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PROFESSOR: Let's look
now at storyboard 25,

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00:00:24,810 --> 00:00:27,660
panels A and B.
Most of the pathways

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00:00:27,660 --> 00:00:30,810
we have studied so far in
5.07 have been catabolic--

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00:00:30,810 --> 00:00:33,270
that is, the breakdown
of molecules, usually

12
00:00:33,270 --> 00:00:35,790
with the objective of producing
energy rich molecules,

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00:00:35,790 --> 00:00:38,550
such as ATP, or
reducing equivalents

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00:00:38,550 --> 00:00:41,850
that ultimately could be used
for reductive biosynthesis.

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00:00:41,850 --> 00:00:45,510
Now, we're going to turn to
anabolism, or biosynthesis.

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00:00:45,510 --> 00:00:48,990
Biosynthetic pathways are
going to require energy input,

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00:00:48,990 --> 00:00:50,820
as well as reducing equivalents.

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00:00:50,820 --> 00:00:54,900
Overall, biosynthesis is
an endergonic process.

19
00:00:54,900 --> 00:00:57,210
Looking at panel B,
you can see that we're

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00:00:57,210 --> 00:01:01,140
going to start with fatty
acid and lipid biosynthesis.

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00:01:01,140 --> 00:01:03,600
Fatty acids are made
in the cytoplasm.

22
00:01:03,600 --> 00:01:06,450
The mitochondrion is going
to play an important role,

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00:01:06,450 --> 00:01:07,290
however.

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00:01:07,290 --> 00:01:10,330
We'll see just how in
a couple of minutes.

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00:01:10,330 --> 00:01:13,240
Let me say a couple of things
about the mitochondrion.

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00:01:13,240 --> 00:01:20,620
First, acetyl CoA, oxaloacetate
and the NADP plus NADPH pair,

27
00:01:20,620 --> 00:01:22,810
as we have described
above, cannot pass through

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00:01:22,810 --> 00:01:24,890
the mitochondrial membrane.

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00:01:24,890 --> 00:01:27,610
However, malate and citrate can.

30
00:01:27,610 --> 00:01:29,900
In fact, they can go
in either direction.

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00:01:29,900 --> 00:01:33,400
Keep these points in
mind as we move ahead.

32
00:01:33,400 --> 00:01:35,380
Now let's look at panel c.

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00:01:35,380 --> 00:01:37,870
We're going to break down
fatty-acid biosynthesis

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00:01:37,870 --> 00:01:39,800
into five steps.

35
00:01:39,800 --> 00:01:42,640
The first step involves
getting acetyl coenzyme A

36
00:01:42,640 --> 00:01:44,290
into the cytoplasm.

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00:01:44,290 --> 00:01:47,620
As I just said, acetyl CoA
cannot directly go past

38
00:01:47,620 --> 00:01:49,450
the mitochondrial membrane.

39
00:01:49,450 --> 00:01:51,490
To overcome this problem,
nature, quote unquote,

40
00:01:51,490 --> 00:01:55,980
packages acetyl CoA as citrate,
which can easily traverse

41
00:01:55,980 --> 00:01:57,670
the mitochondrial membrane.

42
00:01:57,670 --> 00:02:02,040
Hence, we put acetyl CoA
into a molecule of citrate,

43
00:02:02,040 --> 00:02:04,640
bring citrate out
into the cytoplasm,

44
00:02:04,640 --> 00:02:08,840
and then split off the
acetyl CoA once again.

45
00:02:08,840 --> 00:02:10,680
The second step
involves maintaining

46
00:02:10,680 --> 00:02:12,740
oxaloacetate balance.

47
00:02:12,740 --> 00:02:15,170
We just transported
a molecule of citrate

48
00:02:15,170 --> 00:02:17,240
out of the mitochondrial
matrix, thus

49
00:02:17,240 --> 00:02:21,380
depleting the TCA cycle of one
of its important intermediates.

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00:02:21,380 --> 00:02:24,320
The levels of all TCA
cycle intermediates

51
00:02:24,320 --> 00:02:27,830
are going to drop,
including oxaloacetate.

52
00:02:27,830 --> 00:02:29,750
Now think about what's
happened to the citrate

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00:02:29,750 --> 00:02:31,990
in the cytoplasm.

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00:02:31,990 --> 00:02:36,100
It was split into
oxaloacetate and acetyl CoA.

55
00:02:36,100 --> 00:02:38,560
If we could send
that on oxaloacetate

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00:02:38,560 --> 00:02:41,350
back into the
mitochondrion, the TCA cycle

57
00:02:41,350 --> 00:02:43,720
would be replenished,
once again.

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00:02:43,720 --> 00:02:47,800
Step two will show how
oxaloacetate finds its way back

59
00:02:47,800 --> 00:02:50,410
into the mitochondrial matrix.

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00:02:50,410 --> 00:02:52,720
The third step in
fatty-acid biosynthesis

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00:02:52,720 --> 00:02:56,410
involves the synthesis
of malonyl-coenzyme A.

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00:02:56,410 --> 00:02:59,290
This molecule is made by
putting a carbon-dioxide

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00:02:59,290 --> 00:03:03,280
molecule onto the methyl
carbon of acetyl CoA

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00:03:03,280 --> 00:03:05,650
using acetyl coenzyme
A carboxylase--

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00:03:05,650 --> 00:03:08,770
an enzyme we looked at earlier
in the carboxylase module

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00:03:08,770 --> 00:03:10,300
of 5.07.

