl-CoA, note

142
00:06:54,980 --> 00:06:58,280
that I've labeled each of
the atoms with a symbol.

143
00:06:58,280 --> 00:07:00,260
The triangle and
square were from

144
00:07:00,260 --> 00:07:02,690
the original acetyl-CoA
molecule that came

145
00:07:02,690 --> 00:07:05,100
in at the top of the pathway.

146
00:07:05,100 --> 00:07:07,900
The enzyme that processes
succinyl-CoA is succinyl

147
00:07:07,900 --> 00:07:09,950
coenzyme A synthetase.

148
00:07:09,950 --> 00:07:13,070
As JoAnne taught us,
synthetases are enzymes that

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00:07:13,070 --> 00:07:15,470
typically need a nucleotide.

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00:07:15,470 --> 00:07:17,420
In this case, the
nucleotide involved

151
00:07:17,420 --> 00:07:22,550
is GDP in mammalian systems
or ADP in bacterial systems.

152
00:07:22,550 --> 00:07:26,160
In the traditional clockwise
direction of the TCA cycle,

153
00:07:26,160 --> 00:07:32,510
they are phosphorylated to
form GTP or ATP, respectively.

154
00:07:32,510 --> 00:07:36,230
The molecule that you get after
the phosphorylation reaction

155
00:07:36,230 --> 00:07:38,180
in the hydrolysis
of the coenzyme A

156
00:07:38,180 --> 00:07:41,420
is succinate, a
4-carbon compound, which

157
00:07:41,420 --> 00:07:43,790
is also a dicarboxylic acid.

158
00:07:43,790 --> 00:07:46,400
This molecule is
perfectly symmetrical

159
00:07:46,400 --> 00:07:49,370
and can tumble in
three-dimensional space.

160
00:07:49,370 --> 00:07:52,820
The next enzyme in the pathway,
succinate dehydrogenase,

161
00:07:52,820 --> 00:07:56,390
cannot distinguish one arm
of its substrate, succinate,

162
00:07:56,390 --> 00:07:57,500
from the other.

163
00:07:57,500 --> 00:08:00,650
So if there were a radio
label in the acetyl-CoA

164
00:08:00,650 --> 00:08:03,200
at the beginning of the
pathway, that radio label

165
00:08:03,200 --> 00:08:05,390
would become scrambled
at this point,

166
00:08:05,390 --> 00:08:09,740
uniformly distributed among the
two carbons of the two arms.

167
00:08:09,740 --> 00:08:12,810
From this point onward
in the TCA cycle,

168
00:08:12,810 --> 00:08:15,890
you will note that the label
denoted as the triangle

169
00:08:15,890 --> 00:08:19,670
and box is scrambled, as
indicated by triangle divided

170
00:08:19,670 --> 00:08:22,700
by 2 or box divided by 2.

171
00:08:22,700 --> 00:08:26,450
The next enzyme in the pathway
is succinate dehydrogenase.

172
00:08:26,450 --> 00:08:30,680
This is the only membrane-bound
enzyme in the TCA cycle.

173
00:08:30,680 --> 00:08:33,169
It is a dehydrogenase,
and it uses flavin

174
00:08:33,169 --> 00:08:35,240
as a cofactor to
help remove electrons

175
00:08:35,240 --> 00:08:37,390
from the succinate substrate.

176
00:08:37,390 --> 00:08:40,100
Flavin picks up
electrons from succinate,

177
00:08:40,100 --> 00:08:44,250
converting FAD to FADH2 in
the mitochondrial membrane.

178
00:08:44,250 --> 00:08:47,720
The product of the reaction
is the alkene fumarate.

179
00:08:47,720 --> 00:08:52,100
The enzyme fumarase adds water
to fumarate to form the alcohol

180
00:08:52,100 --> 00:08:53,670
product malate.

181
00:08:53,670 --> 00:08:56,300
The hydroxyl group
of the alcohol malate

182
00:08:56,300 --> 00:08:59,850
is primed for oxidation by the
next enzyme in the pathway,

183
00:08:59,850 --> 00:09:02,200
malate dehydrogenase, or MDH.

184
00:09:02,200 --> 00:09:07,510
MDH oxidizes malate, which
is an alcohol, to a ketone.

185
00:09:07,510 --> 00:09:10,490
The ketone product
is oxaloacetate.

186
00:09:10,490 --> 00:09:14,660
The hydride removed from malate
is transferred to NAD+ to form

187
00:09:14,660 --> 00:09:16,010
NADH.

