10/25/21

Lecture 18 (10/25/21)
Enzyme Regulation
“Enzyme” Regulation: Hemoglobin
1. Introduction
1. Roles of Hb
2. Strategies a. Oxygen transport
a. Gene Regulation b. CO2 binding
b. Covalent Modification c. Blood buffer: Bohr effect
1. Xx
2. Xx c. Allosteric Control 2. Oxygen Binding/role of protein
3. Covalent Modification 3. Binding curves
1.Proteolysis a. oxygen
2.Protein modification b. Allosteric effectors (BPG)
i. Phosphorylation c. Bohr effect; (protons)
a) Kinases d. Carbon dioxide
b) Phosphatase 4. Structure-Function; Structural basis for
c) Example: PKA
1.
2.
3.
X
X
X
physiology (T & R states)
4.Allosteric Control 5. Mechanism of Cooperativity
a.Regulation nomenclature • Reading: Ch5; 157-160
i. Example: ATCase Ch6; 213-220
b.Review; binding curves; why sigmoidal
Ch5; 148-149, 152-
c.Hill Equation
d.Physical models; 3° and 4° conf. changes 157, 160-164
i. To vs. Ro • Problems: Ch5; 7,8,9,11
ii.Sequential; KNF
iii.Concerted (symmetry); MWC NEXT
iv.Examples of measuring these parameters
• Reading: Ch4; 128-136
• Problems: Ch4; 13,18
Ch6; 1,4

Hemoglobin (Hb)
Best understood example of an allosteric protein
Evolution of oxygen transporters Hb and myoglobin (Mb):
Ø Serum [Oxygen] ≈ 2.3 mL/L; Blood [Oxygen] ≈ 200 mL/L
Ø Metabolism:
Needs O2 à C6H12O6 + 6O2 D 6CO2 + 6H2O
Oxidation of sugars à metabolic acids and from CO2 + H2O D H2CO3

Roles of Hb:
Ø Oxygen transport
Ø Proton transport-Blood buffer
Ø Carbon dioxide transport 4

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Hemoglobin & Myoglobin

in O2 & CO2 Transport

Hemoglobin & Myoglobin

in O2 & CO2 Transport
(and buffering)

pO2 = 100 torr pO2 = 20 torr

• •
CO2

Carbonic anhydrase Carbonic anhydrase

CO2

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Oxygen transport

Heme
methyl, vinyl & propionate

pyrole
methenyl

Heme
protoporphyrin IX

• Heme is protoporphyrin IX plus Fe2+ Fe2+

• Heme prosthetic group in globins binds oxygen via Fe2+
• Heme itself is not a good oxygen transporter because of
oxidation to Fe3+
7

Oxygen transport
Myoglobin (Mb): O2-binding protein
Val E11 (68)
Phe CD1Histidine
(43) E7 (64)
(Distal)

Histidine F8 (93)
(proximal) Carbon monoxide
Hydrophobic
microenvironment- binds heme 20,000
prevent oxidation better than dioxygen,
to ferric-Fe(III). but in Mb/Hb it only
Provides site for binds 200 times
6th ligand better.
Limits binding by
other analogs
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Oxygen transport

Quantitative measure of O2 binding

[O2 ] Þ pO2
K D Þ p50

M O2 ⇌ MMbbO+2 O2
Mb + O2b
Kd
[Mb] [O2] [Mb] [O2]
Kd = [MbO2] =
[MbO2] Kd
Same as : [Mb] [O2]
[ L] [MbO2] Kd [O2]
Y= YO2 = = =
K D + [ L] [MbO2] + [Mb] [Mb] [O2] Kd + [O2]
+ [Mb]
Kd

Oxygen transport

Oxygen Binding Curve of Hemoglobin

20-30 torr 100 torr

Mb à Kd= 3-4 torr

Hb à Kd= 26 torr

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Cooperativity: Hill coefficient

Hb + n O2 -1
Hb(O2)n
Kd
[Hb] [O2]n (pO2)n
Kd = YO2 =
[Hb(O2)n] (p50)n + (pO2)n
• Positive cooperativity:
(pO2)nn > 1

• Negative
Y O2
cooperativity:
(p50)n + (pOn2)n< 1 (pO2)n
• Non-cooperative:
1-Y
= n=1 n =
(p50)n
O2
(pO2)
1- n n
(p50) + (pO2)
• Theoretical maximum cooperativity = # of
binding sites
Y O2
_
log = n log pO2 n log p50
1 - YO2 11

Cooperativity: Hill coefficient

Hb + n O2 -1
Hb(O2)n
Kd
[Hb] [O2]n (pO2)n
Kd = YO2 =
[Hb(O2)n] (p50)n + (pO2)n
• Positive cooperativity:
(pO2)nn > 1

