1
00:00:00,000 --> 00:00:00,024
The following content is
provided under a Creative

2
00:00:00,024 --> 00:00:00,033
Commons license.

3
00:00:00,033 --> 00:00:00,057
Your support will help MIT
OpenCourseWare continue to

4
00:00:00,057 --> 00:00:00,081
offer high quality educational
resources for free.

5
00:00:00,081 --> 00:00:00,108
To make a donation or view
additional materials from

6
00:00:00,108 --> 00:00:00,132
hundreds of MIT courses, visit
MIT OpenCourseWare at

7
00:00:00,132 --> 00:00:01,382
ocw.mit.edu.

8
00:00:21,260 --> 00:01:46,690
PROFESSOR: All right, so let's
do our first clicker question.

9
00:01:46,690 --> 00:02:03,620
All right, let's just
do 10 more seconds.

10
00:02:03,620 --> 00:02:10,850
Interesting.

11
00:02:10,850 --> 00:02:15,560
So, in this problem you were
given the maximum rate, Vmax,

12
00:02:15,560 --> 00:02:18,710
which, by the way, you
calculated in class last time.

13
00:02:18,710 --> 00:02:22,710
And then said what will the
substrate concentration be

14
00:02:22,710 --> 00:02:25,850
when you have half
of that rate.

15
00:02:25,850 --> 00:02:29,590
And so, when you have half of
the maximum rate, that's the

16
00:02:29,590 --> 00:02:31,310
definition of k m.

17
00:02:31,310 --> 00:02:35,330
So all you have to do is if a
k m is given, you just write

18
00:02:35,330 --> 00:02:37,370
down that value.

19
00:02:37,370 --> 00:02:39,420
So that's the level
of question you'll

20
00:02:39,420 --> 00:02:41,840
get on enzyme kinetics.

21
00:02:41,840 --> 00:02:44,790
All right, so that should be
a good tie breaker, too.

22
00:02:44,790 --> 00:02:48,170
All right, so in this little
review and we talk about the

23
00:02:48,170 --> 00:02:53,110
material from the second half of
the course, and why I think

24
00:02:53,110 --> 00:02:57,110
this material is really
important and cool, and that's

25
00:02:57,110 --> 00:03:00,450
because it all represents the
basic principles of how

26
00:03:00,450 --> 00:03:01,500
enzymes work.

27
00:03:01,500 --> 00:03:04,270
And as a biochemist, I recognize
this material is

28
00:03:04,270 --> 00:03:05,350
really important.

29
00:03:05,350 --> 00:03:08,090
So that's what interests me
particularly about it.

30
00:03:08,090 --> 00:03:10,790
And the point that I want to
make in general is that when

31
00:03:10,790 --> 00:03:14,030
you're studying introductory
material, sometimes it seems

32
00:03:14,030 --> 00:03:16,000
like you're never going to
really need that material

33
00:03:16,000 --> 00:03:18,740
again, but that's because you
haven't seen the connection

34
00:03:18,740 --> 00:03:20,710
between it in other topics.

35
00:03:20,710 --> 00:03:24,310
So, a lot of the things you've
learned will be related to

36
00:03:24,310 --> 00:03:26,340
things that you're going
to see you later on.

37
00:03:26,340 --> 00:03:28,890
So I'm going to give you
examples and review examples

38
00:03:28,890 --> 00:03:31,860
of how the material you've
learned will allow you to

39
00:03:31,860 --> 00:03:35,360
understand an enzyme,
for example.

40
00:03:35,360 --> 00:03:39,090
So, we talked last time that one
reason why you care about

41
00:03:39,090 --> 00:03:42,450
understanding how an enzyme
works is because a lot of

42
00:03:42,450 --> 00:03:43,390
people want to inhibit
enzymes.

43
00:03:43,390 --> 00:03:47,170
Well, why would you want to
inhibit enzymes, and that's

44
00:03:47,170 --> 00:03:50,290
because inhibiting enzymes
is very useful to

45
00:03:50,290 --> 00:03:51,770
treat a bunch of things.

46
00:03:51,770 --> 00:03:55,470
A number of you will, in the
next week, I'm pretty sure, be

47
00:03:55,470 --> 00:03:59,980
interested in ways to inhibit
enzymes to prevent headaches

48
00:03:59,980 --> 00:04:02,000
or to decrease headaches.

49
00:04:02,000 --> 00:04:04,970
And this is true of students
and faculty

50
00:04:04,970 --> 00:04:06,310
during finals week.

51
00:04:06,310 --> 00:04:11,950
And so, some of the treatments
for headaches that a number of

52
00:04:11,950 --> 00:04:14,890
those pharmaceuticals are geared
toward enzymes called

53
00:04:14,890 --> 00:04:18,390
prostaglandin synthesis, and
they inhibit those enzyme from

54
00:04:18,390 --> 00:04:21,220
working and that gets rid
of your headache.

55
00:04:21,220 --> 00:04:24,950
Arthritis, for example, is also
treated by a lot of the

56
00:04:24,950 --> 00:04:27,370
same medications that
treat headaches.

57
00:04:27,370 --> 00:04:33,320
Cancer, often chemotherapy
or you're giving people

58
00:04:33,320 --> 00:04:37,450
inhibitors of enzymes to
decrease tumor growth.

59
00:04:37,450 --> 00:04:41,390
And we talked last time about
inhibiting HIV proteases as

60
00:04:41,390 --> 00:04:46,170
part of a treatment for HIV. So,
understanding enzymes is

61
00:04:46,170 --> 00:04:49,550
very important for the
pharmaceutical industry, and

62
00:04:49,550 --> 00:04:52,160
so that's one example of why
you need to know about

63
00:04:52,160 --> 00:04:55,350
chemical equilibrium, why you
need to know about acid based

64
00:04:55,350 --> 00:05:00,870
oxidation reduction transition
metals, and also kinetics.