188
00:09:16,010 --> 00:09:18,320
As I mentioned, the product
of the overall reaction

189
00:09:18,320 --> 00:09:21,740
is oxaloacetate, and
its ketone functionality

190
00:09:21,740 --> 00:09:26,360
is now primed for attack by
the next molecule of acetyl-CoA

191
00:09:26,360 --> 00:09:28,730
entering the TCA cycle.

192
00:09:28,730 --> 00:09:31,850
As a final point, I've
mentioned several times

193
00:09:31,850 --> 00:09:36,050
that oxaloacetate is present at
a very low concentration, only

194
00:09:36,050 --> 00:09:38,960
in the micromolar range,
inside the mitochondrion

195
00:09:38,960 --> 00:09:40,880
of a mammalian cell.

196
00:09:40,880 --> 00:09:44,660
So it's always at rather
limiting concentration.

197
00:09:44,660 --> 00:09:47,210
The cell has to work very
hard to preserve enough

198
00:09:47,210 --> 00:09:51,410
of the oxaloacetate to enable
the next cycle of the TCA

199
00:09:51,410 --> 00:09:54,960
cycle, that is the acquisition
of the next acetyl coenzyme

200
00:09:54,960 --> 00:09:56,150
A group.

201
00:09:56,150 --> 00:09:59,810
One of the ways that the cell
can generate oxaloacetate

202
00:09:59,810 --> 00:10:05,060
is by deamination, or
transamination of aspartic acid

203
00:10:05,060 --> 00:10:08,870
to the keto acid oxaloacetate.

204
00:10:08,870 --> 00:10:11,020
We typically have
plenty of aspartic acid

205
00:10:11,020 --> 00:10:13,220
and this PLP-mediated
reaction helps

206
00:10:13,220 --> 00:10:17,510
to maintain a critical
level of oxaloacetate.

207
00:10:17,510 --> 00:10:20,390
I want to return to storyboard
7 to make some comments

208
00:10:20,390 --> 00:10:22,700
about the importance
of prochirality

209
00:10:22,700 --> 00:10:25,190
in some enzymatic reactions.

210
00:10:25,190 --> 00:10:27,170
This short interlude
will help explain

211
00:10:27,170 --> 00:10:29,730
how to track a radio
label in a TCA cycle

212
00:10:29,730 --> 00:10:32,210
intermediate as that
intermediate progresses

213
00:10:32,210 --> 00:10:33,830
through the TCA cycle.

214
00:10:33,830 --> 00:10:35,370
As you will note,
at first glance,

215
00:10:35,370 --> 00:10:37,980
the label does some
unexpected things.

216
00:10:37,980 --> 00:10:39,440
But at the end of
the day, the fact

217
00:10:39,440 --> 00:10:41,120
that the label does
surprising things

218
00:10:41,120 --> 00:10:44,270
helped early biochemists
figure out mechanistically how

219
00:10:44,270 --> 00:10:47,540
several enzymes work in
concert during the linear steps

220
00:10:47,540 --> 00:10:49,590
of a pathway.

221
00:10:49,590 --> 00:10:52,830
We're going to look at
storyboards 7, 8, and 9,

222
00:10:52,830 --> 00:10:54,300
starting with the
chemical reaction

223
00:10:54,300 --> 00:10:56,720
at the bottom right
of storyboard 7,

224
00:10:56,720 --> 00:10:59,670
panel D. This is the
chemical reaction that's

225
00:10:59,670 --> 00:11:02,202
catalyzed by citrate synthase.

226
00:11:02,202 --> 00:11:03,660
You'll notice that
I've highlighted

227
00:11:03,660 --> 00:11:08,220
the two carbons with
either a triangle or a box.

228
00:11:08,220 --> 00:11:11,310
As we have seen, the
nucleophile on acetyl-CoA

229
00:11:11,310 --> 00:11:13,680
attacks the
electropositive carbon

230
00:11:13,680 --> 00:11:18,260
of the carbonyl functionality
of oxaloacetate.

231
00:11:18,260 --> 00:11:22,710
The carbonyl functionality is
a flat sp2 hybridized center.

232
00:11:22,710 --> 00:11:25,830
So if this were a typical
organic chemical reaction,

233
00:11:25,830 --> 00:11:29,070
the electrophile could
come in from either the top

234
00:11:29,070 --> 00:11:32,250
or the bottom, and you'd get
two different stereochemistries

235
00:11:32,250 --> 00:11:33,360
in the product.

