Biochemistry

Hanjias Biochemistry Lecture
Essential Questions
Chapter 15
Before this class, ask your self the following

questions:
Enzyme
y
Regulation
g
Reginald H. Garrett
Charles M. Grisham
What are the properties of regulatory enzymes?

How do you know this enzyme is a regulatory enzyme?
?
How do regulatory enzymes sense the momentary

needs
d off cells?
ll ?
How signal is delivered?
Wh
Whatt molecular
l
l mechanisms
h i
are used
d tto regulate
l t
enzyme activity?
http://lms.ls.ntou.edu.tw/course/106
http://lms
ls ntou edu tw/course/106
hanjia@mail.ntou.edu.tw
Outline
15 1 What Factors Influence Enzymatic

15.1
Activity?
Part 1 Factors that influence enzymatic

y
activity
1. The availability of substrates and cofactors!

2. Product
oduct accumulates
accu u ates tthe
e rate
ate will decrease!
dec ease
3. The amount of enzyme present at any moment
Zymogen,
Zymogen isozyme and covalent modification!
Part 2: The general features of allosteric

regulation
Genetic regulation of enzyme synthesis and decay
4. Regulation of Enzyme activity
The mechanisms of allosteric regulation

g
Example of a enzyme controlled by both
allosteric regulation and covalent modification
Part 3: Special focus on hemoglobin and

myoglobin
l bi
Zymogens, isozymes,
Zymogens
isozymes and modulator proteins may play
a role
Enzyme activity can be regulated through covalent
modification
Allosteric Regulation
4
Regulation 1: Zymogen
Zymogen
The proteolytic activation of

chymotrypsinogen
Zymogens are inactive

precursors of enzymes.
Typically,
T i ll proteolytic
t l ti
cleavage produces the
active enzyme.
Figure 15.2 Proinsulin is an 86-residue
precursor to insulin
5
How to stop bleeding?
Proteolytic Enzymes of the

Digestive Tract
How
H
our bl
blood
d clot?
l t?
What is clotted?
Fibrinogen Fibrin
the result of a series of zymogen
y g activations
Ann Ny Acad Sci 2001 Mosesson
Two routes to blood clot formation
Aggregation of Fibrin
The Cascade
activation of seven
clotting factors
make fibrinogen
quickly transformed
into fibrin!
kallikrein,
kallikrein XIIa
XIIa, XIa
XIa,
IXa, VIIa, Xa, and
thrombin.
Thrombin
specifically cleaves
R-G peptide bonds
IIntrinsic
t i i pathway
th
blood physically
contact
t t with
ith
abnormal
surfaces caused
by injury
Extrinsic pathway
factors released
from injured
tissue
Ann Ny Acad Sci 2001 Mosesson
Why Cascade?
10
Example of isozymes:
lactate dehydrogenase (LDH)
Isozymes
Isozymes (also known as isoenzymes)
y
that differ in amino acid
are enzymes
sequence but catalyze the same chemical
reaction These enzymes usually display
reaction.
different kinetic parameters (i.e. different
Km values)
values), or different regulatory
properties.
How different?
Why different?
11
Mammalian lactate dehydrogenase (LDH), which exists

as five different isozymes, depending on the tetrameric
association of two different subunits, A and B: A4, A3B,
A2B2, AB3, and B4
12
15.4
5 What
at Kinds
ds o
of Co
Covalent
a e t Modification
od cat o
Regulate the Activity of Enzymes?
R
Regulation
l ti 2:
2 C
Covalent
l t modification
difi ti
Catalytic
activities of enzymes can also be

altered by
y reversible,, covalent changes
g to
specific amino acid side chains.
Phosphorylation
Most
M
prominent
i
fform off covalent
l
modification
difi i
Reversible by protein kinases / phosphoprotein
phosph______
Target
g specific
p
of kinase ((and p
phosph
p _____))
Regulating proteins are also a target of
regulation
Converter enzyme
Autophosphorylation..
interconvertible enzymes
13
14
Protein Kinases
Classificatin of Kinases
Target residues on target proteins:

Ser (
), Thr (
), and Tyr (
Typically recognize specific amino acid

sequences in their targets
But,
B t allll kinases
ki
share
h
a common catalytic
t l ti
mechanism
Regulation of kinases (usually, intrasteric
control)
A regulatory subunit with a pseudosubstrate
sequence that
th t mimics
i i th
the ttargett sequence
Inhibitor? What kind?
15
16
Example
p of Kinase: PKA
Structure of PKA
A conserved core kinase
domain of about 260
Protein kinase A (Green)
pseudosubstrate
peptide RRGA
peptide,
RRGA*II
(orange)
2
ATP (red) and two Mn2+
ions (y
(yellow))
Cyclic AMP-dependent protein kinase