65
00:05:00,870 --> 00:05:02,610
So, I'm going to give
you a case study.

66
00:05:02,610 --> 00:05:04,440
You've seen some of these
examples before, but we're

67
00:05:04,440 --> 00:05:06,250
going to put them all
together now.

68
00:05:06,250 --> 00:05:11,290
And I try in every lecture, if I
can, to mention vitamin B12,

69
00:05:11,290 --> 00:05:14,010
and here, we're going to tell
you about a vitamin B12

70
00:05:14,010 --> 00:05:18,460
dependent enzyme, and how
knowing what you know now

71
00:05:18,460 --> 00:05:21,600
about these topics would allow
you to understand how this

72
00:05:21,600 --> 00:05:25,580
enzyme works.

73
00:05:25,580 --> 00:05:29,360
So, first, kinetics, this is
easy, methionine synthase,

74
00:05:29,360 --> 00:05:32,620
it's an enzyme.

75
00:05:32,620 --> 00:05:35,960
So, what does this enzyme do?

76
00:05:35,960 --> 00:05:39,340
So this particular enzyme
converts an amino acid called

77
00:05:39,340 --> 00:05:43,440
homocysteine to another amino
acid, methionine.

78
00:05:43,440 --> 00:05:48,070
It also converts another
vitamin, a B vitamin, the

79
00:05:48,070 --> 00:05:50,780
methyltetrahydrofolate form to
tetrahydrofolate, so this

80
00:05:50,780 --> 00:05:55,860
folate is one your B vitamins.

81
00:05:55,860 --> 00:05:57,120
Why do you care?

82
00:05:57,120 --> 00:06:01,220
Well, methionine is needed for
proper formation of brain and

83
00:06:01,220 --> 00:06:06,040
spinal column, and it's
particularly important when

84
00:06:06,040 --> 00:06:09,350
women are pregnant that they
have enough folic acid so that

85
00:06:09,350 --> 00:06:11,480
they can make enough
methionine.

86
00:06:11,480 --> 00:06:14,510
If they do not have enough folic
acid, then you can have

87
00:06:14,510 --> 00:06:17,280
babies born with neural
tube defects.

88
00:06:17,280 --> 00:06:20,600
Also, as I mentioned before,
inhibiting methionine synthase

89
00:06:20,600 --> 00:06:25,140
raises homocysteine levels, so
you can't do this conversion,

90
00:06:25,140 --> 00:06:27,680
and that increases risk
of heart disease that

91
00:06:27,680 --> 00:06:30,540
homocysteine is actually a
really good indicator, high

92
00:06:30,540 --> 00:06:33,180
homocysteine levels of whether
someone's going to have heart

93
00:06:33,180 --> 00:06:35,030
trouble or not.

94
00:06:35,030 --> 00:06:38,770
This is also a target for
chemotherapy, because you need

95
00:06:38,770 --> 00:06:43,820
tetrahydrofolate to do DNA
biosynthesis, and tumor cells

96
00:06:43,820 --> 00:06:47,670
that are growing, tumors that
are growing, need a lot of DNA

97
00:06:47,670 --> 00:06:47,980
biosynthesis.

98
00:06:47,980 --> 00:06:51,740
So if you inhibit this enzyme,
you can inhibit DNA

99
00:06:51,740 --> 00:06:54,300
biosynthesis, so it's
a potential

100
00:06:54,300 --> 00:06:58,030
chemotherapeutic target.

101
00:06:58,030 --> 00:07:00,170
There is some use of
this in Europe.

102
00:07:00,170 --> 00:07:03,590
Actually there is a treatment
that's given that's

103
00:07:03,590 --> 00:07:05,060
specifically supposed to inhibit
methione synthase,

104
00:07:05,060 --> 00:07:11,030
it's not used so much in this
country, but laughing gas will

105
00:07:11,030 --> 00:07:12,920
inhibit methione synthase.

106
00:07:12,920 --> 00:07:15,750
And so, in Europe some cancer
patients are treated with

107
00:07:15,750 --> 00:07:19,580
laughing gas as a way to
decrease their tumors.

108
00:07:19,580 --> 00:07:22,940
I'm not sure it's the most
effective therapy, but they're

109
00:07:22,940 --> 00:07:24,440
at least pretty happy with it.

110
00:07:24,440 --> 00:07:29,950
So that's the counterpart.

111
00:07:29,950 --> 00:07:32,860
So, this is an important
enzyme.

112
00:07:32,860 --> 00:07:39,840
And enzymes, of course, as we
know are catalysts and they

113
00:07:39,840 --> 00:07:44,440
catalyze reactions, so tell me
what you know about catalysts.

114
00:07:44,440 --> 00:07:46,350
Which is the following
statements about

115
00:07:46,350 --> 00:08:37,690
catalysts are true?

116
00:08:37,690 --> 00:08:52,700
OK, let's take 10 more seconds.

117
00:08:52,700 --> 00:08:58,100
Very good.

118
00:08:58,100 --> 00:09:01,120
All right, so going
back to the notes,

119
00:09:01,120 --> 00:09:04,560
statement 2 is true.

120
00:09:04,560 --> 00:09:09,350
So, catalysts work by lowering
the activation energy barrier

121
00:09:09,350 --> 00:09:12,180
for both the forward and
the reverse reaction.

122
00:09:12,180 --> 00:09:15,340
So both rates would
be changed.

