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DR. BOGDAN FEDELES: Hello, and
welcome to 5.07 Biochemistry

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00:00:33,240 --> 00:00:34,300
online.

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00:00:34,300 --> 00:00:36,100
I'm Dr. Bogdan Fedeles.

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00:00:36,100 --> 00:00:38,250
Let's metabolize some problems.

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00:00:38,250 --> 00:00:41,830
Today we're discussing
problem 2 of problem set 6.

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00:00:41,830 --> 00:00:43,990
Here we're going to
explore in more detail

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00:00:43,990 --> 00:00:47,170
the mechanism of
phosphoglycerate mutase, which

15
00:00:47,170 --> 00:00:48,920
is the eighth enzyme
in glycolysis.

16
00:00:48,920 --> 00:00:51,430
It's the enzyme that
catalyzes the conversion

17
00:00:51,430 --> 00:00:54,820
of 3-phosphoglycerate
to 2-phosphoglycerate.

18
00:00:54,820 --> 00:00:57,130
Generally speaking,
mutases are enzymes

19
00:00:57,130 --> 00:00:58,900
that catalyze the
shift of a functional

20
00:00:58,900 --> 00:01:02,540
group between two similar
positions of a molecule.

21
00:01:02,540 --> 00:01:04,540
In the case of
phosphoglycerate mutase,

22
00:01:04,540 --> 00:01:06,820
this enzyme catalyzes
the transfer

23
00:01:06,820 --> 00:01:09,935
of the phosphate group from
the 3 position of glycerate

24
00:01:09,935 --> 00:01:12,190
to the 2 position of glycerate.

25
00:01:12,190 --> 00:01:15,730
In 5.07, you will
encounter several mutases.

26
00:01:15,730 --> 00:01:17,860
Similar to
phosphoglycerate mutase,

27
00:01:17,860 --> 00:01:20,020
there is a
bisphosphoglycerate mutase,

28
00:01:20,020 --> 00:01:22,420
which converts
1,3-bisphosphoglycerate

29
00:01:22,420 --> 00:01:24,480
to 2,3-bisphosphoglycerate.

30
00:01:24,480 --> 00:01:26,350
Now, this reaction
is very important

31
00:01:26,350 --> 00:01:29,020
when it happens in
the red blood cells.

32
00:01:29,020 --> 00:01:31,960
Another mutase
you will encounter

33
00:01:31,960 --> 00:01:34,470
is in the glycogen
breakdown pathway.

34
00:01:34,470 --> 00:01:37,390
It's called phosphoglucomutase
and converts

35
00:01:37,390 --> 00:01:40,780
glucose 1-phosphate to
glucose 6-phosphate.

36
00:01:40,780 --> 00:01:43,720
Now finally, the most
intriguing of them

37
00:01:43,720 --> 00:01:46,540
all is the
methylmalonyl-coa mutase,

38
00:01:46,540 --> 00:01:50,650
which is a fascinating enzyme
that converts methylmalonyl-coA

39
00:01:50,650 --> 00:01:52,530
to succinyl-coA.

40
00:01:52,530 --> 00:01:57,250
In this reaction, it
rearranges this carbon skeleton

41
00:01:57,250 --> 00:02:01,360
of the molecule, and it
requires adenosylcobalamin,

42
00:02:01,360 --> 00:02:04,330
which is a co-factor
derived from vitamin B12.

43
00:02:04,330 --> 00:02:08,139
Back to phosphoglycerate mutase,
this is a fascinating enzyme

44
00:02:08,139 --> 00:02:12,110
because it uses a phosphorylated
histidine in the active site.

45
00:02:12,110 --> 00:02:15,490
And this is actually an example
of a phosphorous-nitrogen bond,

46
00:02:15,490 --> 00:02:18,820
one of the very few
available in biochemistry.

47
00:02:18,820 --> 00:02:21,100
Here is a schematic
of the mechanism

48
00:02:21,100 --> 00:02:23,500
of phosphoglycerate mutase.

49
00:02:23,500 --> 00:02:27,310
Now, the reaction
starts where the enzyme

50
00:02:27,310 --> 00:02:28,870
is already phosphorylated.