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00:03:10,300 --> 00:03:13,330
malonyl-coenzyme A is the
actual precursor to all,

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00:03:13,330 --> 00:03:17,260
but two of the carbons
in the fatty acid chain.

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00:03:17,260 --> 00:03:19,720
The fourth step in
fatty-acid biosynthesis

70
00:03:19,720 --> 00:03:22,600
involves putting the
malonyl-coenzyme A

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00:03:22,600 --> 00:03:27,580
onto a very large protein on
the fatty acid synthase complex.

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00:03:27,580 --> 00:03:32,170
This large protein is called
ACP, or acyl carrier protein.

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00:03:32,170 --> 00:03:34,750
The fifth step involves
the actual reactions

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00:03:34,750 --> 00:03:38,020
of the fatty acid synthase,
sometimes called FAS.

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00:03:38,020 --> 00:03:41,180
This is a large
multi-protein complex.

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00:03:41,180 --> 00:03:42,880
These are the enzymes
by which we're

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00:03:42,880 --> 00:03:46,510
going to see the stepwise
addition of two carbon units,

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00:03:46,510 --> 00:03:49,630
up until we get to the
formation of a 16 carbon

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00:03:49,630 --> 00:03:52,720
long fatty acid
called palmitic acid.

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00:03:52,720 --> 00:03:56,350
Palmitic acid,
abbreviated 16 colon 0,

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00:03:56,350 --> 00:03:59,800
is the default fatty acid length
made by the fatty acid synthase

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00:03:59,800 --> 00:04:02,180
complex.

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00:04:02,180 --> 00:04:05,110
Let's look at panel
D. After the five step

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00:04:05,110 --> 00:04:08,330
synthesis of palmitic
acid the C16 fatty

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00:04:08,330 --> 00:04:10,250
acid can be elongated.

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00:04:10,250 --> 00:04:12,020
It can have double
bonds put into it,

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00:04:12,020 --> 00:04:14,100
and it can be branched.

88
00:04:14,100 --> 00:04:17,160
We're not going to be
looking into those reactions,

89
00:04:17,160 --> 00:04:21,329
but we shall be looking into
the way that either two or three

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00:04:21,329 --> 00:04:25,650
fatty acids will be placed onto
a glycerol backbone in order

91
00:04:25,650 --> 00:04:28,050
to make either, a
membrane lipid that

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00:04:28,050 --> 00:04:32,670
is a diacylglyceride phosphate,
or triacylglycerides.

93
00:04:32,670 --> 00:04:36,150
Triacylglycerides are our
primary storage form of energy.

94
00:04:36,150 --> 00:04:38,580
We're not going to be
looking into those reactions,

95
00:04:38,580 --> 00:04:40,710
but we shall be
looking at the way

96
00:04:40,710 --> 00:04:42,300
that either two or
three fatty acids

97
00:04:42,300 --> 00:04:44,700
can be placed onto a
glycerol backbone in order

98
00:04:44,700 --> 00:04:46,530
to make either a
membrane lipid that

99
00:04:46,530 --> 00:04:50,350
is a diacylglyceride phosphate,
or triacylglycerides.

100
00:04:50,350 --> 00:04:53,370
Triacylglycerides are
our primary storage forms

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00:04:53,370 --> 00:04:55,300
of energy.

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00:04:55,300 --> 00:04:57,930
I'm not going to be
going over another type

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00:04:57,930 --> 00:05:01,879
of fatty-acid biosynthesis,
called polyketide biosynthesis,

104
00:05:01,879 --> 00:05:03,420
but I'll encourage
you to take a look

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00:05:03,420 --> 00:05:04,640
at this pathway in the book.

106
00:05:04,640 --> 00:05:07,950
Polyketide biosynthesis
is a very important source

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00:05:07,950 --> 00:05:10,740
of a host of biologically
active lipids.

108
00:05:10,740 --> 00:05:13,350
Let's look at panel
E. Before we look

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00:05:13,350 --> 00:05:15,510
at a schematic of
lipid biosynthesis,

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00:05:15,510 --> 00:05:18,110
I'd like to introduce NADPH.

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00:05:18,110 --> 00:05:21,390
NADPH is going to be the source
of electrons that are used

112
00:05:21,390 --> 00:05:23,820
for reductive biosynthesis.

113
00:05:23,820 --> 00:05:28,230
NADPH is identical to NADH with
the exception of the phosphate

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00:05:28,230 --> 00:05:29,940
at the lower right
of the molecule,

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00:05:29,940 --> 00:05:32,850
as drawn in panel
E. This phosphate is

116
00:05:32,850 --> 00:05:35,310
a molecular decoration
that's recognized

117
00:05:35,310 --> 00:05:36,900
by biosynthetic enzymes.

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00:05:36,900 --> 00:05:38,490
That is, enzymes
that are looking

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00:05:38,490 --> 00:05:41,880
for a source of
reducing equivalents.

120
00:05:41,880 --> 00:05:44,460
NADPH is the co-factor
that provides

121
00:05:44,460 --> 00:05:46,550
those reducing equivalents.