123
00:09:15,340 --> 00:09:18,850
And also, catalysts are not
going to affect the

124
00:09:18,850 --> 00:09:21,280
thermodynamics, they're not
going to shift the equilibrium

125
00:09:21,280 --> 00:09:23,050
toward products.

126
00:09:23,050 --> 00:09:25,060
What's something that you
could use to shift an

127
00:09:25,060 --> 00:09:28,840
equilibrium, say, to products
or reactants.

128
00:09:28,840 --> 00:09:30,710
Temperature, yeah.

129
00:09:30,710 --> 00:09:33,820
So, just a little review
of kinetics.

130
00:09:33,820 --> 00:09:37,120
Again, a catalyst works by
lowering the activation energy

131
00:09:37,120 --> 00:09:41,140
barrier, or another way to say
that is by stabilizing a

132
00:09:41,140 --> 00:09:47,040
transition state, so it's going
to affect the activation

133
00:09:47,040 --> 00:09:48,640
energy for the forward
reaction.

134
00:09:48,640 --> 00:09:51,950
And for the reverse reaction,
it's going to decrease both of

135
00:09:51,950 --> 00:09:53,760
them by the same amount.

136
00:09:53,760 --> 00:09:56,350
So if you know how much one is
decreased, you also know how

137
00:09:56,350 --> 00:09:58,140
much the other is decreased.

138
00:09:58,140 --> 00:10:02,010
And we also know that it
doesn't affect the

139
00:10:02,010 --> 00:10:02,810
thermodynamics.

140
00:10:02,810 --> 00:10:06,620
So, does it change the
equilibrium constant?

141
00:10:06,620 --> 00:10:10,740
No, it doesn't change the
equilibrium constant.

142
00:10:10,740 --> 00:10:13,420
All right, so a little
review of kinetics.

143
00:10:13,420 --> 00:10:16,390
So now let's talk about
transition metals.

144
00:10:16,390 --> 00:10:19,520
So this particular enzyme needs
2 different kinds of

145
00:10:19,520 --> 00:10:20,680
transition metals.

146
00:10:20,680 --> 00:10:24,830
It needs cobalt in the form of
vitamin B12 and it also needs

147
00:10:24,830 --> 00:10:31,650
zinc. So we talked about
methylcobalamin, so cobalamin

148
00:10:31,650 --> 00:10:34,680
is another name for
vitamin B12.

149
00:10:34,680 --> 00:10:37,900
And here we have a methyl
ligand, c h 3 is a methyl

150
00:10:37,900 --> 00:10:42,140
ligand coordinated to the cobalt
in the center, and we

151
00:10:42,140 --> 00:10:47,160
saw before that when you have a
thing that forms attachments

152
00:10:47,160 --> 00:10:50,810
at more than one point, it's
called a chelate, so the corn

153
00:10:50,810 --> 00:10:54,270
ring here is a chelating agent,
and it's a tetradentate

154
00:10:54,270 --> 00:10:58,020
ligand because there are
4 points of attachment.

155
00:10:58,020 --> 00:10:59,680
So we can have monodentate,
bidentate, tetradentate,

156
00:10:59,680 --> 00:11:05,100
hexadentate, and tell you
the number of points of

157
00:11:05,100 --> 00:11:06,720
attachment.

158
00:11:06,720 --> 00:11:11,260
And again, this is a chelate,
and what do you know about a

159
00:11:11,260 --> 00:11:12,220
chelate effect?

160
00:11:12,220 --> 00:11:15,450
I want someone to tell me what
the chelate effect is, and

161
00:11:15,450 --> 00:11:18,550
there's a special bonus
prize of a teeshirt

162
00:11:18,550 --> 00:11:19,690
for the best answer.

163
00:11:19,690 --> 00:11:21,620
Who wants to give it a try
and tell me what the

164
00:11:21,620 --> 00:11:25,860
chelate effect is.

165
00:11:25,860 --> 00:11:26,750
Any volunteers?

166
00:11:26,750 --> 00:11:39,640
Bonus teeshirt.

167
00:11:39,640 --> 00:11:43,290
Who wants to give it a try.

168
00:11:43,290 --> 00:11:50,500
Not very brave.

169
00:11:50,500 --> 00:11:55,760
STUDENT: [INAUDIBLE]

170
00:11:55,760 --> 00:11:58,440
PROFESSOR: OK, so chelate, it's
something that's going to

171
00:11:58,440 --> 00:12:03,490
be binding to a metal at
multiple points of attachment.

172
00:12:03,490 --> 00:12:07,820
And what do we know about how
stable or how favorable that

173
00:12:07,820 --> 00:12:08,940
kind of interaction is?

174
00:12:08,940 --> 00:12:17,240
STUDENT: It makes it more stable
because [INAUDIBLE].

175
00:12:17,240 --> 00:12:27,810
PROFESSOR: Are you reading
from your notes?

176
00:12:27,810 --> 00:12:28,830
Well, you know what, I didn't
say that was not allowed, so

177
00:12:28,830 --> 00:12:28,850
congratulations.

178
00:12:28,850 --> 00:12:31,640
Right, so chelates are unusually
stable due to

179
00:12:31,640 --> 00:12:33,480
entropic affects.

180
00:12:33,480 --> 00:12:35,740
So it's an entropy affect.

181
00:12:35,740 --> 00:12:38,390
So if you have a metal that's
free in solution, it's going

182
00:12:38,390 --> 00:12:41,750
to bind to a lot of water
molecules, and often, metals

183
00:12:41,750 --> 00:12:43,210
like to bind to thick things.