(also known as protein kinase A, PKA
150150 to 170-kD
170 kD R2C2 tetramer
The two R (regulatory) subunits bind cAMP
R subunits
b it released
l
d (A
(Activated)
ti t d) ffrom th
the C
(catalytic) subunits after cAMP binding.
In other kinases, the regulatory sequence may
on the same peptide chain.
17
Phosphorylation is Not the Only

Form of Covalent Modification
End of Part 1
Ask yourself..
Types of Protein chemical modification > 100

Only
O l a ffew off these
th
are used
d to
t regulation!
l ti !
reversible conversion
18
Related to the energy state of cell
19
How many
y ways
y an enzyme
y
could be
regulated?
What is a cascade reaction? Is it any good?
What is an isozyme? Where is an isozyme
came from?
What is kinase? What kinds of properties
ki
kinases
h
have?
?
20
Regulatory Enzymes Have Certain

Exceptional Properties
R
Regulation
l ti 3:
3 Allosteric
All t i Regulation
R
l ti
Action at another
another site
site
Most key enzymes in metabolic pathways are
regulated in this way!
Kinetics are sigmoid ("S-shaped")
Substrate binding is cooperative

Allosteric enzymes are usually oligomeric
Regulation of allosteric enzyme involved
protein conformation change
Regulated by allosteric effectors
usually produced elsewhere in the pathway
may be feed-forward activators or feedback
inhibitors
21
22
Mechanism of allosteric enzymes
U d
Understand
t d MWC model:
d l St
Step 1
MWC Concerted Model (1965, Monod,

y
& Changeux)
g
):
Wyman
The enzyme exists in only two

interconvertable states.
R State relaxed form
T State taut form
The enzyme exists in only two

interconvertable states or conformations and
all subunits must be in the same state or
conformation:
M
Molecules
l
l off mixed
i d conformation
f
ti (h
(having
i
subunits of both R and T states) are not
allowed
ll
db
by this
hi model
d l ((symmetry model)
d l)
KNF model (Koshland, Nemethy, and

Filmer )
Ligand binding triggers a conformation change
in a protein
23
24
Understand MWC model: Step 2
Most of the enzyme oligomers will assume the

homogenous T state in the absence of bound
effector molecules
If no effector molecule is bound, then the R and T
states are indicated as: R0 and T0
The R and the T states have different

affinities for substrate.
The
Th substrate
b t t di
dissociation
i ti constant
t t ffor th
the
R state is defined as KR (so as KT).
ERS
KR =
KR
ER+S
[ER][S]
[ERS]
The equilibrium constant L is assumed to be

large!
Assuming KT>>KR KR/KT = 0

It means most S
Substrates
bstrates bind onl
only to R
25
26
Most enzyme in T state

Most substrates bind to R state
Key point is the [R0] and [T0]
2 ways to increase [R0]
27
28
KNF model
Heterotropic effectors
Based on ligand-induced conformation

g of enzymes
y
changes
If the protein is oligomeric, ligand-induced
conformation changes in one subunit may
lead to conformation changes in adjacent
subunits
Explains negative cooperativity
Also known as sequential model
Molecules that influence the binding of

something other than themselves
Homotropic effectors
Ligands
Li
d such
h as S are positive
iti h
homotropic
t i
effectors (homotropic activators)
S bi
binding
di increases
i
the
th population
l ti off R
R,
which increases the sites available to S
Cooperativity
29
30
Enzymes Controlled by Both Allosteric

Regulation and Covalent Modification
Explanation of KNF model

(a) A subunit changes its
conformation after S
binding (induced fit)
(b) Binding of S to one
subunit may cause the
other subunit changing its
conformation to (*
Use Glycogen phosphorylase (GP) as an

example!
GP cleaves glucose units from nonreducing
ends
d off glycogen
l
The product,
glucose-1-phosphate
having a greater affinity

for S (positive cooperativity)
g a less affinity
y for
having
S (negative cooperativity)
is a readily usable
fuel
31
32
Glucose-1-phosphate
Glucose-6-phosphate is the real metabolite for
energy production.
You will learn the detail in Chapter 18
G-1-P can easily

y be converted to G-6-P by
y the
phosphoglucomutase
Glucose 6 phosphate
Glucose-6-phosphate
In muscle glucose-6-phosphate is used to
produce energy
In the liver
li er it is ultimately
ltimatel transported to other
tissues via the circulatory system
Str ct re of glycogen
Structure
gl cogen phosphor
phosphorylase
lase
Allosteric Regulation of GP
Muscle GP is a homodimer
Each 842 amino acids, ~97
kD
Each subunit contains
1. a pyridoxal phosphate* cofactor,
covalently linked as a Schiff base**
to Lys680.
2. an active site (at the center of the
subunit)
b it)
3. an allosteric effector site near the
subunit interface .
4. a regulatory phosphorylation site
is located at Ser14 on each
subunit
*
See chap 17
* * Schiff bases are of the general formula R1R2C=N-R
Phosphate (Pi) shows strong positive cooperativity.

