Reginald H.

Garrett
Charles M. Grisham
[Link]/chemistry/garrett

Chapter 15
Enzyme Regulation

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Essential Questions

• How do enzymes sense the momentary needs

of cells and change activity accordingly?

• What molecular mechanisms are used to

regulate enzyme activity?

2
 What Factors Influence Enzymatic Activity
in Cells?
1. Rate of enzyme synthesis and decay
Transcription – eg., lac operon
Translation – eg., Shine-Dalgano sequence,
Kozak sequence
Post-translation – eg., ubiquitination, sumoylation

The response time may take hours in higher

eukaryotes.

What Factors Influence Enzymatic Activity

in Cells?
2. Concentration of substrates and cofactors

The KM value and enzyme concentration commonly

approximate the concentration of the substrate in
vivo

Not enough cofactors  low enzyme activity

4
 What Factors Influence Enzymatic Activity
in Cells?
3. Product accumulation and removal affect the
reaction rate – (a) many enzymes catalyze
reversible reactions; (b) products may exert
feed-back inhibition enzymes

A B

A B C

What Factors Influence Enzymatic Activity

in Cells?
4. Enzyme regulation by “reversible” covalent
modification (e.g., acetylation, methylation,
phosphorylation)
“irreversible” modification is more energy costly

The phosphorylation residues may be at

Ser/Thr, Tyr
His, Asp (primarily in bacteria and fungi)

6
 What Factors Influence Enzymatic Activity
in Cells?
5. Activation of zymogens (= inactive precursors of
enzymes), typically through proteolytic cleavage to
form an active enzyme)

Example 1 - Proteolytic Enzymes of the Digestive

Tract

The proteolytic activation of chymotrypsinogen

8
 What Factors Influence Enzymatic Activity
in Cells?
5. Activation of zymogens
Example 2 - The Cascade of Activation Steps Leading to Blood
Clotting
Mostly serine proteases
Activated by traumas Factors are in the tissue
inside the vascular
system. Factors are in
the blood. Shorter pathway

EC

The Cascade of Activation Steps Leading to Blood

Clotting

Activated by thrombin
As a cofactor in Xa complex

Conversion of fibrinogen into fibrin, which aggregates into

ordered filamentous arrays that crosslink to form the clot.

10
 What Factors Influence Enzymatic Activity
in Cells?

Example 3 - activation of insulin

Preproinsulin: with a signal peptide

Proinsulin: an 86-residue precursor to
insulin

Manufacturing insulin is not a simple

task. Why?

11

What Factors Influence Enzymatic Activity

in Cells?
６. Regulation through forming different isozymes eg.,
lactate dehydrogenase
More efficient in
pyruvate  lactate
(NAD regeneration)

More efficient in
lactate  pyruvate
(for fuel)
12
 What Factors Influence Enzymatic Activity
in Cells?
７. Allosteric regulation - Binding of small effector molecule
non-covalently to domain other than the active site.
[Link]

13

General Features of Allosteric Regulation

1. A common mechanism of enzyme regulation

2. Effectors can be activators or inhibitors
3. The effector acts at a site different from catalytic
site
4. Effectors may be feed-forward activators or
feedback inhibitors
5. The enzyme “typically” have an oligomeric
structure, each subunit has one substrate and one
effector binding site The effectors are usually
produced elsewhere in the pathway.

19
Non-competitive inhibition vs. Allosteric
regulation
Competitive Noncompetitive Uncompetitive

What is the difference between allosteric regulation

and non-competitive regulation?

20

Non-competitive inhibition vs. Allosteric

regulation
Allosteric regulation:
Any enzyme activity regulation in which the effector
acts at a site different from the catalytic site
Include both single- and multi-subunit enzymes
Interplay between subunits may occur
e.g. substrate binding may be cooperative
May or may not follow Michaelis-Menten kinetics
Non-competitive inhibition:
A subtype of allosteric regulation
Discuss primarily “inhibition”
Use “single subunit” as a model
Follow Michaelis-Menten kinetics

21
Non-competitive inhibition vs. Allosteric
regulation
Non-competitive inhibition
1. Vmax decreases; Km unchanged
2. Follow typical hyperbolic M-M kinetics
3. Reciprocal plots are linear

Allosteric regulation “discussed in this lecture”

Multi-subunit enzymes
Two different substrate-binding states
1. Vmax remains unchanged.
2. Does not obey M-M kinetics. Results in sigmoid
kinetics. Reciprocal plot is not linear.