184
00:12:43,210 --> 00:12:45,790
You see a lot of octahedral
complexes.

185
00:12:45,790 --> 00:12:49,240
So if you have a metal bound to
6 waters, and you bring in

186
00:12:49,240 --> 00:12:52,430
a chelate that will blind with
multiple points of attachment,

187
00:12:52,430 --> 00:12:57,010
such as EDTA, which binds at 6
points of attachment, that 1

188
00:12:57,010 --> 00:13:01,230
molecule of EDTA will displace
6 water molecules.

189
00:13:01,230 --> 00:13:04,110
So that is going to be
entropically favorable.

190
00:13:04,110 --> 00:13:07,450
6 things floating around in
solution have a lot more

191
00:13:07,450 --> 00:13:10,360
entropy than 1 thing floating
around in solution.

192
00:13:10,360 --> 00:13:13,770
And this makes chelates
very stable.

193
00:13:13,770 --> 00:13:16,720
And so, if you want to get --
if someone is poisoned with

194
00:13:16,720 --> 00:13:19,350
the metal and you want to get
that metal out of your system,

195
00:13:19,350 --> 00:13:23,150
you give them a chelate such as
EDTA, hospitals have EDTA.

196
00:13:23,150 --> 00:13:26,410
It's also good, as we learned,
for cleaning bath tubs.

197
00:13:26,410 --> 00:13:31,370
All right, so that's
the chelate effect.

198
00:13:31,370 --> 00:13:35,190
All right, so what about zinc.
This enzyme has a zinc and it

199
00:13:35,190 --> 00:13:38,360
has zinc in the plus
-- zinc plus 2 ion.

200
00:13:38,360 --> 00:13:43,910
So, if you know that, you
can calculate a d count.

201
00:13:43,910 --> 00:13:47,230
And so again, we can go
to our periodic table.

202
00:13:47,230 --> 00:13:49,640
What group is zinc in?

203
00:13:49,640 --> 00:13:50,450
12.

204
00:13:50,450 --> 00:13:53,570
So what would be the d count?

205
00:13:53,570 --> 00:13:54,560
10.

206
00:13:54,560 --> 00:13:58,290
So 12 minus 2 equals 10.

207
00:13:58,290 --> 00:14:01,520
So what would you think would
be true about the color of

208
00:14:01,520 --> 00:14:33,810
this d 10 system?

209
00:14:33,810 --> 00:14:48,710
All right, so let's just
take 10 more seconds.

210
00:14:48,710 --> 00:14:55,310
All right, so it would
be colorless.

211
00:14:55,310 --> 00:14:59,700
And so, all of the d orbitals
are going to be full, and so

212
00:14:59,700 --> 00:15:03,190
there's no transition from one
set of d orbitals to the

213
00:15:03,190 --> 00:15:07,120
other, and so it would be
a colorless compound.

214
00:15:07,120 --> 00:15:11,590
And that is, in fact, true that
it took years and years

215
00:15:11,590 --> 00:15:14,100
of studying methionine synthase
before people

216
00:15:14,100 --> 00:15:17,150
realized that it had a zinc in
it, no one knew that there was

217
00:15:17,150 --> 00:15:19,450
a zinc, because they
couldn't see it.

218
00:15:19,450 --> 00:15:23,330
It didn't add a color, and so
no one knew that a metal was

219
00:15:23,330 --> 00:15:24,660
there for a long time.

220
00:15:24,660 --> 00:15:31,750
All right, so what about
oxidation reduction?

221
00:15:31,750 --> 00:15:34,900
So, this enzyme does
a lot of different

222
00:15:34,900 --> 00:15:37,440
oxidation reduction reactions.

223
00:15:37,440 --> 00:15:41,280
So, when it reacts with
homocysteine to form

224
00:15:41,280 --> 00:15:44,960
methionine, and the vitamin
B12, which is shown in a

225
00:15:44,960 --> 00:15:48,950
cartoon form down here, goes
from a plus 3 state that has

226
00:15:48,950 --> 00:15:52,360
the methyl associated, it gives
the methyl group, the c

227
00:15:52,360 --> 00:15:55,900
h 3 group, up to homocysteine
to form methionine, and then

228
00:15:55,900 --> 00:15:58,750
you're in a plus 1 state
of cobalt here.

229
00:15:58,750 --> 00:16:03,780
But this state can lose an
electron, and you can have a

230
00:16:03,780 --> 00:16:06,570
what's called Co(II) form of the
enzyme, which you need to

231
00:16:06,570 --> 00:16:10,870
put another electron in and
reduce it to go back, and you

232
00:16:10,870 --> 00:16:12,790
also need to methylate
it again.

233
00:16:12,790 --> 00:16:16,220
And the methyl group comes from
s adenosylmethionine.

234
00:16:16,220 --> 00:16:19,320
So, we need to think about how
we're going to reduce the

235
00:16:19,320 --> 00:16:23,990
vitamin to get it back in its
primary turnover state.

236
00:16:23,990 --> 00:16:26,910
so, we know the redox
potentials for this.

237
00:16:26,910 --> 00:16:29,460
The standard reduction
potential for vitamin

238
00:16:29,460 --> 00:16:30,800
B12 is minus 0 .

239
00:16:30,800 --> 00:16:32,670
5 2 6 volts.

240
00:16:32,670 --> 00:16:35,640
The potential reduction
potential for flavodoxin,

241
00:16:35,640 --> 00:16:38,420
which is the protein that gives
it the 1 electron, and

242
00:16:38,420 --> 00:16:41,090
it's a flavin protein, which
is another B vitamin.