122
00:05:46,550 --> 00:05:48,810
The hydrides shown
in the blue ellipse,

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00:05:48,810 --> 00:05:50,910
at the top of the
molecule, represents

124
00:05:50,910 --> 00:05:52,650
the reducing equivalents
that were going

125
00:05:52,650 --> 00:05:54,960
to be used in biosynthesis.

126
00:05:54,960 --> 00:05:57,060
The two main
pathways that result

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00:05:57,060 --> 00:06:00,030
in the production of
NADPH, for biosynthesis,

128
00:06:00,030 --> 00:06:02,160
are the pentose
phosphate pathway,

129
00:06:02,160 --> 00:06:04,590
which I'm going to deal
with in a separate lecture,

130
00:06:04,590 --> 00:06:06,240
and the malic enzyme,
which I'm going

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00:06:06,240 --> 00:06:08,320
to introduce in a few minutes.

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00:06:08,320 --> 00:06:11,430
Now turn to panel
F. This schematic

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00:06:11,430 --> 00:06:14,790
shows a high-level view of
fatty-acid biosynthesis.

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00:06:14,790 --> 00:06:17,550
I think it's useful to put
fatty-acid biosynthesis

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00:06:17,550 --> 00:06:20,880
in the context of one of
our physiological scenarios.

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00:06:20,880 --> 00:06:24,540
The scenario is
eat sugar, get fat.

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00:06:24,540 --> 00:06:26,700
Put more scientifically,
this scenario

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00:06:26,700 --> 00:06:29,130
is going to show
how the sugar we eat

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00:06:29,130 --> 00:06:32,820
can end up being converted,
very efficiently, into fat.

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00:06:32,820 --> 00:06:36,060
I think that, probably, most of
us want to avoid getting fat.

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00:06:36,060 --> 00:06:38,730
But keep in mind that
converting sugar to fat

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00:06:38,730 --> 00:06:42,300
is one way of converting
energy into a very compact

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00:06:42,300 --> 00:06:44,430
and mobile form.

144
00:06:44,430 --> 00:06:46,340
Let's start with
the glucose molecule

145
00:06:46,340 --> 00:06:48,170
over on the left of the panel.

146
00:06:48,170 --> 00:06:51,170
Starting with step one, follow
the horizontal hatched line

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00:06:51,170 --> 00:06:52,060
to the right.

148
00:06:52,060 --> 00:06:54,830
The glucose passes
through glycolysis,

149
00:06:54,830 --> 00:06:58,730
produces pyruvate, pyruvate
goes into the mitochondrion

150
00:06:58,730 --> 00:07:01,310
and is converted to acetyl CoA.

151
00:07:01,310 --> 00:07:04,220
Once again, following
the hatched line,

152
00:07:04,220 --> 00:07:07,070
acetyl CoA condenses
with oxaloacetate.

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00:07:07,070 --> 00:07:10,400
This is the citrate synthase
reaction to form citrate.

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00:07:10,400 --> 00:07:13,550
Rather than progressing
further into the TCA cycle

155
00:07:13,550 --> 00:07:16,650
where it would lose its
carbons as carbon dioxide,

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00:07:16,650 --> 00:07:19,820
the citrate at step two is
exported by a transporter

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00:07:19,820 --> 00:07:22,190
out into the cytoplasm.

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00:07:22,190 --> 00:07:24,140
Step three is
catalyzed by an enzyme

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00:07:24,140 --> 00:07:27,140
called ATP citrate lyase.

160
00:07:27,140 --> 00:07:31,100
This enzyme splits the citrate
into acetyl CoA, which is what

161
00:07:31,100 --> 00:07:33,370
we want, and leave a residue--

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00:07:33,370 --> 00:07:35,660
a molecule of oxaloacetate.

163
00:07:35,660 --> 00:07:38,120
I'm not going to go through
the mechanism of this enzyme,

164
00:07:38,120 --> 00:07:41,690
but I think it's worthwhile to
look back at storyboard seven,

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00:07:41,690 --> 00:07:43,550
look at the citrate
synthase reaction

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00:07:43,550 --> 00:07:46,430
and then imagine it
running backwards.

167
00:07:46,430 --> 00:07:50,120
Now, it's not going to be
exactly the reverse reaction as

168
00:07:50,120 --> 00:07:52,550
drawn, but at this
point in 5.07 you

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00:07:52,550 --> 00:07:56,180
should be able to look at the
cosubstrates for the reaction,

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00:07:56,180 --> 00:08:00,440
and be able to figure out how
ATP citrate lyase liberates

171
00:08:00,440 --> 00:08:02,960
the oxaloacetate residue.

172
00:08:02,960 --> 00:08:07,100
I'm going to come back to the
oxaloacetate, which is labeled

173
00:08:07,100 --> 00:08:09,410
by a star, in a few minutes.

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00:08:09,410 --> 00:08:11,510
Keep in mind that we
have to find a way

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00:08:11,510 --> 00:08:15,590
to return oxaloacetate to the
mitochondrial matrix or else

176
00:08:15,590 --> 00:08:17,900
we won't have enough
oxaloacetate to enable

177
00:08:17,900 --> 00:08:20,030
the next molecule
of citrate to be

178
00:08:20,030 --> 00:08:23,416
formed from an incoming
acetyl coenzyme A.