22

General Features of Allosteric Regulation

v is proportional to
[S]n, where n >1
Hill coefficient
Positive
cooperativity

In the regions, v is very

sensitive to [S]

Dotted line: normal Michaelis-Menten kinetics

Red line: allosteric reaction

23
 Why is allosteric regulation important?

It permits a more dynamic and complex control of

biochemical activity using identical enzymes thereby
conserving its resources.

Change the enzyme activity more readily to meet the

availability of the substrate and physiological needs

24

Allosteric Regulation – Two Models, Both

Involved Conformational Changes
1. MWC, Concerted, or Symmetry Model
Monad & Jacob, 1963;
Monod, Wyman, Changeux, 1965

2. KNF or Sequential Model

Pauling, 1935
Koshland, Nemethy, and Filmer. 1966

Both models can be used to explain oxygen binding in

hemoglobin (discussed later in this chapter)

25
Jacob, foreground, and Jacques Monod in their lab at
the Pasteur Institute in Paris in 1971
26

27
28

1. Symmetry (Concerted, MWC) Model

- Allosteric enzymes exist in two states:

R (relaxed) and T (taut or tense)
- All the subunits of an oligomer are in the same state
- T-state predominates in the absence of substrate S
[T] >> [R]
- S binds primarily to R

29
1. The Symmetry Model
k

S and A bind R
to increase [R]
I binds T to
increase [T]
The Ligand-induced
conformational change is ignored
Ｔwo interchangeable enzyme conformational states: R (relaxed) and T
(taut or tense)
- All the subunits of an oligomer are in the same state
- T state predominates in the absence of substrate S
- S and A (activator) bind primarily to the R state. S and A binding favors
more S binding (cooperative).
30

1. The Symmetry Model

Allosteric effects
Binding A (or S) to R
 increase [R]
 increase S binding sites
 decrease in cooperativity
(K0.5) of S saturation curve

K0.5 = [S] giving ½ maximal

response

Negative effector I raises K0.5

S is a positive “homotropic” effector, A is a “heterotropic” effector.

The model explains primarily positive cooperativity
31
2. Koshland, Nemethy, and Filmer (the KNF or
sequential) model
Ligand binding triggers a conformation change in a
protein, which leads to conformation changes in
adjacent subunits

Explains how ligand-induced conformation changes

could cause subunits to adopt conformations with
little affinity for the ligand
 Both positive cooperative and negative
cooperativity can be explained

32

2. The Sequential Model

(a) S binding can, by induced

fit, cause a conformational
change in the subunit to
which it binds

(b) Binding of S to one subunit

may cause the other subunit
to assume a conformation T R
having a greater or lesser
affinity for S

33
 2. The Sequential Model

Relative velocity

Binding of a ligand to
an enzyme having four
identical subunits,
each with one binding
site for the ligand.

Relative [S]
34

Difference between MWC and KNF models

• Symmetry model: (1) imposes functional and
structural symmetry (= all subunits of the oligomer
share the same structure/state); (2) Focus on
conformational equilibrium and population selection
by the ligand ; (3) Ignore ligand-induced
conformational changes
e.g., glycogen phosphorylase
• Sequential model: (1) postulates asymmetry in the
partially liganded enzyme; (2) focus on ligand-
induced conformation changes; (3) ignore
conformational equilibrium
e.g, tyrosyl tRNA synthetase

35
 Example of allosteric enzymes
Some allosteric enzymes contain a regulatory subunit
1. Glucokinase (GK): controls blood glucose
homeostasis
fructose-1-phosphate  GKRP  GK
2. Ribonucleotide reductase: controls NTPs and
dNTPs balance
Effectors: NTPs/dNTPs
３．Aspartate Transcarbamoylase (ATCase)
Effectors: ATP/CTP
Some of the regulatory proteins may be subject to
covalent modification to modulate their activity