• Negative
Y O2
cooperativity:
(p50)n + (pOn2)n< 1 (pO2)n
• Non-cooperative:
1-Y
= n=1 n =
(p50)n
O2
(pO2)
1- n n
(p50) + (pO2)
• Theoretical maximum cooperativity = # of
binding sites
Y O2
_
log = n log pO2 n log p50
1 - YO2 12

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Hill Plot: Myoglobin & Hemoglobin

for Mb & Hb

Eqn for line à y = ax + b

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Mb and Hb-subunits: structural

similarity, differential binding

• Mb, Hb-a, Hb-

b overlay

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Structure of Hemoglobin (Hb)

b2 b1

a2 a1

Tetramer has 2a and 2b subunits

But, the a & b have more affinity for each other than either aa or bb
Therefore, Hb is best described as a dimer of ab dimers = (ab)2
Notice nice pocket at b-b interface

This view is looking down from

the top

Structure of Hemoglobin (Hb)

Changes during binding!
De-oxy Hemoglobin Oxy Hemoglobin

a2 b1 a2 b1

b2 a1 b2 a1

Lets look at this

conformational change from
Notice residue 97 (His) the top of one ab dimer
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Structure of Hemoglobin (Hb)

b2 b1

a1 a2

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What binds to Hb in addition to O2?

1.

2. Protons

3. Carbon Dioxide

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Structure of BPG

BPG Binds to Deoxyhemoglobin

a2 b1 a2 b1

b2 a1 b2 a1
Binds to deoxy-Hb Does not bind to oxy-Hb

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BPG Binds to Deoxyhemoglobin

b1

b2

[BPG] is 5 mM in blood cells

What binds to Hb in addition to O2?

1.

2. Protons

3. Carbon Dioxide

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Proton transport-Blood buffer

Bohr Effect: pH dependence of O2 binding

Christian Bohr-1904

Hb H+ + O2 Hb O2 + H+

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What binds to Hb in addition to O2?

1.

2. Protons

3. Carbon Dioxide

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Carbon Dioxide binds via Carbamate

formation

Carbon dioxide binds better to the form of

Hb that is not bound to oxygen; deoxy-Hb
Therefore, oxygen binding releases CO2,
and CO2 binding releases oxygen

–
H+ 2+ O2
Hb CO H+ 2
Hb O2 + CO

Consequences of carbamate:
charge change
contributes to acidity: when CO2 increases, carbamate
formation increases, which is conducive to the Bohr effect

CO2 Binds to Deoxyhemoglobin

b2 b1

a1 Arg-141
(of a 2)
Arg-141
(of a 1) a2
Val-1 26 –
Val-1 & Arg-141 further away
Val-1 (of a1) weaker electrostatic bond
(of a2)

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What binds to Hb in addition to O2?

1.

2. Protons

3. Carbon Dioxide

So, BPG, protons, and CO2 bind specifically to deoxy-Hb.

Do these stabilize a possible T-state? Lets look at these states more closely….

T-state and R-state of Hemoglobin

a2 b1

b2 a1

• T state • R state
• Less active • More active
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R and T States of Hemoglobin

(146)

(40)

Look at where this

His-146 (HC3) is
after binding of O2
(R-state).

+
InfluencesHon T→R Transition
H

+
b
H

O2
+

BPG
b +
+

+
H
H +H
H
O2
H

+
b

O2 O
H

2CO2
O 22 BPG
+

BPG O O
2+H O
Hb H+ +HHb H+ O2 + H+
2 2

O2 Ob 2 H+ + Hb b2 b1 2

O2 O2 + Hb
+

H +H
+
H

a2 a1
+
O2

+ CO CO
2 2
BPG
O2

H BPG
2CO2
b 2+H
+

H O 2
H
H

+ CO
+

+ CO
b

2 2
O2

HT-state R-state
b
+

H Recall: L ( [To]
[Ro]
) = 300,000
H

and
+

C ( [K R]
[KT]
) = 0.01

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+
InfluencesHon T→R Transition

H
+
b
H
O2
+
BPG
b +
+
+
H
H +H
H
O2
H

+
b

O2 O
H

2CO2
O 22 BPG
+

BPG O O
2+H O
+ b + b2
H+ O2 + H+
b1 2 2

O2
2
Hb H +HHb
O2 H + Hb O2 O2 + Hb
+

H +H
+

H
a2 a1
+
O2

b
+ CO CO
2 2
BPG

O2
H BPG
2CO2
b 2+H

+
H O 2

H
H

+ CO
+
+ CO
b

2 2
O2

HT-state R-state
b
+

H Recall: L ( [To]
[Ro]
) = 300,000
H

and
+

C ( [K R]
[KT]
) = 0.01

Effects of BPG & CO2 on

Hb’s O2 Dissociation Curve

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O2 binds to Hb

1.