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00:08:23,416 --> 00:08:26,520
At step five we have
acetyl coenzyme A

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00:08:26,520 --> 00:08:30,270
in the cytoplasm, where we want
it for fatty-acid biosynthesis.

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00:08:30,270 --> 00:08:33,636
But we can also do other
things with this molecule.

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00:08:33,636 --> 00:08:35,010
In the last lecture,
for example,

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00:08:35,010 --> 00:08:37,260
I talked about ketone
body formation.

184
00:08:37,260 --> 00:08:40,919
So look at the vertical arrow--
that is the one going up.

185
00:08:40,919 --> 00:08:44,039
You can see we can
produce HMG CoA, hydroxy

186
00:08:44,039 --> 00:08:46,440
methyl glutaryl
coenzyme A. Which,

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00:08:46,440 --> 00:08:49,770
as I said in the last lecture,
is a biosynthetic precursor

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00:08:49,770 --> 00:08:54,240
to, for example, cholesterol,
as well as ketone bodies.

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00:08:54,240 --> 00:08:56,490
Then, follow the
arrows through step 5A

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00:08:56,490 --> 00:08:59,940
to the deposition of cholesterol
into the plasma membrane.

191
00:08:59,940 --> 00:09:03,030
Keep in mind, cholesterol is
a plasticizer of the membrane.

192
00:09:03,030 --> 00:09:05,880
Therefore, it's an
essential molecule.

193
00:09:05,880 --> 00:09:08,720
Now let's go back and
pick up the acetyl CoA

194
00:09:08,720 --> 00:09:11,270
as it progresses through
the fatty acid synthase,

195
00:09:11,270 --> 00:09:14,805
or FAS Reactions of 5B.

196
00:09:14,805 --> 00:09:17,810
Acetyl CoA is built
up in eight steps

197
00:09:17,810 --> 00:09:21,230
to form the 16 carbon
fatty-acid palmytate.

198
00:09:21,230 --> 00:09:23,210
Following the upward
arrow, palmytate

199
00:09:23,210 --> 00:09:25,760
can go on to form a
phospholipid, which

200
00:09:25,760 --> 00:09:28,400
will become an integral
part of the membrane,

201
00:09:28,400 --> 00:09:31,340
or it can go
horizontally, to the left,

202
00:09:31,340 --> 00:09:33,290
to form a
triacylglyceride, which

203
00:09:33,290 --> 00:09:35,300
will become embedded
in a lipid globule

204
00:09:35,300 --> 00:09:38,690
where it can serve as a
storage depot for energy.

205
00:09:38,690 --> 00:09:40,760
So at this point,
through the series

206
00:09:40,760 --> 00:09:44,540
of reactions numbered
5A and 5B, we've

207
00:09:44,540 --> 00:09:48,270
made the membrane plasticizer,
cholesterol, phospholipids,

208
00:09:48,270 --> 00:09:50,030
which are the main
structural elements

209
00:09:50,030 --> 00:09:53,150
of a biological membrane,
and a triacylglycerides,

210
00:09:53,150 --> 00:09:56,870
which is our principal
energy storage molecule.

211
00:09:56,870 --> 00:10:00,500
Now let's go back to the
asterisked oxaloacetate,

212
00:10:00,500 --> 00:10:02,000
at step 3.

213
00:10:02,000 --> 00:10:04,790
This orphaned molecule
of oxaloacetate

214
00:10:04,790 --> 00:10:07,580
has to be able to get back
into the mitochondrion.

215
00:10:07,580 --> 00:10:09,860
There are two pathways by
which this can happen--

216
00:10:09,860 --> 00:10:11,690
4A and 4B.

217
00:10:11,690 --> 00:10:14,000
Oxaloacetate is a ketone.

218
00:10:14,000 --> 00:10:18,590
It can be reduced by NADH
to form the alcohol, malate.

219
00:10:18,590 --> 00:10:20,600
This is the reverse
of the reaction

220
00:10:20,600 --> 00:10:23,240
that we're used to
seeing in the TCA cycle.

221
00:10:23,240 --> 00:10:26,510
Nevertheless, this is indeed
the thermodynamically favorable

222
00:10:26,510 --> 00:10:28,490
direction for this reaction.

223
00:10:28,490 --> 00:10:30,920
The malate dehydrogenase
enzyme used,

224
00:10:30,920 --> 00:10:33,680
here, is closely related
to the one that's

225
00:10:33,680 --> 00:10:35,510
in the mitochondrial matrix.

226
00:10:35,510 --> 00:10:38,360
Keep in mind, however, that
this is the cytoplasmic version

227
00:10:38,360 --> 00:10:39,620
of the enzyme.

228
00:10:39,620 --> 00:10:41,420
I mentioned earlier
that malate can

229
00:10:41,420 --> 00:10:44,870
go in either direction across
the mitochondrial membrane.

230
00:10:44,870 --> 00:10:49,250
In this case, path 4A, malate
goes into the mitochondrian,

231
00:10:49,250 --> 00:10:52,280
becomes part of the
mitochondrial malate pool,

232
00:10:52,280 --> 00:10:54,920
and then the mitochondrial
malate dehydrogenase

233
00:10:54,920 --> 00:10:57,350
will convert it to oxaloacetate.