243
00:16:41,090 --> 00:16:42,810
And that's minus 0 .

244
00:16:42,810 --> 00:16:46,290
2 3 0 volts.

245
00:16:46,290 --> 00:16:49,120
So, what do we know
about these?

246
00:16:49,120 --> 00:17:34,540
Tell me which is the better
reducing agent.

247
00:17:34,540 --> 00:17:37,110
All right, let's just
do 10 more seconds.

248
00:17:37,110 --> 00:17:50,730
Click in your responses.

249
00:17:50,730 --> 00:17:55,450
OK.

250
00:17:55,450 --> 00:18:00,860
So, vitamin B12 is a better
reducing agent.

251
00:18:00,860 --> 00:18:05,610
If you have a large negative
number, then the reduced

252
00:18:05,610 --> 00:18:08,110
species is very reducing.

253
00:18:08,110 --> 00:18:11,390
So it wants to reduce
other things and

254
00:18:11,390 --> 00:18:14,030
get oxidized itself.

255
00:18:14,030 --> 00:18:16,440
So, that's interesting.

256
00:18:16,440 --> 00:18:20,120
It seems like vitamin B12 should
be reducing flavodoxin

257
00:18:20,120 --> 00:18:22,940
and not the other way around.

258
00:18:22,940 --> 00:18:27,040
So we can look at how
unfavorable this process is,

259
00:18:27,040 --> 00:18:30,850
and we can calculate a standard
potential for this,

260
00:18:30,850 --> 00:18:34,040
and we can use this equation
where we have reduction minus

261
00:18:34,040 --> 00:18:39,080
oxidation and plug the values
in, and so we have a minus 0 .

262
00:18:39,080 --> 00:18:45,050
5 2 6 minus a minus of the
floating potential, and that

263
00:18:45,050 --> 00:18:48,050
gives us a negative
value for delta e.

264
00:18:48,050 --> 00:18:51,750
So is this reaction
spontaneous?

265
00:18:51,750 --> 00:18:56,640
No, and that's because if you
have a negative standard

266
00:18:56,640 --> 00:18:58,890
reduction potential, then
you're going to have a

267
00:18:58,890 --> 00:19:03,280
positive value for delta g, so
it will not be spontaneous.

268
00:19:03,280 --> 00:19:06,720
And again, you can calculate the
value for delta g, so we

269
00:19:06,720 --> 00:19:09,330
have minus n, number of moles
of electrons, Faraday's

270
00:19:09,330 --> 00:19:13,640
constant times the difference
in potential, and it's a one

271
00:19:13,640 --> 00:19:17,520
electron process, we put in
Faraday's constant and the

272
00:19:17,520 --> 00:19:21,760
calculated value for delta e,
and we get a positive 28 .

273
00:19:21,760 --> 00:19:22,730
6.

274
00:19:22,730 --> 00:19:26,150
And as I mentioned before in
class, the way this reaction

275
00:19:26,150 --> 00:19:30,010
goes is that at the same time an
electron goes in, you also

276
00:19:30,010 --> 00:19:33,040
cleave s adenosylmethionine,
which gives the methyl group

277
00:19:33,040 --> 00:19:36,000
to also return to the catalytic
cycle, and that

278
00:19:36,000 --> 00:19:39,580
process is very favorable
minus 37 .

279
00:19:39,580 --> 00:19:41,430
6 kilojoules per mole.

280
00:19:41,430 --> 00:19:44,650
So it drives the unfavorable
reaction.

281
00:19:44,650 --> 00:19:48,300
So in the body you will have a
favorable oxidation reductions

282
00:19:48,300 --> 00:19:53,780
and you'll have unfavorable
ones.

283
00:19:53,780 --> 00:19:56,680
All right, so what do you a cell
that requires energy to

284
00:19:56,680 --> 00:20:01,990
catalyze a non-spontaneous
reaction?

285
00:20:01,990 --> 00:20:03,070
So, that's an electrolitic
cell.

286
00:20:03,070 --> 00:20:08,170
And a cell that catalyzes
a spontaneous reaction?

287
00:20:08,170 --> 00:20:10,880
Galvanic, right.

288
00:20:10,880 --> 00:20:13,100
So again, with oxidation
reduction, it doesn't matter

289
00:20:13,100 --> 00:20:15,610
if you're talking about a
battery, or if you're talking

290
00:20:15,610 --> 00:20:20,040
about a cellular process, all
the same equations apply.

291
00:20:20,040 --> 00:20:26,260
All right, what about acid
based equilibrium.

292
00:20:26,260 --> 00:20:30,560
So in this particular -- in this
catalytic cycle, we are

293
00:20:30,560 --> 00:20:34,780
converting homocysteine to
methionine, and that chemistry

294
00:20:34,780 --> 00:20:38,670
involves acid base.

295
00:20:38,670 --> 00:20:41,280
So what is true about the
reaction is that the

296
00:20:41,280 --> 00:20:43,310
protonation state of
the substrate,

297
00:20:43,310 --> 00:20:45,750
homocysteine, matters.

298
00:20:45,750 --> 00:20:49,120
So, you can have a protonated
homocysteine, and here's the

299
00:20:49,120 --> 00:20:51,790
structure of homocysteine, a
deprotonated homocysteine

300
00:20:51,790 --> 00:20:55,880
where the sulfur does not
have a proton anymore.

301
00:20:55,880 --> 00:20:57,900
So we have an s minus here.

302
00:20:57,900 --> 00:21:01,920
And this protonated form of
homocysteine can be converted

303
00:21:01,920 --> 00:21:05,760
to methionine where there's a
methyl group attached to the

304
00:21:05,760 --> 00:21:07,970
sulfur here.