36

Glucokinase and Its Regulatory Protein

37
 Example of allosteric enzymes
high affinity
Aspartate Transcarbamoylase

38

Covalent Modification Regulates the Activity of

Enzymes
• Reversible phosphorylation is the most prominent
form of covalent modification of enzyme in cellular
regulation
• Accomplished by distinct protein kinases and
phosphatases
• ~500 protein kinases in humans
• Kinases and phosphatases themselves are common
targets of regulation

42
 Effects of Protein Phosphorylation

Conformational changes, Enzyme activity, Stability,

Interaction, Subcellular localization, Activation of a
Pathway, …

43

Covalent Modification Regulates the

Activity of Enzymes
• Common phosphorylation sites in enzyme: Ser, Thr, and
Tyr residues (Asp/His in bacteria) in target proteins

• Adding 2 negatively charged groups to protein

• All kinases share a common catalytic mechanism based
on a conserved core kinase domain of ~260 residues
• Target phosphorylation sequence is specific (may or
may not be contiguous sequence)
44
 Protein Kinases and Phosphatases

45

Protein Kinases and Phosphatases

46
 Protein Kinases and Phosphatases

47

48
 Eight Families of Eukaryotic Protein Kinases
• Classified by Sequence Similarity)

49

Eight Families of Eukaryotic Protein Kinases

50
 The Activity of Some Kinases Are Regulated by
Intrasteric Control.
• Certain important kinases are regulated by
intrasteric control, in which a regulatory subunit (or
domain) has a pseudosubstrate sequence that
mimics the target sequence but w/o the
phosphorylatable residue (ie., not ser or thr)
• E.g., cyclic AMP-dependent protein kinase (aka.
protein kinase A, PKA)
Pseudo
peptide Target seq.

Substrate
Enzyme

51

cAMP-Dependent Protein Kinase (PKA)

• PKA phosphorylates many important metabolic
enzymes. For example:
- acetyl-CoA carboxylase
- pyruvate dehydrogenase
- glycogen phosphorylase

 Regulate lipogenesis, glycogen formation, and

gluconeogenesis…

52
Structure of cAMP-Dependent Protein
Kinase
PKA is a 170-kD R2C2 tetramer in mammalian cells.
C: catalytic; R: regulatory subunits

The two R subunits bind two cAMP each (Kd ~ 30-300 nM);
cAMP binding releases the R subunits from the C subunits.
C subunits are enzymatically active as monomers.
*Recent literatures argue against this C-R separation model

53

Structure of cAMP-Dependent Protein Kinase

A C2R2 tetramer.
Red: C subunits
Blue: R subunits

C-R Interacting site

54
 Autoregulation of cAMP-dependent Protein
Kinase
Protein kinase A is shown
complexed with a pseudosubstrate
peptide (orange).
ATP (red) and two Mn2+ ions
(yellow) bound at the active site.

Pseudosubstrate in R (RRGAI)
binds the active site of C

The sequence is similar to the target

sites of PKA (RRGSI)

55

Other Forms of Covalent Modification that

Regulate Protein Function
• Several hundred different chemical modifications of
proteins have been discovered

• Only a few of these are used to achieve metabolic

regulation through reversible conversion of an
enzyme between active and inactive forms

60
 Forms of Covalent Modification that Regulate
Protein Function

Also Ser and Thr

Many aa

61

Examples:
GTPases; Gln dehydrogease

Wikipedia
62
 • Many bacterial toxins are ADP-ribosyltransferase
• Altered activity, subcellular localization, protein-
protein interaction…
• Apoptosis, gene regulation at any level, DNA repair
• Poly(ADP-ribose)polymerases (PARPs) are
important cancer therapy targets
63

Acetylation in Enzyme Regulation

• A prominent modification for the regulation of
metabolic enzymes
• Acetylation of an ε-NH3+ group on a Lys residue
changes it from a positively charged amino group to
a neutral amide
• This change may have consequences for protein
structure and thus functions
• Controlled by acetyl-CoA-dependent lysine
acetyltransferase (KAT) and KDACs (lysine
deacetylases)
• More than 30 KATs are known in mammals