234
00:10:57,350 --> 00:11:00,470
And thus, we have restored
the molecule of oxaloacetate

235
00:11:00,470 --> 00:11:04,340
that we borrowed from
the mitochondrial matrix.

236
00:11:04,340 --> 00:11:06,710
Path 4B represents
an alternative way

237
00:11:06,710 --> 00:11:08,810
to return the
oxaloacetate molecule

238
00:11:08,810 --> 00:11:10,910
to the mitochondrial matrix.

239
00:11:10,910 --> 00:11:13,850
4B involves the
malic enzyme, ME,

240
00:11:13,850 --> 00:11:19,460
which uses NADP plus to oxidize
malate back to oxaloacetate.

241
00:11:19,460 --> 00:11:23,300
In the process, NADP
plus is reduced to NADPH,

242
00:11:23,300 --> 00:11:25,370
the biosynthetic precursor.

243
00:11:25,370 --> 00:11:27,860
This is one of
the two major ways

244
00:11:27,860 --> 00:11:31,880
that a cell produces the NADPH
that it needs for biosynthesis.

245
00:11:31,880 --> 00:11:36,350
The other way to make NADPH is
the pentose phosphate pathway.

246
00:11:36,350 --> 00:11:40,490
It may seem kind of useless
to have taken an oxaloacetate,

247
00:11:40,490 --> 00:11:43,790
and then, by using malate
dehydrogenase convert

248
00:11:43,790 --> 00:11:48,230
that oxaloacetate to malate, and
then, convert the malate back

249
00:11:48,230 --> 00:11:49,670
to oxaloacetate.

250
00:11:49,670 --> 00:11:51,740
But by doing this
series of reactions,

251
00:11:51,740 --> 00:11:57,470
you are effectively
converting NADH to an NADPH.

252
00:11:57,470 --> 00:12:01,530
And NADPH is desperately needed
for biosynthesis, as we'll see.

253
00:12:01,530 --> 00:12:05,480
Lipid biosynthesis is
very NADPH intensive.

254
00:12:05,480 --> 00:12:07,470
Let's look at a couple
of more details.

255
00:12:07,470 --> 00:12:10,320
Decyl acetate shown
in brackets in step 4B

256
00:12:10,320 --> 00:12:12,920
exists on the malic enzyme.

257
00:12:12,920 --> 00:12:15,620
The enzyme facilitates
the decarboxylation

258
00:12:15,620 --> 00:12:16,430
of that molecule.

259
00:12:16,430 --> 00:12:20,000
Remember, oxaloacetate
is a beta keto acid.

260
00:12:20,000 --> 00:12:24,740
Decarboxylation by malic enzyme,
at step 4B, produces pyruvate.

261
00:12:24,740 --> 00:12:29,390
Now we can follow the pyruvate,
by the continuation of step 4B,

262
00:12:29,390 --> 00:12:30,890
into the mitochondrion.

263
00:12:30,890 --> 00:12:34,280
The pyruvate becomes a substrate
for pyruvate carboxylase,

264
00:12:34,280 --> 00:12:35,790
which we've looked at before.

265
00:12:35,790 --> 00:12:39,860
Pyruvate carboxylase will
put a CO2 back into pyruvate

266
00:12:39,860 --> 00:12:43,410
and convert it
into oxaloacetate.

267
00:12:43,410 --> 00:12:46,760
Pyruvate carboxylase is
an ATP requiring enzyme.

268
00:12:46,760 --> 00:12:50,840
Hence, path 4B has a
finite energy requirement.

269
00:12:50,840 --> 00:12:53,360
It would seem as though
this might be a problem.

270
00:12:53,360 --> 00:12:56,030
Nevertheless, when a cell
is doing biosynthesis,

271
00:12:56,030 --> 00:13:00,120
it usually has a lot of energy
around in the form of ATP.

272
00:13:00,120 --> 00:13:02,330
So this small investment
is a good one,

273
00:13:02,330 --> 00:13:05,690
in order to, first of all,
restore the oxaloacetate

274
00:13:05,690 --> 00:13:09,110
balance of the mitochondria,
and secondly, keep in mind,

275
00:13:09,110 --> 00:13:12,440
that 4B also gives you
an NADPH, which you

276
00:13:12,440 --> 00:13:14,870
need for biosynthesis, anyway.

277
00:13:14,870 --> 00:13:16,430
Let's summarize this figure.

278
00:13:16,430 --> 00:13:18,910
At the upper left, we
start with glucose,

279
00:13:18,910 --> 00:13:21,680
and by the hatched lines,
that glucose is converted

280
00:13:21,680 --> 00:13:23,780
to citrate in the cytoplasm.

281
00:13:23,780 --> 00:13:26,330
Citrate then yields
an acetyl CoA molecule

282
00:13:26,330 --> 00:13:29,810
that is the precursor to
fatty acids and cholesterol.

283
00:13:29,810 --> 00:13:33,350
Step two dealt with the problem
of getting oxaloacetate back

284
00:13:33,350 --> 00:13:35,150
into the mitochondrion.

285
00:13:35,150 --> 00:13:36,950
Remember, fatty-acid
biosynthesis

286
00:13:36,950 --> 00:13:38,930
happens in the cytoplasm.