305
00:21:07,970 --> 00:21:12,410
So, we want to know at
physiological p h 7 .

306
00:21:12,410 --> 00:21:16,620
4, how much of the homocysteine
is deprotonated?

307
00:21:16,620 --> 00:21:20,510
And here, if you're asking how
much of something is in a

308
00:21:20,510 --> 00:21:24,610
protonated state, how much of
something is in a deprotonated

309
00:21:24,610 --> 00:21:30,050
state, what you are asking
is what is the p k a of

310
00:21:30,050 --> 00:21:32,810
homocysteine.

311
00:21:32,810 --> 00:21:34,660
So what is the p k a
of homocysteine?

312
00:21:34,660 --> 00:21:37,870
That's what you need to know to
figure out how much would

313
00:21:37,870 --> 00:21:40,220
be in the protonated state,
how much would be in the

314
00:21:40,220 --> 00:21:41,860
deprotonated state.

315
00:21:41,860 --> 00:21:45,250
So here, the p k a is 10.

316
00:21:45,250 --> 00:21:48,870
So now, without doing any
calculations, I want you to

317
00:21:48,870 --> 00:21:53,460
tell me what you expect about
how much is protonated and how

318
00:21:53,460 --> 00:21:57,070
much is deprotonated at
physiological p h if

319
00:21:57,070 --> 00:22:36,190
the p k a is 10.

320
00:22:36,190 --> 00:22:57,480
All right, let's just take
10 more seconds.

321
00:22:57,480 --> 00:23:00,720
I hope that I mentioned
that acid base will be

322
00:23:00,720 --> 00:23:02,800
on the final exam.

323
00:23:02,800 --> 00:23:05,740
So good thing we're reviewing
it right now.

324
00:23:05,740 --> 00:23:13,850
All right, so now let's
look at the math.

325
00:23:13,850 --> 00:23:17,630
So if you're given a p k a and p
h and asked about a ratio of

326
00:23:17,630 --> 00:23:20,620
protonated to deprotonated
it's OK to pull out your

327
00:23:20,620 --> 00:23:23,820
favorite Henderson Hasselbalch
equation, which gives you a

328
00:23:23,820 --> 00:23:27,010
sense of the ratio, can predict
the ratio, again it's

329
00:23:27,010 --> 00:23:29,950
approximation, but predict the
ratio of protonated to

330
00:23:29,950 --> 00:23:30,070
deprotonated.

331
00:23:30,070 --> 00:23:34,300
H a, of course, being an
abbreviation for protonated, a

332
00:23:34,300 --> 00:23:36,440
minus for deprotonated.

333
00:23:36,440 --> 00:23:40,550
And so we can calculate here
that if you have a p h of 7 .

334
00:23:40,550 --> 00:23:45,220
4, p k a of 10, you get
a ratio of 400:1.

335
00:23:45,220 --> 00:23:48,150
So that's the math, but you can
also just think about it

336
00:23:48,150 --> 00:23:49,540
in terms of what you know.

337
00:23:49,540 --> 00:23:52,430
So let's just do
a brief review.

338
00:23:52,430 --> 00:23:54,560
So the answer here is that
free homocysteine is

339
00:23:54,560 --> 00:23:58,220
protonated and non-reactive
at physiological p h.

340
00:23:58,220 --> 00:24:00,260
But let's just think about this
question a little more.

341
00:24:00,260 --> 00:24:02,500
So this is not a figure in
today's handout, but you've

342
00:24:02,500 --> 00:24:04,710
seen this a few times,
so let's just

343
00:24:04,710 --> 00:24:06,400
look at it for a minute.

344
00:24:06,400 --> 00:24:09,140
So, we talked a lot about
acid based titrations.

345
00:24:09,140 --> 00:24:11,920
We talked about so here we have
a weak acid tritrated

346
00:24:11,920 --> 00:24:13,070
with a strong base.

347
00:24:13,070 --> 00:24:15,650
In the beginning it's a weak
acid problem, at the

348
00:24:15,650 --> 00:24:18,540
equivalence point it's a weak
base problem, because we've

349
00:24:18,540 --> 00:24:21,650
added enough moles of our strong
acid to convert all of

350
00:24:21,650 --> 00:24:24,580
our weak acid to its
conjugate base.

351
00:24:24,580 --> 00:24:27,800
And in the middle when you've
added half the number of the

352
00:24:27,800 --> 00:24:32,340
moles, you've converted half
of your weak acid to its

353
00:24:32,340 --> 00:24:34,990
conjugate, then the p
h is equal to the

354
00:24:34,990 --> 00:24:36,560
p k a at this point.

355
00:24:36,560 --> 00:24:38,300
So that would be right
in the middle of

356
00:24:38,300 --> 00:24:39,760
this buffering region.

357
00:24:39,760 --> 00:24:42,780
Again, in a buffering region
you have quite a bit -- you

358
00:24:42,780 --> 00:24:45,740
have quite a bit of your weak
acid and quite a bit of a

359
00:24:45,740 --> 00:24:47,880
conjugate, so it can buffer.

360
00:24:47,880 --> 00:24:51,630
If strong acid is added, then
it can be used up, if strong

361
00:24:51,630 --> 00:24:54,000
base is added, it can be used
up without changing

362
00:24:54,000 --> 00:24:55,170
the p h very much.

363
00:24:55,170 --> 00:24:59,180
That's a buffer, so the p h
curve is flat in here in that

364
00:24:59,180 --> 00:25:00,520
buffering region.