51
00:02:28,870 --> 00:02:30,430
We'll call it a phospho enzyme.

52
00:02:30,430 --> 00:02:32,260
And the histidine
in the active site

53
00:02:32,260 --> 00:02:34,380
contains the phosphate group.

54
00:02:34,380 --> 00:02:38,480
Then the enzyme binds the
substrate 3-phosphoglycerate.

55
00:02:38,480 --> 00:02:41,470
And then it's going to transfer
this phosphate group onto the 2

56
00:02:41,470 --> 00:02:45,400
position, the 2-hydroxyl of the
3-phosphoglycerate to generate

57
00:02:45,400 --> 00:02:47,860
the 2,3-bisphosphoglycerate.

58
00:02:47,860 --> 00:02:50,800
Then the phosphate
at the 3 position

59
00:02:50,800 --> 00:02:53,740
is transferred to the
histidine to generate

60
00:02:53,740 --> 00:02:56,440
the product of the
reaction 2-phosphoglycerate

61
00:02:56,440 --> 00:03:01,130
and regenerate
the phosphoenzyme.

62
00:03:01,130 --> 00:03:04,430
Note that the phosphate group
is in fact not transferred.

63
00:03:04,430 --> 00:03:06,890
This phosphate group
here is not the same

64
00:03:06,890 --> 00:03:09,050
that ends up on the 2 position.

65
00:03:09,050 --> 00:03:12,740
But rather, this phosphate group
gets transferred to the enzyme.

66
00:03:12,740 --> 00:03:15,080
And the phosphate
group from the enzyme

67
00:03:15,080 --> 00:03:18,872
ends up on the second
position of glycerate.

68
00:03:22,570 --> 00:03:25,260
As we just mentioned, the
active form of the enzyme

69
00:03:25,260 --> 00:03:28,632
has already the phosphate
bound to histidine.

70
00:03:28,632 --> 00:03:30,840
Now the question is, how
did this phosphate get there

71
00:03:30,840 --> 00:03:32,160
in the first place?

72
00:03:32,160 --> 00:03:35,370
Presumably, the enzyme is
first synthesized in a form

73
00:03:35,370 --> 00:03:38,850
that we call apo form, which
does not have the phosphate.

74
00:03:38,850 --> 00:03:41,580
And the phosphate is then
added as a post-translational

75
00:03:41,580 --> 00:03:43,170
modification.

76
00:03:43,170 --> 00:03:46,710
Now, our problem suggests that
one source for this phosphate

77
00:03:46,710 --> 00:03:48,780
is phosphoenolpyruvate.

78
00:03:48,780 --> 00:03:50,540
And there's some
experimental evidence

79
00:03:50,540 --> 00:03:53,580
that phosphoenolpyruvate
can transfer their phosphate

80
00:03:53,580 --> 00:03:56,970
and phosphorylate the
histidine in this enzyme.

81
00:03:56,970 --> 00:03:59,190
Now, we are asked
to comment on how

82
00:03:59,190 --> 00:04:01,500
reasonable this proposal is.

83
00:04:01,500 --> 00:04:04,260
We're going to evaluate
the proposed transformation

84
00:04:04,260 --> 00:04:07,770
between phosphoenolpyruvate
and phosphoglycerate mutase

85
00:04:07,770 --> 00:04:09,400
from two points of view.

86
00:04:09,400 --> 00:04:11,400
First of all, is
this transformation

87
00:04:11,400 --> 00:04:13,830
thermodynamically accessible?

88
00:04:13,830 --> 00:04:17,990
And second, is this
structurally feasible?

89
00:04:17,990 --> 00:04:20,690
We know that
phosphoenolpyruvate contains

90
00:04:20,690 --> 00:04:23,150
a high-energy
phosphate bond that

91
00:04:23,150 --> 00:04:26,540
can release a lot of
energy upon hydrolysis.

92
00:04:26,540 --> 00:04:30,050
Now, if we look
in our book, this

93
00:04:30,050 --> 00:04:32,400
is the Voet & Voet,
Third Edition.