64
 Lysine Acetylation

Nonenzymatic
process

65

Lysine De-Acetylation

Nicotinamide +

66
 Acetylation in Enzyme Regulation
• Can be detected by MS-Spectrometer
• Nearly all enzymes involved in glycolysis,
gluconeogenesis, the TCA cycle, fatty acid oxidation,
the urea cycle, nitrogen metabolism, glycogen
metabolism, oxidative phosphorylation, and amino
acid metabolism are regulated by acetylation.
• Acetylation activates some enzymes and inhibits
others
• Cellular levels of major metabolic fuels such as
glucose, fatty acids, and amino acids influence the
degree of acetylation

67

Acetylation in Enzyme Regulation

• Malate dehydrogenase (MDH) activity is regulated
by acetylation
• Glucose enhances MDH acetylation

Inhibitor of KDAC glucose

68
Most sirtuins are NAD+-dependent KDAC that
implicated in energy metabolism and longevity

• [Link]

69

• Sirtuins, mTOR, and AMP (activated protein) kinase

are major players in aging. (mTOR is a Ser/Thr
kinase. A rapamycin target)

70
 NMN

MAPK

71

NMN NAD
Genetically modified mice with enhanced sirtuin gene expression
have shown extended lifespan. Also related to fastening.
72
 Some Enzymes Are Controlled by Both Allosteric
Regulation and Covalent Modification
• Glycogen phosphorylase (GP) cleaves glucose units
from nonreducing ends of glycogen
• Form readily usable fuel (glc-1-phosphate)
• A phosphorolysis (lysed by the attack of phosphate)
reaction
• Muscle GP is a dimer of identical subunits, each with
a covalently linked pyridoxal phosphate
• There is an allosteric effector site at the subunit
interface

76

GP Reaction

77
 Phosphoglucomutase converts glc-1-P into the
glycolytic substrate, glc-6-P

Required for polysaccharide, Muscle: glycolysis

proteoglycan, and glycogen Liver: glucose exportation
synthesis, galactose utilization

78

The structure of glycogen phosphorylase

dimerization
• A homodimer
• Each subunit Also for ATP
contains an
allosteric effector
site near the subunit
interface (a “tower
helix, residues 262
to 278).
• A regulatory
phosphorylation site
is located at Ser14

Assist Pi protonation
79
 Glycogen Phosphorylase Activity is Regulated
Allosterically

• Different GP isozyme in different tissues

• Muscle GP shows cooperativity in substrate binding
• ATP and glc-6-P are allosteric inhibitors
• AMP is an allosteric activator
• When ATP and glc-6-P are abundant, glycogen
breakdown is inhibited
• When cellular energy reserves are low (i.e., high
[AMP] and low [ATP] and [glc-6-P]), glycogen
catabolism is stimulated

80

Glycogen Phosphorylase Activity is Regulated

Allosterically
Sigmoid curve

v versus [S] curves for glycogen phosphorylase.

(a) The response to the concentration of the substrate (Pi).
(b) ATP is a feedback inhibitor.
(c) AMP is a positive effector. It binds at the same site as ATP.

81
 Glycogen phosphorylase conforms to the
MWC model
• Active form (R state) Inactive
form （T state）
• AMP promotes the
conversion of T to R
• ATP, glc-6-P, and caffeine
favor the conversion of R to T
• A conformation difference at
the active site between the T
and R states:
T state (Asp283; repels Pi)
R state (Arg569; favors Pi)

82

Glycogen Phosphorylase is Controlled by Both

Allosteric Regulation and Covalent Modification
The enzyme is allosterically
activated by AMP and inhibited
by ATP and glc-6-P.