365
00:25:00,520 --> 00:25:05,310
And so, when you think about
this, if you're at p h's that

366
00:25:05,310 --> 00:25:09,580
are below the p k a, then
you're going to be more

367
00:25:09,580 --> 00:25:14,140
protonated, and p h is above
the p k a, you'll be more

368
00:25:14,140 --> 00:25:14,290
deprotonated.

369
00:25:14,290 --> 00:25:18,060
So let's look at the figure now
that's in today's handout

370
00:25:18,060 --> 00:25:19,440
and think about this.

371
00:25:19,440 --> 00:25:24,980
So when the p h equals the p k
a, you will have equal number

372
00:25:24,980 --> 00:25:30,090
of moles, of something that's
protonated as deprotonated.

373
00:25:30,090 --> 00:25:33,590
And if you're at p h's above
the p k a, you'll be more

374
00:25:33,590 --> 00:25:34,850
deprotonated.

375
00:25:34,850 --> 00:25:39,290
P h's below the p k a, you'll
be more protonated.

376
00:25:39,290 --> 00:25:43,300
And in the particular example
I gave, we have a p k a

377
00:25:43,300 --> 00:25:45,140
of 10, so at 7 .

378
00:25:45,140 --> 00:25:50,650
4 we're at a p h that is below
the p k a, and so here they'd

379
00:25:50,650 --> 00:25:52,200
be more protonated.

380
00:25:52,200 --> 00:25:54,600
So if you do the math
it's 400:1.

381
00:25:54,600 --> 00:25:57,810
But even if you aren't doing
any math, you should think

382
00:25:57,810 --> 00:26:01,950
about the fact that that would
have more of the protonated

383
00:26:01,950 --> 00:26:05,130
form than the deprotonated
form.

384
00:26:05,130 --> 00:26:09,160
All right, but the enzyme has
a problem, because only the

385
00:26:09,160 --> 00:26:11,630
deprotonated form is active.

386
00:26:11,630 --> 00:26:15,320
The p k a of the free
homocysteine is 10, and you're

387
00:26:15,320 --> 00:26:18,630
at physiological p h, so this
reaction doesn't seem

388
00:26:18,630 --> 00:26:19,980
like it should go.

389
00:26:19,980 --> 00:26:23,310
But again, it's an enzyme, and
enzymes catalyze reactions.

390
00:26:23,310 --> 00:26:26,630
So they do things to make
that reaction go faster.

391
00:26:26,630 --> 00:26:30,800
And what this particular enzyme
does is it lowers the p

392
00:26:30,800 --> 00:26:33,390
k a of homocysteine.

393
00:26:33,390 --> 00:26:37,110
So when homocysteine is bound
to the enzyme, it's p k a is

394
00:26:37,110 --> 00:26:42,460
no longer 10, but now
its p k a is 6.

395
00:26:42,460 --> 00:26:44,080
So, how does the
enzyme do this?

396
00:26:44,080 --> 00:26:46,590
Well, that's where the
zinc comes in.

397
00:26:46,590 --> 00:26:50,100
So, the zinc binds to the
homocysteine and it acts as a

398
00:26:50,100 --> 00:26:54,350
Lewis acid as it binds
to the homocysteine.

399
00:26:54,350 --> 00:26:56,620
So the homocysteine is your
donor ligand, and you have

400
00:26:56,620 --> 00:26:59,450
your metal as the Lewis
acid, your acceptor.

401
00:26:59,450 --> 00:27:05,290
And that alters the p k a, so
it actually associates, zinc

402
00:27:05,290 --> 00:27:09,580
associates with this sulfur,
and that changes the p k a.

403
00:27:09,580 --> 00:27:13,280
So, I just wanted to mention
again, I was having dinner

404
00:27:13,280 --> 00:27:16,390
with some of my faculty
colleagues who teach organic

405
00:27:16,390 --> 00:27:17,400
chemistry here.

406
00:27:17,400 --> 00:27:21,040
We were interviewing another job
candidate, and they looked

407
00:27:21,040 --> 00:27:23,290
at me and said, they're teaching
organic this semester

408
00:27:23,290 --> 00:27:25,950
and they said we asked our class
about p k a's, and they

409
00:27:25,950 --> 00:27:29,215
all insisted that they had never
heard about p k a's in

410
00:27:29,215 --> 00:27:30,740
their freshman chemistry
course.

411
00:27:30,740 --> 00:27:35,280
And I, of course, said, well,
they did not take 511-1 then.

412
00:27:35,280 --> 00:27:39,440
So just remember, especially
when it's Barbara Imperiali

413
00:27:39,440 --> 00:27:43,870
asking, did you hear about
p k a's, the answer is?

414
00:27:43,870 --> 00:27:45,130
STUDENT: Yes.

415
00:27:45,130 --> 00:27:46,450
PROFESSOR: Thank you.

416
00:27:46,450 --> 00:27:50,740
OK, so just this week.

417
00:27:50,740 --> 00:27:55,740
So it alters the p k a here.

418
00:27:55,740 --> 00:27:57,550
So now what's our situation?

419
00:27:57,550 --> 00:28:02,890
Well, now at physiological p h,
we have a p k a of 6, and

420
00:28:02,890 --> 00:28:05,230
so that gives us a very
different ratio.

421
00:28:05,230 --> 00:28:08,790
Instead of 400:1,
we have 1:25.

422
00:28:08,790 --> 00:28:13,560
So most of the homocysteine is
deprotonated at physiological

423
00:28:13,560 --> 00:28:17,110
p h, which means that
it can react.

424
00:28:17,110 --> 00:28:22,410
So, if we go back to this now,
our p k a is now 6, and so

425
00:28:22,410 --> 00:28:26,860
physiological p h is now
above that p k a.