236
00:11:33,360 --> 00:11:36,000
That is the citrate
at the very bottom

237
00:11:36,000 --> 00:11:39,330
would have equally labeled
acetyl arms at the top

238
00:11:39,330 --> 00:11:41,930
and bottom of the
molecule as I've drawn it.

239
00:11:41,930 --> 00:11:45,480
You would have a delta divided
by 2 for the blue methylene

240
00:11:45,480 --> 00:11:47,460
group, and a box
or square divided

241
00:11:47,460 --> 00:11:50,760
by 2 for the red
carboxylate group in each

242
00:11:50,760 --> 00:11:53,010
of the two acetyl arms.

243
00:11:53,010 --> 00:11:56,610
Again, these arms are at the
bottom and top of the molecule

244
00:11:56,610 --> 00:11:58,800
as drawn in the
lower right of panel

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00:11:58,800 --> 00:12:03,360
D. You'll note, however, that
only the top acetyl group

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00:12:03,360 --> 00:12:05,520
of citrate has the labels.

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00:12:05,520 --> 00:12:09,390
Initially, the observation that
only the top arm acquired label

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00:12:09,390 --> 00:12:12,350
was a puzzle to
early biochemists.

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00:12:12,350 --> 00:12:15,090
One way to think about the
citrate synthase reaction

250
00:12:15,090 --> 00:12:17,970
is to think about the
oxaloacetate laying

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00:12:17,970 --> 00:12:21,180
on the surface of the
citrate synthase enzyme.

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00:12:21,180 --> 00:12:25,080
Now imagine that the enzyme
precludes, or blocks, access

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00:12:25,080 --> 00:12:29,010
to the carbonyl from the
bottom and allows access only

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00:12:29,010 --> 00:12:31,290
to the top, giving
rise to only one

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00:12:31,290 --> 00:12:34,980
stereochemical outcome, the one
that I've shown in the citrate

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00:12:34,980 --> 00:12:37,030
to the right.

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00:12:37,030 --> 00:12:39,790
Now move ahead to
the storyboard number

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00:12:39,790 --> 00:12:43,810
9, panel B. This panel
shows a more cartoon-like

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00:12:43,810 --> 00:12:46,390
representation of the
molecule of citrate.

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00:12:46,390 --> 00:12:51,070
You can see the acetyl arm
on the top, the pro-S arm,

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00:12:51,070 --> 00:12:52,600
as having the labels.

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00:12:52,600 --> 00:12:56,290
And the pro-R arm, the one
that came from oxaloacetate,

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00:12:56,290 --> 00:12:58,840
at the bottom, is label free.

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00:12:58,840 --> 00:13:04,280
So while the pro-R and pro-S
arms are chemically identical,

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00:13:04,280 --> 00:13:06,430
they're going to be
handled by the next enzyme

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00:13:06,430 --> 00:13:09,250
in the series, aconitase,
as being chemically

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00:13:09,250 --> 00:13:11,260
different from one another.

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00:13:11,260 --> 00:13:13,540
All biochemistry is
going to be occurring

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00:13:13,540 --> 00:13:16,300
on the pro-R arm,
that is the arm that

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00:13:16,300 --> 00:13:19,690
came in from oxaloacetate
and not the arm that

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00:13:19,690 --> 00:13:23,480
came in for acid 2
with acetyl coenzyme A.

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00:13:23,480 --> 00:13:25,650
In the bottom right
of panel B, I've

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00:13:25,650 --> 00:13:30,400
sketched out an imaginary active
site for the aconitase enzyme.

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00:13:30,400 --> 00:13:32,670
I show a base
picking up a proton

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00:13:32,670 --> 00:13:35,880
from the pro-R arm of
the citrate molecule.

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00:13:35,880 --> 00:13:38,460
And you can see the elimination
of the water molecule

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00:13:38,460 --> 00:13:40,600
from the 3 carbon.

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00:13:40,600 --> 00:13:43,930
So despite the fact that the
citrate molecule is chemically

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00:13:43,930 --> 00:13:48,250
symmetrical, aconitase, the next
enzyme in the reaction series,

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00:13:48,250 --> 00:13:54,060
is able to distinguish between
the pro-R and the pro-S arms.

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00:13:54,060 --> 00:13:57,240
At this point, I want you to
look back at storyboard 8,

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00:13:57,240 --> 00:14:00,030
panel A. Look once again
at the citrate that

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00:14:00,030 --> 00:14:03,910
is at the upper left-hand
corner of storyboard number 8,

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00:14:03,910 --> 00:14:07,290
and let's imagine that
the aconitase chemistry

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00:14:07,290 --> 00:14:11,500
has happened on the pro-S arm,
that is the one in the box.