When abundant ATP is needed

urgently, phosphorylation
converts the enzyme from
b form: less-active, allosterically
regulated
to
a form: more active,
allosterically unresponsive form

83
 Regulation of GP by Covalent Modification

• In 1950s, Edwin Krebs and Edmond Fischer

showed that phosphorylase kinase converted
phosphorylase b to phosphorylase a through
covalent phosphorylation

• 1992 Nobel Prize in Physiology and Medicine

84

Dimeric form
-AMP (yellow)
-Ser14 (orange)
-Glycogen binding (blue)
-Catalytic site (red)

Ser14 Phosphorylation
 aa 10 to 22
disordered
 -helix

85
 A Conformation Change Regulates Activity of
Glycogen Phosphorylase
N-terminal conformation of Ser14
(red) phosphorylated (a form,
yellow) and unphosphorylated
( b form, cyan) phosphorylase
 [from disordered into -helix]
 Rotation of the tower helix
tower
and subunit in the dimer
 Better access of the active
site
 Enhance ~25% activity

Phosphorylation converts the

enzyme from allosteric Glycogen
regulation to constantly binding
activation form site
Active site

86

Summary of glycogen phosphorylase

regulation
• Allosteric effector binding affects active site structure
T state (Asp283; repels Pi)
R state (Arg569; favors Pi)
• Reversible phosphorylation results in significant N-
terminal conformation changes, which in turn affects
the accessibility of the active site

87
 Regulation of glycogen, sugar, and lipid
metabolism
7 TM domains

92

The Adenylyl Cyclase Reaction

The reaction is driven forward by subsequent hydrolysis of

pyrophosphate by the enzyme inorganic pyrophosphatase.

94
 The Adenylyl Cyclase Reaction

The reaction is driven forward by subsequent hydrolysis of

pyrophosphate by the enzyme inorganic pyrophosphatase
95

cAMP is a 2nd messenger - intracellular

agent of extracellular hormones
Ligand binding activates the
guanine nucleotide exchange
activity of GPCR

GEF

96
 GPCR are major drug targets
 100 G protein coupled receptors (GPCR, guanine nucleotide
exchange factors (GEF)) and > 21 G are known
 Different Gcoupled to different protein and exert a different
function: Metabolism, secretion, proliferation, invasion and
motility

97

Examples of drugs target to GPCR

98
 Myoglobin (Mb) and hemoglobin (Hb)
• Exemplifies the relationship between quaternary
structure (multi-subunits) and allosteric regulation
(cooperate binding)

• Both are heme-binding proteins

• Hb: oxygen-transport, tetrameric (in adults, two α

chains of 141 residues each, 2 β chains of 146
residues), must bind oxygen in lungs and release it
in capillaries of peripheral tissues

• Mb: oxygen-storage, monomeric, 153 aa, has a

greater affinity for oxygen at all oxygen pressures

103

pO2 - reflects the amount of oxygen gas

dissolved in the blood

in arterial blood: 75 - 100 mmHg (torr).

in venous blood: 30 - 40 mmHg

104
 1. Why % (oxygen saturation)? Why not using pO2 ?
2. Why 2% difference is critical?
3. What is the relationship between % (oxygen
saturation) and pO2
92% O2 saturation (pneumonia) = pO2 ? torr
pO2 200 Torr (w/ oxygen mask) = ?%O2 saturation

105

Pulse oxymetry

• [Link]

106
 Pulse oxymetry

Measured w/ 2 wavelengths
Ratio of the 2 signals reflects
the oxygenation state
Provide % O2 saturation data
(pO2 requires blood drawing)

107

%O2 saturation vs. pO2

Not a linear relationship
90% O2 saturation = pO2 ~60 torr; 94% O2 saturation = pO2 ~90
torr; 200 torr =….

108
 Oxygen Binding to Hb is Cooperative and the
Property Influences Hb Function
• Hb becomes saturated with O2 in the lungs, where pO2 ~100 torr
• In capillaries, pO2 is about 40 torr, and oxygen is released from Hb
• The binding of O2 to Hb is cooperative – binding of oxygen to the
first subunit makes binding to the other subunits more favorable

The curve
correlates with
physiological
functions

109

Mb and Hb Have a Similar Structure

114
 Mb and Hb Use Heme to Bind Fe2+

Heme: protoporphyrin IX binds Fe2+

115

Fe2+, Heme, Hb, Ｍｂ，and O2

Iron interacts with ６ ligands
#1-4: N atoms of the porphyrin
#5: imidazole side chain of His F8
the 8th residue in the “F” (sixth) helix
#６：O2