426
00:28:26,860 --> 00:28:30,420
So when you're above the p
k a, you should have more

427
00:28:30,420 --> 00:28:32,460
deprotonated than protonated.

428
00:28:32,460 --> 00:28:37,700
So that's how you can
rationalize these things.

429
00:28:37,700 --> 00:28:41,340
All right, now I have to ask,
all right, we do not need a

430
00:28:41,340 --> 00:28:42,620
tie breaker.

431
00:28:42,620 --> 00:28:46,610
So at the end of the lecture,
we will announce the winner.

432
00:28:46,610 --> 00:28:51,000
But let's first do chemical
equilibrium.

433
00:28:51,000 --> 00:28:54,510
Chemical equilibrium.

434
00:28:54,510 --> 00:28:57,540
So we didn't talk too much
about this in chemical

435
00:28:57,540 --> 00:29:01,170
equilibrium, but enzymes can
have alternate conformation,

436
00:29:01,170 --> 00:29:05,150
so the enzyme can change its
shape during chemistry.

437
00:29:05,150 --> 00:29:08,740
And that those conformation
of the enzyme can be in

438
00:29:08,740 --> 00:29:09,760
equilibrium.

439
00:29:09,760 --> 00:29:13,220
So the enzyme itself can be an
equilibrium with different

440
00:29:13,220 --> 00:29:16,960
conformational states.

441
00:29:16,960 --> 00:29:19,240
So here, there are a lot of
states of the enzyme.

442
00:29:19,240 --> 00:29:20,870
It needs to react with
homocysteine, it needs to

443
00:29:20,870 --> 00:29:22,760
react with
methyltetrahydrofolate and

444
00:29:22,760 --> 00:29:23,610
with s adenosylmethionine.

445
00:29:23,610 --> 00:29:30,360
And when the structure was
determined to this enzyme,

446
00:29:30,360 --> 00:29:33,360
here in green is the vitamin
B12, and red

447
00:29:33,360 --> 00:29:34,790
is the methyl group.

448
00:29:34,790 --> 00:29:36,980
You see that the methyl group
is pretty buried, you can

449
00:29:36,980 --> 00:29:39,970
barely see it, but yet it
needs to interact with

450
00:29:39,970 --> 00:29:42,790
homocysteine to give it a methyl
group, it needs to take

451
00:29:42,790 --> 00:29:45,470
a methyl group off
methyltetrahydrofolate, it

452
00:29:45,470 --> 00:29:47,370
also needs to take a
methyl group off s

453
00:29:47,370 --> 00:29:48,730
adenosylmethionine.

454
00:29:48,730 --> 00:29:50,780
But it doesn't seem like there's
any room for any of

455
00:29:50,780 --> 00:29:52,740
them to actually get in there.

456
00:29:52,740 --> 00:29:56,080
So, this was a sign that there
has to be some kind of change

457
00:29:56,080 --> 00:29:57,360
in the structure.

458
00:29:57,360 --> 00:29:59,680
So here's another picture
of the structure.

459
00:29:59,680 --> 00:30:02,090
Here's the vitamin B12 in green,
the methyl group in

460
00:30:02,090 --> 00:30:05,400
red, and there's this whole
protein part up here that has

461
00:30:05,400 --> 00:30:08,350
to get out of the way
to do the chemistry.

462
00:30:08,350 --> 00:30:11,550
And, in fact, we do know from
experimental data that it does

463
00:30:11,550 --> 00:30:14,720
move so you can do
the chemistry.

464
00:30:14,720 --> 00:30:17,780
So that means that the enzyme
has to have a number of

465
00:30:17,780 --> 00:30:19,130
different structures.

466
00:30:19,130 --> 00:30:20,720
It's modular.

467
00:30:20,720 --> 00:30:23,950
Here's the vitamin B12 binding
domain, the vitamin B12,

468
00:30:23,950 --> 00:30:27,130
there's this region that have to
move called the methyl cap.

469
00:30:27,130 --> 00:30:30,060
It needs to interact, the B12
here needs to interact with a

470
00:30:30,060 --> 00:30:32,680
folate domain, a homocysteine
domain, and

471
00:30:32,680 --> 00:30:35,530
an activation domain.

472
00:30:35,530 --> 00:30:39,250
So you need to have at least
these four different

473
00:30:39,250 --> 00:30:42,490
structures, and these four
structures will be in

474
00:30:42,490 --> 00:30:45,010
equilibrium with each other.

475
00:30:45,010 --> 00:30:47,680
So you need to have a structure
with folate, a

476
00:30:47,680 --> 00:30:51,340
binding domain on top of the
red B12, you need to have

477
00:30:51,340 --> 00:30:55,050
homocysteine in yellow on top of
the B12, you need to have a

478
00:30:55,050 --> 00:30:58,530
resting state where those
helices are on top of the B12,

479
00:30:58,530 --> 00:31:00,660
and you need to have an
activation state where the

480
00:31:00,660 --> 00:31:04,450
ado-meth domain is on top of
the B12, and all of these

481
00:31:04,450 --> 00:31:07,810
states are going to be in
equilibrium with each other,

482
00:31:07,810 --> 00:31:10,100
and they're going to be moving,
which means that

483
00:31:10,100 --> 00:31:12,880
enzymes are dynamic,
which means that

484
00:31:12,880 --> 00:31:15,960
chemistry is dynamic.

485
00:31:15,960 --> 00:31:19,580
Chemistry in the body is
not in the solid state.

486
00:31:19,580 --> 00:31:24,170
Chemistry in the body
is in solution.