287
00:13:38,930 --> 00:13:42,290
We borrowed an oxaloacetate
from the mitochondrion, hence,

288
00:13:42,290 --> 00:13:44,480
we have to get it back
to where it belongs

289
00:13:44,480 --> 00:13:46,400
in the mitochondrial matrix.

290
00:13:46,400 --> 00:13:49,970
Paths 4A and 4B represent
alternative pathways

291
00:13:49,970 --> 00:13:53,210
for getting the oxaloacetate
back where it belongs.

292
00:13:53,210 --> 00:13:56,270
Once the oxaloacetate is
back in the mitochondrion,

293
00:13:56,270 --> 00:13:58,580
then the next
molecule of acetyl CoA

294
00:13:58,580 --> 00:14:01,130
can come in, get
converted to citrate,

295
00:14:01,130 --> 00:14:04,340
and follow the hatched line
path, ultimately resulting

296
00:14:04,340 --> 00:14:06,560
in synthesis of lipids.

297
00:14:06,560 --> 00:14:11,450
Let's turn now to story board
26, panel A. At this point,

298
00:14:11,450 --> 00:14:15,240
we have a pool of acetyl
coenzyme A in the cytoplasm.

299
00:14:15,240 --> 00:14:18,350
It's now ready to start
making a fatty acid.

300
00:14:18,350 --> 00:14:20,180
In the carboxylase
module, I talked

301
00:14:20,180 --> 00:14:23,450
about how acetyl
CoA carboxylase is

302
00:14:23,450 --> 00:14:26,700
one of the carboxylases
took an active form of CO2

303
00:14:26,700 --> 00:14:28,850
and placed it onto
the methyl carbon

304
00:14:28,850 --> 00:14:31,950
at the end of the acetyl
coenzyme A molecule.

305
00:14:31,950 --> 00:14:33,920
The product of the
carboxylase reaction

306
00:14:33,920 --> 00:14:37,760
is malonyl coenzyme
A. This carboxylase

307
00:14:37,760 --> 00:14:41,720
is a biotin containing
enzyme, and requires ATP.

308
00:14:41,720 --> 00:14:43,880
As I've said
earlier, malonyl CoA

309
00:14:43,880 --> 00:14:46,250
is going to be the source of
all, but two of the carbons

310
00:14:46,250 --> 00:14:48,090
that make up the fatty acid.

311
00:14:48,090 --> 00:14:50,180
The initial fatty acid
that we're going to make

312
00:14:50,180 --> 00:14:52,940
is the C16 fatty
acid, palmitate.

313
00:14:52,940 --> 00:14:55,130
Palmitate will be
synthesized entirely

314
00:14:55,130 --> 00:14:57,640
on the fatty acid synthase.

315
00:14:57,640 --> 00:15:00,130
Let's look at panel B.
Fatty-acid synthesis

316
00:15:00,130 --> 00:15:02,470
in bacteria is carried
out by a series

317
00:15:02,470 --> 00:15:04,900
of enzymes that work as
discrete, or independent,

318
00:15:04,900 --> 00:15:05,670
units.

319
00:15:05,670 --> 00:15:07,990
Collectively, this
ensemble of enzymes

320
00:15:07,990 --> 00:15:11,320
is called the fatty
acid synthase complex.

321
00:15:11,320 --> 00:15:14,560
In a million cells, all of
the enzymatic activities--

322
00:15:14,560 --> 00:15:17,830
the same enzymatic activities--
are present in a single, very

323
00:15:17,830 --> 00:15:19,124
long peptide.

324
00:15:19,124 --> 00:15:21,040
At this point, I want
to go over the chemistry

325
00:15:21,040 --> 00:15:24,520
behind each of the activities
in the fatty acid synthase

326
00:15:24,520 --> 00:15:26,550
complex.

327
00:15:26,550 --> 00:15:29,300
One of the peptides that I
introduced, briefly, above

328
00:15:29,300 --> 00:15:33,080
is called ACP, or
acyl carrier protein.

329
00:15:33,080 --> 00:15:35,390
It contains assisting
sulfhydryl residue that

330
00:15:35,390 --> 00:15:38,420
will attack the carbonyl
group of malonyl CoA,

331
00:15:38,420 --> 00:15:42,470
forming a malonyl ACP adduct.

332
00:15:42,470 --> 00:15:44,340
Keep in mind that
the product is still

333
00:15:44,340 --> 00:15:46,951
a thioester with all of
the chemical properties

334
00:15:46,951 --> 00:15:48,367
you would find in
acetyl coenzyme.

335
00:15:48,367 --> 00:15:51,830
A The enzymatic subunit
that does this chemistry

336
00:15:51,830 --> 00:15:57,110
is called MAT, or mat, for
malonyl acetyl transferase.

337
00:15:57,110 --> 00:16:00,080
Another domain, shown at
the bottom of the fatty acid

338
00:16:00,080 --> 00:16:02,120
synthase, has a
cysteine residue that

339
00:16:02,120 --> 00:16:04,940
attacks the carbonyl
group of acetyl CoA.

340
00:16:04,940 --> 00:16:07,670
This reaction is also
catalyzed by MAT.

341
00:16:07,670 --> 00:16:09,920
Hence, in this case,
the MAT subunit

342
00:16:09,920 --> 00:16:14,750
forms an acetyl thioester
with the fatty acid synthase.