The O2 molecule is tilted relative to a

perpendicular to the heme plane

116
 O2 Binding Alters Mb Conformation
• In deoxymyoglobin, Fe2+ lies 0.055 nm above the heme plane
• When O2 binds to Fe, the heme Fe is drawn toward the plane
to ~0.026 nm. Fe2+ pulls its His F8 along with it (0.029 nm)
• For Mb: little consequence
• For Hb: initiates a series of conformational changes that are
transmitted to adjacent subunits

117

Mｂ, Fe2+ and Fe3+

• Fe in Mb is Fe2+ (ferrous iron)  binds oxygen

• Oxidation of Fe2+ yields Fe3+ (ferric iron)
Mb with Fe3+ is called metmyoglobin  does not bind
oxygen

• Living muscle can reduce metmyoglobin by

metmyoglobin reductase and NADH to maintain
normal function
• Dead muscle accumulates metmyoglobin with time
 undesired brown color of meat and fish

118
 Mｂ, Fe2+ and Fe3+

119

Mｂ Structure

120
 Oxygen binding curves of Mb
MbO2  Mb + O2
K: dissociation constant
[Mb][O2]
K=
[MbO2]
Y: fraction of O2 bound Mb; 1 – Y: fraction of unbound
[MbO2] [O2]
Y = [MbO ] + [Mb] = [O ] + K
2 2

pO2 Y pO2
Y= =
pO2 + K 1 - Y K
(when gas O2 in equilibrium with solution O2)

121

O2-Binding Curves of Mb

The oxygen binding curve of

Mb resembles a [ES]
saturation curve

(Not allosterically regulated)

P50 = 1 torr
1 torr is sufficient for half saturation

122
 O2 Binding Curve of Hb Is More Complex

Hb(O2)4  Hb + 4O2
Suppose 4 O2 occupy Hb simultaneously
[Hb][O2]4
K=
[Hb(O2)4]

 (pO2)4
Y=
(pO2)4 + K

Y: fraction of O2 bound Hb

123

A Hypothetical O2-Binding Curve for Hb

O2 saturation curve for

Hb in the form of Y vs.
pO2, assuming n = 4
and P50 = 26 torr.

Y is the fractional
saturation of Hb:

[ pO 2 ]4
Y=
[ pO2 ]4 + K

The plot does not fit

the experimental
result

124
 Binding of O2 to Hb - Hill-Langmuir Eq.
• Archibald Hill (1913): The O2 binding behavior of Hb could be
described by the Hill-Langmuir Eq.

Y [ pO 2 ]n
=
1- Y K
• Y: the ratio of oxygenated heme groups
• 1-Y: O2-free heme (1-Y)
• K : dissociation constant
• n (Hill constant) is related to the number of O2-binding sites
on Hb and the degree of interaction between ligand-binding
sites

125

An Alternative O2-Binding Curve for Hb

n = 4 4 O2 bind
simultaneously
(fully cooperative)

n = 1 O2 binds
independently
(no cooperative)

n = 2.8 Experimentally
observed curve
(partially cooperative)

126
 The Physiological Significance of the Hb:O2
Interaction
Hb must be able to
bind O2 in the lungs
and release O2 in
capillaries

In capillaries (eg., pO2

= 10 torr)

n = 1 curve : 30% Hb
is oxygenated

n = 2.8 curve: 7% Hb is
oxygenated

127

Hb Has an α2β2 Tetrameric Structure

Blue: αβ dimer, with packing
contacts

Yellow: sliding contacts made

with the other αβ dimer

132
 Oxygen Binding by Hb Induces a Quaternary
Structure Change
• A large-scale structural change was observed
when deoxy-Hb crystals are exposed to oxygen
• One αβ dimer moves relative to the other by 15
degrees upon oxygen binding

Subunit motion in Hb when the molecule goes from the (a)

deoxy form to the (b) oxy form. Some atoms may move 0.6 nm.
133

Fe2+ Movement Induces the Conformation

Change in Hb
In deoxy-Hb, iron atom to
heme plane: ~0.06 nm

Upon O2 binding, Fe2+

atom moves ~0.039 nm
closer
(O2 is drawing the heme 87 92)
iron into the plane)