94
00:04:32,400 --> 00:04:35,240
If we look here,
phosphoenolpyruvate,

95
00:04:35,240 --> 00:04:39,260
it says, releases about
62 kilojoules per mole

96
00:04:39,260 --> 00:04:41,060
upon hydrolysis.

97
00:04:41,060 --> 00:04:44,510
Now this is significantly
more than what

98
00:04:44,510 --> 00:04:47,210
energy is released by
the hydrolysis of ATP

99
00:04:47,210 --> 00:04:52,150
going to ADP, which is only
about 31 kilojoules per mole.

100
00:04:52,150 --> 00:04:54,260
Now, this should not
be surprising to you

101
00:04:54,260 --> 00:04:57,530
because PEP,
phosphoenolpyruvate,

102
00:04:57,530 --> 00:04:59,790
is used in the last
step of glycolysis,

103
00:04:59,790 --> 00:05:03,620
the pyruvate kinase, to
phosphorylate ADP and generate

104
00:05:03,620 --> 00:05:04,710
ATP.

105
00:05:04,710 --> 00:05:07,640
So the fact it has a higher
energy of hydrolysis,

106
00:05:07,640 --> 00:05:09,830
it just makes that
transformation

107
00:05:09,830 --> 00:05:11,900
thermodynamically accessible.

108
00:05:11,900 --> 00:05:14,600
Let's now take a look at
the arrow pushing mechanism

109
00:05:14,600 --> 00:05:17,860
of how phosphoenolpyruvate
can phosphorylate

110
00:05:17,860 --> 00:05:20,180
phosphoglycerate mutase.

111
00:05:20,180 --> 00:05:22,880
Here is the
phosphoenolpyruvate molecule,

112
00:05:22,880 --> 00:05:26,600
and here is the histidine in
the active site of the enzyme.

113
00:05:26,600 --> 00:05:31,330
Now, as you know, histidine
has a pKa of about 6,

114
00:05:31,330 --> 00:05:34,150
so an important fraction
of the histidine

115
00:05:34,150 --> 00:05:36,670
will be protonated
at physiological pH.

116
00:05:36,670 --> 00:05:38,560
However, for this
reaction to work

117
00:05:38,560 --> 00:05:40,600
we need the histidine
to act as a nucleophile

118
00:05:40,600 --> 00:05:41,950
to attack the phosphate.

119
00:05:41,950 --> 00:05:45,830
Therefore, we're going to
consider it deprotonated.

120
00:05:45,830 --> 00:05:48,100
Now, the reaction
starts by assuming

121
00:05:48,100 --> 00:05:50,470
there's a base in the
active site that's

122
00:05:50,470 --> 00:05:53,100
going to deprotonate
the histidine,

123
00:05:53,100 --> 00:05:56,170
and then it's going to
attack the phosphate.

124
00:05:56,170 --> 00:05:57,640
And finally, the
phosphate group is

125
00:05:57,640 --> 00:06:02,110
going to leave with the
assistance of a general acid.

126
00:06:02,110 --> 00:06:06,180
So these are the
products that we obtain.

127
00:06:06,180 --> 00:06:09,240
This is the phosphoenzyme with
the histidine that now has

128
00:06:09,240 --> 00:06:11,070
the phosphate group attached.

129
00:06:11,070 --> 00:06:15,810
And this is the enol
that is released

130
00:06:15,810 --> 00:06:17,880
from the
phosphoenolpyruvate, which is

131
00:06:17,880 --> 00:06:20,200
the enol form of the pyruvate.

132
00:06:20,200 --> 00:06:24,900
Now, if we evaluate the starting
material and the product

133
00:06:24,900 --> 00:06:27,150
in terms of their
ability to stabilize

134
00:06:27,150 --> 00:06:30,030
negative charge, such as the
charges on the phosphate,

135
00:06:30,030 --> 00:06:31,170
by resonance--

136
00:06:31,170 --> 00:06:34,440
We notice that there is
no significant difference.