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00:14:11,500 --> 00:14:13,680
Keep in mind that these
experiments have shown

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00:14:13,680 --> 00:14:15,540
that this does not happen.

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00:14:15,540 --> 00:14:18,940
This is just a
hypothetical scenario.

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00:14:18,940 --> 00:14:21,820
In this hypothetical
case, the hydroxyl group

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00:14:21,820 --> 00:14:24,550
would end up on the
number 2 carbon,

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00:14:24,550 --> 00:14:26,470
the one with the blue triangle.

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00:14:26,470 --> 00:14:28,450
You have to draw
it out, but if you

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00:14:28,450 --> 00:14:31,720
traced this molecule, in
which the chemistry happened

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00:14:31,720 --> 00:14:36,280
on the pro-S arm, all the way
around to alpha-ketoglutarate,

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00:14:36,280 --> 00:14:38,780
you would find out that
the alpha-ketoglutarate

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00:14:38,780 --> 00:14:42,970
dehydrogenase would liberate
CO2 from the carboxylate that

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00:14:42,970 --> 00:14:44,890
has the red box on it.

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00:14:44,890 --> 00:14:48,070
This is in contrast to the
molecule succinate that appears

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00:14:48,070 --> 00:14:51,640
on storyboard 8, panel
B. Succinate is another

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00:14:51,640 --> 00:14:54,460
symmetrical molecule,
but for some reason,

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00:14:54,460 --> 00:14:58,380
succinate dehydrogenase cannot
distinguish between the two

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00:14:58,380 --> 00:15:00,160
acetyl arms.

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00:15:00,160 --> 00:15:03,200
Because the enzyme is unable
to distinguish the two arms,

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00:15:03,200 --> 00:15:05,710
in this case, unlike
that of aconitase,

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00:15:05,710 --> 00:15:08,680
the radio label would
become scrambled.

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00:15:08,680 --> 00:15:10,780
The reason that I'm
belaboring this point

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00:15:10,780 --> 00:15:13,660
is because one of the ways
that biochemists work out

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00:15:13,660 --> 00:15:16,560
the chemical reactions involved
in a biochemical pathway

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00:15:16,560 --> 00:15:19,360
is by putting in some
kind of labeled molecule.

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00:15:19,360 --> 00:15:22,750
It could be a radio
label or a heavy isotope.

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00:15:22,750 --> 00:15:26,590
And then they trace the position
of the label in the molecule

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00:15:26,590 --> 00:15:30,830
as you move from molecule to
molecule along the pathway.

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00:15:30,830 --> 00:15:33,340
So label tracer studies are
ones that are absolutely

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00:15:33,340 --> 00:15:36,340
central to all of biochemistry.

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00:15:36,340 --> 00:15:38,200
And as I mentioned
earlier, you'll

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00:15:38,200 --> 00:15:40,930
get lots of experience
in the problem sets

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00:15:40,930 --> 00:15:44,290
in using labels to work out
the details of a pathway.

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00:15:44,290 --> 00:15:46,240
Before leaving the
TCA cycle, there

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00:15:46,240 --> 00:15:49,540
are a couple of big picture
points that I want to make.

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00:15:49,540 --> 00:15:52,940
You put in two
carbons as acetyl-CoA,

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00:15:52,940 --> 00:15:56,450
deposit them into
oxaloacetate to form citrate,

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00:15:56,450 --> 00:15:59,260
a 6-carbon compound,
and then in the cycle,

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00:15:59,260 --> 00:16:02,770
you lose 2 carbons
as carbon dioxide.

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00:16:02,770 --> 00:16:05,530
That means that
there's no loss or gain

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00:16:05,530 --> 00:16:07,870
of carbon in this cycle.

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00:16:07,870 --> 00:16:10,720
If you had just one
molecule of oxaloacetate,

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00:16:10,720 --> 00:16:13,600
you'd be able to
complete the TCA cycle.

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00:16:13,600 --> 00:16:15,940
What happens,
however, when the cell

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00:16:15,940 --> 00:16:20,470
is in an, let's say, "energy
needed" situation, where

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00:16:20,470 --> 00:16:22,750
it needs to buff
up this cycle, that

331
00:16:22,750 --> 00:16:25,270
is increase the number
of molecules cycling

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00:16:25,270 --> 00:16:28,660
to be able to accommodate the
processing of more and more

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00:16:28,660 --> 00:16:31,510
molecules of acetyl-CoA?