As Fe2+ moves, it drags His

F8 and the F helix with it

134
Salt bridges (noncovalent, electrostatic interactions)
that stabilize deoxy-Hb are broken in oxy-Hb
(a) The salt bridges and H (b) The salt bridges and H
bonds between N- and C- bonds involving C-terminal
terminal residues in the α- residues of β-chains
chains

Salt bridges stabilize deoxHb (T form)

Oxygen binding in one subunit  salt bridges broken  R form

135

Partially MWC, partially KNF

Real observation:
• In the deoxy Hb (T state), only heme in the -chains
is accessible to O2
• O2 biding to one -chain
 sequential conformation changes in the other -chain
 at Hb: 2O2 T to R state occurs
 O2 binds -subunits

136
 Factors influencing O2 binding by Hb

140

H+ Promotes Dissociation of O2 from Hb

• Deoxy-Hb has a higher affinity for H+ than oxy-Hb

• As pH decreases, dissociation of O2 from
hemoglobin is enhanced
• An effect discovered by Christian Bohr

HbO2 + H+  HbH+ + O2
• Binding of H+ diminishes O2 binding
• Binding of O2 diminishes H+ binding

• Actively working tissue produces acid [H+]

 Promote O2 release from Hb for O2 needing tissue

141
 H+ Promotes Dissociation of O2 from Hb
The O2 saturation curves for Mb and Hb at different pH values

Arterial blood
~pH 7.41
Venous blood
~pH 7.37

May result in ~10%

difference in O2
binding

142

CO2 Promotes the Dissociation of O2 from Hb

• Hydration of CO2 in tissues and extremities leads to

proton production:
CO2 + H2O ⇄ H+ + HCO3–
catalyzed by carbonic anhydrase
• These protons are taken up by Hb as O2 dissociates
• The reverse occurs in the lungs
• CO2 is also transported by (deoxy)Hb through
carbamylation in high [CO2]
R-NH2 + CO2  R-NH-COO- + H+
(at N-termini of Hb)
CO2 is released in lung when Hb becomes oxygenated

143
His146 is key
pKa = 6.5
Also N-termini
of  subunits
pKa = 8.0

144

Summary of the Physiological Effects of H+ and

CO2 on O2 Binding by Hemoglobin
• At the tissue-capillary interface, CO2 hydration and
glycolysis produce extra H+, promoting additional
dissociation of O2 where it is needed most

• At the lung-artery interface, bicarbonate dehydration

consumes extra H+, promoting CO2 release (CO2
exhalation) and O2 binding

146
 BPG Promotes the Dissociation of O2 from Hb

O2 binding curves of blood and of

purified Hb w/ or w/o CO2 and BPG

Purified Hb w/o BPG is saturated with

O2 at pO2 of 20 torr. Cannot release O2
at 40 torr (typical value in human
tissue)

What is BPG?

152

2,3-Bisphosphoglycerate
• Erythrocytes contain ~4.5 mM BPG (same as
[tetrameric Hb])
• BPG binds in the central cavity of Hb; one
tetrameric Hb has one BPG binding site
• An allosteric effector of Hb, binds at a site distant
from the Fe where oxygen binds
 w/o BPG, O2 binding to Hb follows a rectangular
hyperbola
 w/ BPG, a sigmoid binding curve

153
 BPG Binding to Hb Has Important Physiological
Significance

The ionic form of BPG

The negative charges of BPG interact with 8 positive charges
of Hb: 2 Lys, 4 His, 2 N-termini

154

The ionic binding of BPG to the two β-subunits of

Hb
BPG lies at the center Yellow: N-Val
of the Hb cavity. Green: Lys
Purple: His
Links two β-subunits.
Aid in stabilizing deoxy
Hb, favoring O2
dissociation.

The center in oxyHb is

too small for BPG.

BPG and O2 are

mutual exclusive
allosteric effectors.

155
 Fetal Hb Has a Higher Affinity for O2

• The fetus depends on its mother for O2

• Gas exchange takes place across the placenta
• Fetal Hb: γ-chains in place of β-chains  α2γ2 , has
a higher affinity for O2, partly due to the lower affinity
for BPG
Fetal γ-chains have Ser instead of His at position
143 and thus lack two of the positive charges in the
BPG-binding cavity
BPG binds less tightly and Fetal Hb behaves more like
Mb in its O2-binding behavior

156

Fetal Hb Has a Higher Affinity for O2 Because it

has a Lower Affinity for BPG

Comparison of the oxygen

saturation curves of Hb-A and Hb-F
under similar pH and [BPG].