343
00:16:14,750 --> 00:16:18,290
At this point, we have a malonyl
group at the-- let's call it,

344
00:16:18,290 --> 00:16:20,150
northern domain of the
fatty acid synthase,

345
00:16:20,150 --> 00:16:21,490
as I've drawn it--

346
00:16:21,490 --> 00:16:25,220
and an acetyl group at what
I'll call, the southern domain.

347
00:16:25,220 --> 00:16:27,230
Let's look at panel
C. At this point,

348
00:16:27,230 --> 00:16:29,510
the fatty acid synthase
is fully primed.

349
00:16:29,510 --> 00:16:33,290
That is, it is loaded with a
malonyl group and acetyl group.

350
00:16:33,290 --> 00:16:34,850
Now we can start
doing some chemistry

351
00:16:34,850 --> 00:16:36,920
to join these two
groups together.

352
00:16:36,920 --> 00:16:40,310
The malonyl group has a beta
keto acid functionality so

353
00:16:40,310 --> 00:16:43,910
can easily lose CO2, and
generate a nucleophile that

354
00:16:43,910 --> 00:16:46,490
will then go down and
attack the carbonyl group

355
00:16:46,490 --> 00:16:49,280
of the acetyl residue that
is on the southern domain

356
00:16:49,280 --> 00:16:50,180
of the enzyme.

357
00:16:50,180 --> 00:16:52,940
This reaction results
in the acetyl group,

358
00:16:52,940 --> 00:16:56,510
from the southern domain,
replacing the CO2 moiety

359
00:16:56,510 --> 00:16:58,070
on the northern domain.

360
00:16:58,070 --> 00:17:01,100
That is, the carbonyl group
from the southern domain

361
00:17:01,100 --> 00:17:04,609
ends up covalently
attached to the CH2 group,

362
00:17:04,609 --> 00:17:07,640
with the box over it,
in the northern domain.

363
00:17:07,640 --> 00:17:12,220
The ketoacyl synthase, that
is KS, domain of the protein,

364
00:17:12,220 --> 00:17:13,819
does this reaction.

365
00:17:13,819 --> 00:17:17,450
The product of the reaction
shows a beta keto acyl group

366
00:17:17,450 --> 00:17:19,700
attached to the northern domain.

367
00:17:19,700 --> 00:17:21,829
Keep in mind, that
the beta keto acyl

368
00:17:21,829 --> 00:17:25,980
group is attached, specifically,
to the ACL carrier protein.

369
00:17:25,980 --> 00:17:28,820
The next step is catalyzed
by the ketoacyl ACP

370
00:17:28,820 --> 00:17:30,590
reductase, or KR.

371
00:17:30,590 --> 00:17:34,490
In this case, NADPH will
reduce the keto group

372
00:17:34,490 --> 00:17:36,410
that is next to the
terminal carbon,

373
00:17:36,410 --> 00:17:40,120
forming an alcohol,
specifically, beta hydroxy

374
00:17:40,120 --> 00:17:42,330
acyl carrier protein, or ACP.

375
00:17:42,330 --> 00:17:47,120
This beta hydroxyl four
carbon compound is now primed

376
00:17:47,120 --> 00:17:48,920
for dehydration
by a dehydratase,

377
00:17:48,920 --> 00:17:55,130
which removes water to form
the trans-delta-2-enoyl ACP.

378
00:17:55,130 --> 00:17:58,190
This molecule is an alkyne
with the double bond

379
00:17:58,190 --> 00:18:02,680
between the second and third
carbons from the thioester.

380
00:18:02,680 --> 00:18:05,560
Let's turn now to storyboard 27.

381
00:18:05,560 --> 00:18:09,520
The reaction continues
with enoyl reductase, ER,

382
00:18:09,520 --> 00:18:12,790
which uses a second
molecule of NADPH

383
00:18:12,790 --> 00:18:17,050
in order to saturate the
double bond of the enoyl ACP.

384
00:18:17,050 --> 00:18:21,110
The product is a butyryl
ACP at this point,

385
00:18:21,110 --> 00:18:23,500
it should be clear as
to what we've done.

386
00:18:23,500 --> 00:18:26,230
We've taken two
acetyl CoA molecules

387
00:18:26,230 --> 00:18:28,090
and fused them together.

388
00:18:28,090 --> 00:18:30,580
In the process we've done
a number of reductions,

389
00:18:30,580 --> 00:18:32,680
and the product is
a pure hydrocarbon.

390
00:18:32,680 --> 00:18:36,400
In this case, the four
carbon butyryl coenzyme A.

391
00:18:36,400 --> 00:18:38,470
In order for the cycle
to repeat itself,

392
00:18:38,470 --> 00:18:40,840
we have to vacate the
ACP, or northern site

393
00:18:40,840 --> 00:18:43,390
on fatty acid synthase,
in order to make

394
00:18:43,390 --> 00:18:45,910
it available for the next
molecule of malonyl CoA

395
00:18:45,910 --> 00:18:46,990
to come in.

396
00:18:46,990 --> 00:18:50,710
That's done by the enzyme, or
subunit, called translocase,

397
00:18:50,710 --> 00:18:53,890
which simply moves the butyryl
group from the northern site

398
00:18:53,890 --> 00:18:55,420
down to the southern site.