137
00:06:34,440 --> 00:06:37,980
Here we have two negative
charges and one set

138
00:06:37,980 --> 00:06:40,440
of phosphorus oxygen bonded.

139
00:06:40,440 --> 00:06:42,910
The charge can delocalize
on this oxygen.

140
00:06:42,910 --> 00:06:47,830
We also have this carboxylate
group, which we also have here.

141
00:06:47,830 --> 00:06:52,110
So there's not a lot of resonant
stabilization between starting

142
00:06:52,110 --> 00:06:54,010
materials and products so far.

143
00:06:54,010 --> 00:06:57,870
Therefore, this reaction
is thermodynamically

144
00:06:57,870 --> 00:07:00,550
close to neutral.

145
00:07:00,550 --> 00:07:03,960
However, notice the
enol form of pyruvate.

146
00:07:03,960 --> 00:07:07,230
Now this is in fact a
very unstable product.

147
00:07:07,230 --> 00:07:11,460
And it likes to
tautomerize, basically

148
00:07:11,460 --> 00:07:13,590
isomerize in acid
base conditions

149
00:07:13,590 --> 00:07:16,260
to the keto form of a pyruvate.

150
00:07:16,260 --> 00:07:19,300
The mechanism would
be, as such, the base

151
00:07:19,300 --> 00:07:21,130
can deprotonate the enol.

152
00:07:21,130 --> 00:07:26,960
And then, the general acid
can protonate CH2 group

153
00:07:26,960 --> 00:07:29,120
to generate the keto
form of the pyruvate.

154
00:07:29,120 --> 00:07:31,880
It turns out the delta G
for this transformation

155
00:07:31,880 --> 00:07:33,580
is very negative.

156
00:07:33,580 --> 00:07:40,810
Delta G here is approximately
minus 40 kilojoules per mole.

157
00:07:40,810 --> 00:07:45,490
So that means this reaction is
strongly going to the right,

158
00:07:45,490 --> 00:07:47,770
and strongly favors
the keto form.

159
00:07:47,770 --> 00:07:51,910
So that means per ensemble the
transformation going from PEP

160
00:07:51,910 --> 00:07:53,950
in our histidine
in the active site

161
00:07:53,950 --> 00:07:57,850
is going to be strongly
driven to the right because

162
00:07:57,850 --> 00:08:00,240
of this keto equilibrium.

163
00:08:00,240 --> 00:08:02,830
Therefore, the entire
process shown here

164
00:08:02,830 --> 00:08:04,930
is expected to be
thermodynamically very

165
00:08:04,930 --> 00:08:06,190
favorable.

166
00:08:06,190 --> 00:08:09,130
Now let's take a look at some
structural considerations.

167
00:08:09,130 --> 00:08:11,560
In order for PEP to
phosporylate the enzyme,

168
00:08:11,560 --> 00:08:14,380
it has to be able to
reach the histidine that's

169
00:08:14,380 --> 00:08:16,070
deep in the active site.

170
00:08:16,070 --> 00:08:18,190
Notice that the
2-phosphoglycerate

171
00:08:18,190 --> 00:08:22,040
is one of the products or
substrates of the enzyme.

172
00:08:22,040 --> 00:08:25,480
And therefore, it fits very
nicely in the active site.

173
00:08:25,480 --> 00:08:29,650
Now phosphoenolpyruvate looks
a lot like 2-phosphoglycerate.

174
00:08:32,380 --> 00:08:36,840
Going to sketch it here, going
to have the phosphate there,

175
00:08:36,840 --> 00:08:40,200
and then there's the double
bond in this position.

176
00:08:40,200 --> 00:08:44,159
So because
phosphoenolpyruvate looks

177
00:08:44,159 --> 00:08:47,820
a lot like 2-phosphoglycerate it
should have no problem fitting

178
00:08:47,820 --> 00:08:49,830
inside the active
site of the enzyme

179
00:08:49,830 --> 00:08:53,120
and reaching the
active site histidine.

180
00:08:53,120 --> 00:08:56,010
Therefore, the chemical reaction
proposed in this problem

181
00:08:56,010 --> 00:08:57,330
is quite reasonable.