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00:16:31,510 --> 00:16:34,450
Those molecules of
acetyl-CoA might flood in

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00:16:34,450 --> 00:16:37,530
from carbohydrate metabolism
or, as we'll see later,

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00:16:37,530 --> 00:16:39,760
from catabolism of lipids.

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00:16:39,760 --> 00:16:42,850
Let's look at panel
A of storyboard 11.

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00:16:42,850 --> 00:16:45,730
As shown in this figure,
one way to accomplish

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00:16:45,730 --> 00:16:49,330
increasing the carbon
content of the TCA cycle

340
00:16:49,330 --> 00:16:51,550
is to take an amino
acid, such as glutamate,

341
00:16:51,550 --> 00:16:55,870
and remove its amino group
to form alpha-ketoglutarate.

342
00:16:55,870 --> 00:16:59,740
Note that if you increase
the concentration of any one

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00:16:59,740 --> 00:17:03,580
molecule in the TCA cycle, for
example, alpha-ketoglutarate,

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00:17:03,580 --> 00:17:06,160
you're effectively
increasing the concentrations

345
00:17:06,160 --> 00:17:08,920
of all molecules in the cycle.

346
00:17:08,920 --> 00:17:11,290
Because it's a cycle,
many of the molecules

347
00:17:11,290 --> 00:17:13,450
are in equilibrium
with one another.

348
00:17:13,450 --> 00:17:17,230
Going clockwise around the TCA
cycle from alpha-ketoglutarate,

349
00:17:17,230 --> 00:17:19,540
we come to succinyl-CoA.

350
00:17:19,540 --> 00:17:23,290
Succinyl-CoA is an entry
point into the TCA cycle

351
00:17:23,290 --> 00:17:26,240
from odd-chain fatty acids
in certain amino acids,

352
00:17:26,240 --> 00:17:27,640
such as methionine.

353
00:17:27,640 --> 00:17:32,020
So these molecules can
give rise to succinyl-CoA

354
00:17:32,020 --> 00:17:34,570
that itself will then
increase the concentration

355
00:17:34,570 --> 00:17:37,570
of all molecules
in the TCA cycle.

356
00:17:37,570 --> 00:17:41,570
Our third primary input
point is at oxaloacetate.

357
00:17:41,570 --> 00:17:46,930
It involves aspartic acid being
deaminated into oxaloacetate.

358
00:17:46,930 --> 00:17:49,450
This is a very common
way to increase

359
00:17:49,450 --> 00:17:53,110
the amount of oxaloacetate
available for the TCA cycle.

360
00:17:53,110 --> 00:17:56,320
This reaction can dramatically
increase the rate of processing

361
00:17:56,320 --> 00:17:59,230
of molecules by the TCA cycle.

362
00:17:59,230 --> 00:18:01,600
Finally, there's an
enzyme we'll look at later

363
00:18:01,600 --> 00:18:04,960
called pyruvate
carboxylase, or PC,

364
00:18:04,960 --> 00:18:08,770
which can take pyruvate in the
mitochondrion, add CO2 to it,

365
00:18:08,770 --> 00:18:10,864
and form oxaloacetate.

366
00:18:10,864 --> 00:18:12,280
We're going to
come to this enzyme

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00:18:12,280 --> 00:18:16,370
later when we talk about
carboxylase enzymes as a class.

368
00:18:16,370 --> 00:18:21,020
This is an important enzyme in
the pathway of gluconeogenesis.

369
00:18:21,020 --> 00:18:23,950
So overall, I think you can
see that there are several ways

370
00:18:23,950 --> 00:18:26,500
that a cell can increase
the overall concentration

371
00:18:26,500 --> 00:18:29,680
of the intermediates
of the TCA cycle.

372
00:18:29,680 --> 00:18:33,700
Increasing any one intermediate
increases all of them.

373
00:18:33,700 --> 00:18:35,660
And that will increase
the rate by which

374
00:18:35,660 --> 00:18:38,500
acetyl-CoA molecules can
be processed ultimately

375
00:18:38,500 --> 00:18:40,300
to generate energy.

376
00:18:40,300 --> 00:18:44,290
The general word describing
this buffing up of the TCA cycle

377
00:18:44,290 --> 00:18:48,220
is anapleurosis, which comes
from the Greek word "filling

378
00:18:48,220 --> 00:18:49,770
up."