2. Protons

3. Carbon Dioxide

How are all these related to the cooperative

binding mechanism ?
Lets look at binding of oxygen to the T-state, which its most likely to encounter
(L=300,000).

Structural Basis of Cooperativity

Helix F

Deoxy High-spin (paramagnetic)(larger) Deoxy

Oxygen bound Oxygen bound
Low-spin (diamagnetic)(smaller with Fe-N bonds 0.1 Å shorter)

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Conformational Change Is Triggered

by Oxygen Binding

Structural Basis of Cooperativity

What causes the

conformational
change?

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Structural Basis of Cooperativity

Changes in H-bonds
when an oxygen binds Changes within
to a single subunit bY145
each subunit
b The change at the F-
V98
helix changes the C-
aV93
term stability within
each subunit

a
Y140

Changes
between dimers
(a1 and b2)
The change
at the F-helix
changes the
bD99 C-term
stability
b between
N102
aY42 subunits
a
D94

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Structural Basis of Cooperativity

Changes in H-bonds
when an oxygen binds Changes within
to a single subunit bY145
each subunit
b The change at the F-
V98
helix changes the C-
aV93
term stability within
each subunit

a
Y140

Changes
between dimers
(a1 and b2)
The change
at the F-helix
changes the
bD99 C-term
stability
b between
N102
aY42 subunits
a
D94

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Structural Basis of Cooperativity

Asp94
Asp99

Asn102

Tyr42

a2 b1 a2 b1

b2 a1 b2 a1

Notice residue 97 (His)

Human Deoxyhemoglobin &

Human Oxyhemoglobin
PDBids 2HHB & 1HHO 40

Structural Basis of Cooperativity

Asp94
Asp99

Asn102

Tyr42

How do these slight

changes due to movement
of the F-helix upon O2
binding destabilize the C-
termini and lead to
conformational change?
Recall H-bonds broken
(aY140 & bY145)

Human Deoxyhemoglobin &

Human Oxyhemoglobin
PDBids 2HHB & 1HHO 41

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Carbamate formation favors the T-state

Changes at C-term of a1 Recall H-bonds broken
(aY140)

H+–2+ O2
Hb CO H+ 2
Hb O2 + CO

V-FG5
Changes between dimers (Salt-
bridge interactions at C-term of a1)

Recall, this is the

first thing we
saw destabilized

Protonation favors the T state

Changes at C-term of b2

Hb H+ + O2 Hb O2 + H+ Recall H-bonds broken

(bY145)

Changes between dimers (Salt-

bridge interactions at C-term of b2)

V-FG5

Recall, this is the

first thing we
saw destabilized

The Bohr effect: when

these interactions are
But, what actually causes the dimers to rotate the 15°? lost, the proton is
released (pKa drops)

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Oxygen-Binding affects Bonds to

C-terminus

• Hemoglobin Dynamics at C-term of beta-subunit

See:
• O2 binds
• The salt-bridge between His-146 and Asp-94 on the same b-subunit breaks
• The salt-bridge between the C-term carboxylate of b-subunit loses contact with
Lys-40 of a-subunit
• “Anchor” is lost and subunits move

DON’T See:
• Fe moving into plane of heme when O2 binds
• Helix F and FG loop moving when His-91 (F8) on helix-F moves
• H-bond with Tyr-145 on and Val-98 (on FG loop) on b-subunit breaking
• NONE of the comparable changes at the C-term of the a-subunit, due to binding the b-subunit
• E.g., the H-bond between the Asp-99 of b-subunit and Tyr-42 of a-subunit breaking

• The T- and R- states of Hb

• O2 binds b in gray
• The salt-bridge between His-146 and Asp-94 on the same b-subunit breaks a in blue
• The salt-bridge between the C-term carboxylate of b-subunit loses contact with Lys-40 of a-subunit
• “Anchor” is lost and subunits move

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Summary of changes in Hb T-state

to R-state

Fetal Hemoglobins
Hb is always a tetramer of a-type and b-type subunits (a-b)2

The g-subunit of HbF does not bind BPG as well as the b-subunit

This shifts the TßàR equilibrium to the right.

This shifts the oxygen binding curve to the left

such that at all pO2, HbF
binds oxygen better than HbA

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High altitude adaptation

[BPG] goes up
to 8 mM
This shifts the TßàR equilibrium
to the left.

This shifts the oxygen binding

curve to the right such that at all
pO2, Hb binds oxygen worse and
compensates for the lower pO2 at
high altitudes.
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