182
00:08:57,330 --> 00:08:59,460
First of all, the
thermodynamics are

183
00:08:59,460 --> 00:09:02,640
excellent because the hydrolysis
of phosphoenolpyruvate

184
00:09:02,640 --> 00:09:03,990
gives a lot of energy.

185
00:09:03,990 --> 00:09:07,650
And then the sterics are
also favorable because PEP

186
00:09:07,650 --> 00:09:10,080
resembles
2-phosphoglycerate, one

187
00:09:10,080 --> 00:09:11,420
of the products of the enzyme.

188
00:09:14,410 --> 00:09:18,030
Part 2 of this problem asks us
to evaluate the consequences

189
00:09:18,030 --> 00:09:21,310
on the major function of
glycolysis of this reaction

190
00:09:21,310 --> 00:09:24,370
that we just discussed--
of phosphoenolpyruvate

191
00:09:24,370 --> 00:09:27,030
with phosphoglycerate mutase.

192
00:09:27,030 --> 00:09:29,160
Here is the second
half of glycolysis,

193
00:09:29,160 --> 00:09:31,980
going from glyceraldehyde
phosphate, or GAP, all the way

194
00:09:31,980 --> 00:09:33,520
to pyruvate.

195
00:09:33,520 --> 00:09:35,530
As you know, the main
function of glycolysis

196
00:09:35,530 --> 00:09:37,850
is to generate ATP.

197
00:09:37,850 --> 00:09:39,640
And for each
molecule of glucose,

198
00:09:39,640 --> 00:09:42,595
we have a net generation
of two molecules of ATP.

199
00:09:42,595 --> 00:09:45,400
Now, ATP is produced
in two places.

200
00:09:45,400 --> 00:09:49,080
First, at the
phosphoglycerate kinase when

201
00:09:49,080 --> 00:09:52,590
1,3-bisphosphoglycerate can
phosphorylate ADP to generate

202
00:09:52,590 --> 00:09:53,580
ATP.

203
00:09:53,580 --> 00:09:55,920
And then the
pyruvate kinase where

204
00:09:55,920 --> 00:09:57,720
phosphoenolpyruvate
phosphorylates

205
00:09:57,720 --> 00:10:00,150
ADP to generate ATP.

206
00:10:00,150 --> 00:10:03,060
Now since we start glycolysis
by investing some ATP,

207
00:10:03,060 --> 00:10:07,590
we need two molecules of ATP
to phosphorylate glucose.

208
00:10:07,590 --> 00:10:10,170
We recover those
two molecules of ATP

209
00:10:10,170 --> 00:10:12,540
at the phosphoglycerate
kinase step.

210
00:10:12,540 --> 00:10:15,750
So all the net production of
ATP that we get in glycolysis

211
00:10:15,750 --> 00:10:20,466
comes from the pyruvate
kinase reaction shown here.

212
00:10:20,466 --> 00:10:23,880
Now, if
phosphoenolpyruvate is used

213
00:10:23,880 --> 00:10:27,190
to phosphorylate
phosphoglycerate mutase,

214
00:10:27,190 --> 00:10:30,210
basically, it's going
to react to give

215
00:10:30,210 --> 00:10:32,700
the phosphate group here.

216
00:10:32,700 --> 00:10:38,080
It's going to generate pyruvate
but without generating ATP,

217
00:10:38,080 --> 00:10:38,580
right?

218
00:10:38,580 --> 00:10:41,910
So the phosphate group goes to
this phosphoglycerate mutase,

219
00:10:41,910 --> 00:10:45,010
and it generates
pyruvate, but we

220
00:10:45,010 --> 00:10:47,150
get no net production of ATP.

221
00:10:47,150 --> 00:10:49,540
Now if PEP is used
to phosphorylate

222
00:10:49,540 --> 00:10:51,760
phosphoglycerate
mutase, it's not

223
00:10:51,760 --> 00:10:55,500
going to be available for
the pyruvate kinase step.

224
00:10:55,500 --> 00:10:59,230
But we do generate pyruvate,
so the whole transformation

225
00:10:59,230 --> 00:11:04,230
reaches pyruvate, but without
producing a net amount of ATP.