399
00:18:55,420 --> 00:18:59,200
The northern sulfhydryl on ACP,
of the fatty acid synthase,

400
00:18:59,200 --> 00:19:02,770
is now available to connect
with the next incoming molecule

401
00:19:02,770 --> 00:19:06,250
of malonyl coenzyme A. The
biosynthetic cycle will now

402
00:19:06,250 --> 00:19:10,090
repeat itself six times in
order to form the 16 carbon long

403
00:19:10,090 --> 00:19:11,980
hydrocarbon, palmitate.

404
00:19:11,980 --> 00:19:14,680
In the figure, its shown
attached to the northern site

405
00:19:14,680 --> 00:19:16,840
of the fatty acid synthase.

406
00:19:16,840 --> 00:19:19,690
Let's look back at the
overall synthesis scheme.

407
00:19:19,690 --> 00:19:22,000
What you'll see is that the
terminal two carbons, that

408
00:19:22,000 --> 00:19:24,850
is the two carbons furthest
to the right in the molecule,

409
00:19:24,850 --> 00:19:26,680
came from acetyl CoA.

410
00:19:26,680 --> 00:19:28,720
But all the other
carbons originally

411
00:19:28,720 --> 00:19:32,380
came from alanyl
coenzyme A. The last step

412
00:19:32,380 --> 00:19:35,590
in the overall reaction
scheme involves thioesterase--

413
00:19:35,590 --> 00:19:39,460
transferring the thioester bound
palmitate to a water molecule,

414
00:19:39,460 --> 00:19:42,860
forming palmitic acid,
which is released.

415
00:19:42,860 --> 00:19:45,110
Now let's turn to storyboard 28.

416
00:19:45,110 --> 00:19:47,900
As I mentioned earlier, the
default length of a fatty acid

417
00:19:47,900 --> 00:19:49,160
is 16 carbons.

418
00:19:49,160 --> 00:19:50,259
That is palmitic acid.

419
00:19:50,259 --> 00:19:51,800
You can look in the
book for pathways

420
00:19:51,800 --> 00:19:53,930
leading to elongation,
desaturation,

421
00:19:53,930 --> 00:19:56,372
and branching of the
parent fatty acid chain.

422
00:19:56,372 --> 00:19:58,580
I'd like to give you, however,
a little bit of detail

423
00:19:58,580 --> 00:20:00,620
on how membrane
lipids are formed,

424
00:20:00,620 --> 00:20:02,900
as well as triacylglycerides.

425
00:20:02,900 --> 00:20:05,420
Let's look at panel A. To
make these higher order

426
00:20:05,420 --> 00:20:08,900
lipids, fatty acids will become
connected to a three carbon

427
00:20:08,900 --> 00:20:10,220
glycerol backbone.

428
00:20:10,220 --> 00:20:11,900
The glycerol
backbone is made from

429
00:20:11,900 --> 00:20:13,880
dihydroxyacetone
phosphate, which

430
00:20:13,880 --> 00:20:16,910
is one of the intermediates
in the glycolytic pathway.

431
00:20:16,910 --> 00:20:20,240
To make the lipid backbone,
dihydroxyacetone phosphate

432
00:20:20,240 --> 00:20:23,570
is reduced by glycerol 3
phosphate dehydrogenase

433
00:20:23,570 --> 00:20:26,180
using NADH as the co-factor.

434
00:20:26,180 --> 00:20:29,900
The product of this reaction
is glycerol 3 phosphate.

435
00:20:29,900 --> 00:20:34,010
An acyltransferase will then
take a fatty acyl coenzyme A

436
00:20:34,010 --> 00:20:36,770
and place it on one
of the hydroxyl groups

437
00:20:36,770 --> 00:20:41,030
of the glycerol backbone to form
a monoacylglyceride phosphate.

438
00:20:41,030 --> 00:20:44,840
Then, as shown in panel B,
a second fatty acid group

439
00:20:44,840 --> 00:20:47,780
will be placed on the
only available hydroxyl

440
00:20:47,780 --> 00:20:51,260
to form a diacylglyceride
phosphate.

441
00:20:51,260 --> 00:20:55,640
This is otherwise known as
a membrane phospholipid.

442
00:20:55,640 --> 00:20:59,660
If the biosynthetic objective
is to make a triacylglyceride,

443
00:20:59,660 --> 00:21:02,660
which is of course our major
storage form of energy,

444
00:21:02,660 --> 00:21:05,360
then a phosphatase will
come in and hydrolyze off

445
00:21:05,360 --> 00:21:07,040
the phosphate from
the three carbon

446
00:21:07,040 --> 00:21:09,320
of the diacylglyceride
phosphate to form

447
00:21:09,320 --> 00:21:12,350
what's called a diacylglycerol.

448
00:21:12,350 --> 00:21:14,390
Then an acyltransferase,
as above,

449
00:21:14,390 --> 00:21:17,900
will place a third fatty acid
onto the glycerol backbone

450
00:21:17,900 --> 00:21:21,320
to form a
triacylglyceride, or TAG.

451
00:21:21,320 --> 00:21:23,930
As I said above, this is
our principal storage form

452
00:21:23,930 --> 00:21:25,600
of energy.