Hb-F is suitable for transferring O2

from the mother

157
 Hb genes

158

159
 Thalassemia
Affect ~280 million people globally.
~439,000 in severe forms and 16,800 deaths in 2015.
6% Taiwanese are carrier (400 severe cases and 23
deaths)

• Two copies of  gene on chromosome 16

The severity of the α-thalassemias is correlated with
the number of affected α-globin
• One copy of  gene on chromosome 11
The severity of the disease depends on the nature of
the mutation and on the presence of mutations in one
or both alleles.
160

Sickle-Cell Anemia is a Molecular Disease

• Sickle-cell anemia patients have abnormally-shaped
red blood cells (RBC, erythrocytes)
• Crescent-shaped vs Disc-shaped
• The sickle cells pass less freely through the
capillaries, impairing circulation and causing tissue
damage
• Caused by a single amino acid substitution Glu6Val
in the β-chains of Hb
• As a result, Hb S molecules aggregate into long,
chain-like polymeric structures

164
 Sickle cell anemia

165

Sickle cell anemia

Why?

166
 Sickle-Cell Anemia is a Molecular Disease
Glu6Val substitution on the surface of Hb β forms a “block”
extending from Hb that can insert into hydrophobic pockets in
neighboring Hb S molecules.

167

Sickle-Cell Anemia is a Molecular Disease

168
 Molecular Mechanism of Sickle-Cell
Anemia

A dimer of Hb S tetramers.

Blue: Val at position 6 of

the β-chains
Red: Hemes

Only the 2 Val protrusions

are shown. (The 1 Val is
not involved in filament
formation.)

169

Sickle-Cell Anemia is a Molecular Disease

Structure of the polymerized Hb S filament.

Polymerization is easily to occur due to high [Hb] in RBC

170
 Nitric Oxide (NO·)
• A gaseous molecule that acts as a neurotransmitter
and as a second messenger in signal transduction
• Plays many physiological roles including regulation
of blood vessel relaxation (aka, endothelium-derived
relaxing factor)
• Molecule of the year 1992
• Discovery of NO· was awarded 1998 Nobel Prize in
Physiology or Medicine

175

Significance of Nitric Oxide

176
NO regulates muscle tone of blood vessels

• [Link]
[Link]/watch?v=8Z
bzSLFuLdY

177

Reaction catalyzed by NO synthase

178
 Hb and NO·
• NO· is a high-affinity ligand for Hb, binding to the heme
iron 10,000 times more tightly than O2
• Why is NO· not bound instantaneously to Hb, preventing
its physiological effects?
• NO· reacts with the –SH of Cys93β, forming an S-nitroso
derivative:

• Cys93β Is more exposed and reactive in oxy-Hb (R form).

Its binding with NO precludes reaction of NO to heme-
Fe2+ . When O2 is released from Hb (T form), NO is also
released  allosteric regulation.
• Artificial blood absorbs NO, causing BP fluctuation.

179

Hb and NO·
• The S-nitroso group is in equilibrium with other S-nitroso
compounds formed by reaction of NO· with small-
molecule thiols (eg., free Cys or glutathione)
• GSH : γ-L-Glutamyl-L-cysteinylglycine

• These small-molecule thiols transfer NO· from

erythrocytes to endothelial receptors, where it exerts its
physiological effects (extend NO half-life from s to h)

180
 Carbon monoxide (CO) poisoning
• Produced during incomplete burning of organic
matters
• Ｈｂ ＋ ＣＯ  HbCO (carboxyhemoglobin)
• CO binds to the Hb at the same site as O2
• The affinity between Hb and CO is ~240X greater
than Hb and O2
• Prevent the blood from carrying O2 and expelling
CO2 as carbaminohemoglobin.

184

CO shows allosteric regulation

The binding of CO at any
one of the subunits
increases the O2 affinity of
the remaining three sites
 Hb to retain O2 (not
released to the tissue)

185
 Application of CO in Food

186