226
00:11:04,230 --> 00:11:07,230
Of course, this should not
be a significant problem,

227
00:11:07,230 --> 00:11:10,020
as in glycolysis we
only require the enzymes

228
00:11:10,020 --> 00:11:11,520
in catalytic amounts.

229
00:11:11,520 --> 00:11:16,980
So initially, we're not going to
be generating net amount of ATP

230
00:11:16,980 --> 00:11:19,530
until we phosphorylate
the entire pool

231
00:11:19,530 --> 00:11:21,240
of phosphoglycerate mutase.

232
00:11:21,240 --> 00:11:23,505
After that, now we have
phosphoglycerate mutase,

233
00:11:23,505 --> 00:11:27,210
so PEP is once again available
for the pyruvate kinase

234
00:11:27,210 --> 00:11:29,760
reaction to generate ATP.

235
00:11:29,760 --> 00:11:33,210
I hope you noticed that there
is a more subtle question here.

236
00:11:33,210 --> 00:11:36,310
If we're going to use
PEP to phosphorylate

237
00:11:36,310 --> 00:11:39,390
phosphoglycerate
mutase, how are we

238
00:11:39,390 --> 00:11:42,490
going to get to produce
PEP in the first place

239
00:11:42,490 --> 00:11:45,750
since we need
phosphoglycerate mutase to go

240
00:11:45,750 --> 00:11:48,570
from 3-phosphoglycerate
to 2-phosphoglycerate,

241
00:11:48,570 --> 00:11:50,490
which then produces PEP.

242
00:11:50,490 --> 00:11:53,040
Again, it's kind of like one
of these chicken and the egg

243
00:11:53,040 --> 00:11:54,690
problems.

244
00:11:54,690 --> 00:11:57,300
As you'll find
out, many pathways

245
00:11:57,300 --> 00:12:01,050
feed into or intersect
with glycolysis.

246
00:12:01,050 --> 00:12:03,180
And therefore,
phosphoenolpyruvate

247
00:12:03,180 --> 00:12:06,390
could in principle be
made in other ways.

248
00:12:06,390 --> 00:12:08,550
For example, in
gluconeogenesis you'll

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00:12:08,550 --> 00:12:14,790
see that pyruvate can lead
to oxaloacetate, which then

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00:12:14,790 --> 00:12:19,000
can lead to phosphoenolpyruvate
using this enzyme called

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00:12:19,000 --> 00:12:22,560
PEP carboxykinase, or PEPCK.

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00:12:22,560 --> 00:12:26,070
So there are ways to produce
phosphoenolpyruvate, which

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00:12:26,070 --> 00:12:28,920
can then, say, phosphorylate
phosphoglycerate mutase, which

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00:12:28,920 --> 00:12:34,290
then allows the glycolysis to
flow through in the normal way.

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00:12:34,290 --> 00:12:36,120
Well, that's it
for this problem.

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00:12:36,120 --> 00:12:38,430
I hope you found
pretty intriguing how

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00:12:38,430 --> 00:12:40,690
phosphoglycerate mutase works.

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00:12:40,690 --> 00:12:43,020
Now remember, this is one
of the very few enzymes

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00:12:43,020 --> 00:12:46,950
in biochemistry that utilizes
the phosphorylated histidine.

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00:12:46,950 --> 00:12:48,720
And this is one of
the few examples

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00:12:48,720 --> 00:12:52,470
of a phosphorus-nitrogen
bond we have in biochemistry.

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00:12:52,470 --> 00:12:54,990
Also remember, the
phosphoenolpyruvate

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00:12:54,990 --> 00:12:58,980
is the highest high-energy
phosphate compound

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00:12:58,980 --> 00:13:00,730
we have in the body.

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00:13:00,730 --> 00:13:03,180
But all that hydrolysis
energy really

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00:13:03,180 --> 00:13:05,610
comes from the keto
enol tautomerization

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00:13:05,610 --> 00:13:10,730
equilibrium of the pyruvate that
gets released upon hydrolysis.