ATP,
ATP glucose-6-P are a feedback inhibitor that
affects the affinity of glycogen phosphorylase for its
substrates but does not affect Vmax.
Vmax
AMP is a positive heterotropic effector for glycogen
phosphorylase (enhances the binding of substrate
to glycogen phosphorylase)
36
Covalent Modification of Glycogen

Phosphorylase
MWC Model of Glycogen Phosphorylase

AMP p
promotes the conversion to the active R state
Two forms of GP
ATP, glucose-6-P, and caffeine favor conversion to

the inactive T state
Phosphorylase a (more active,

phosphorylated)
Structural explanations:
T state, the negatively charged carboxyl group of
faces the active site.
Phosphorylase b (less active,

unphosphorylated)
Asp283
Converting enzymes
R state, Asp283 is displaced from the active site and

replaced by Arg569.
Phosphorylase kinase stimulates

GP byy p
phosphorylation
p y
of Ser14
that turns b a
Physiological explanataions:
ATP and glucose-6-P are abundant, GP activity
Cellular energy reserves are low (i.e., high [AMP] and low [ATP]
and [G-6-P]), GP activity .
Phosphoprotein phosphatase 1
(PP1) inactivates GP
38
A Conformation Change of Glycogen

Phosphorylase by Phosphorylation
Glycogen phosphoryase is
activated by a cascade of reactions
After phosphorylation of
Ser14(red), the major
conformational change
occurs in the N-terminal
residues.
Secondary messenger
N-terminal conformation of
phosphorylated enzyme
(phosphorylase a): yellow.
N-terminal conformation
f
off
unphosphorylated enzyme
(phosphorylase b): cyan.
cyan
Figure
g
15.17 The hormone-activated enzymatic
y
cascade that leads to activation
of glycogen phosphorylase.
39
40
The Adenylyl Cyclase Reaction
Ad
Adenylyl
l l cyclase
l
Adenylyl cyclase, a membrane-bound enzyme that
converts ATP to adenosine-3',5' -cyclic monophosphate
(cyclic AMP or cAMP)
The hormonal stimulation of adenylyl cyclase is effected
by a transmembrane signaling pathway comprising three
components
Figure 15.18 The adenylyl cyclase reaction. The reaction is driven forward by
subsequent hydrolysis of pyrophosphate by the enzyme inorganic pyrophosphatase.
( ) membrane-bound hormone receptor

(a)
p
(b) GTP-binding protein (G protein ) :have a heterotrimeric ()
quaternary structure
(c) Adenylate cyclase
41
Signaling pathway
End of Part 2
Ask yourself..
Step1. G protein is stimulated by a hormonereceptor complex, GDP dissociates and GTP binds
to G , causing it to dissociate from Gband to
associate with adenylyl cyclase
Step2 Binding of G(GTP) activates adenylyl
Step2.
cyclase to form cAMP from ATP
Step3. Intrinsic GTPase activity of G eventually
hydrolyzes GTP to GDP, leading to dissociation of
G a (GDP) from adenylyl cyclase and reassociation
with Gb to form the inactive Gb complex
Guanosine triphosphate (GTP),

Guanosine diphosphate (GDP)
How to identify
y an allosteric regulated
g
enzyme?
How to explain the mechanism of allosteric
regulation?
What is MWC model?
Still remember the regulation of glycogen
phosphorylase?
h
h l
? Wh
Why it need
d more th
than one
regulation system?
43
44
Special Focus: Hemoglobin
Hemoglobin & myoglobin

Myoglobin,
Myoglobin as an oxygen storage protein
A classic
l
i example
l off allostery
ll t
Greater affinity for O2 than Hb at all oxygen

pressures.
Hemoglobin (Hb) and myoglobin

(Mb) are oxygen transport and
storage
g p
proteins
Myoglobin
Hemoglobin,
g
, as the oxygen
yg carrier
Monomeric
153 aa, 17,200 MW
Hemoglobin
Tetrameric
2 chains of 141 residues,
2 chains of 146 residues
45
Saturated with O2 in the lungs (pO2 ~100 torr.)

Oxygen released from Hb in the capillaries of
tissues (pO2 ~40 torr)
Some
S
oxygen are b
bound
db
by Mb iin muscle.
l It will
ill
release in case of severe oxygen deprivation,
such
h as d
during
i strenuous
t
exercise
i (pO2
( O2 iis lless
20 torr)
46
Hemoglobin & myoglobin
Myoglobin
Hemoglobin displays sigmoid

sigmoid-shaped
shaped O2binding curves
Myoglobin
Myoglobins
s interaction with oxygen obeys
classical Michaelis - Menten-type substrate
saturation behavior
behavior.
Structure of Mb
consists
i t off 8 helical
h li l segments
t (d
(designated
i
t dA
A~H)
H)
unordered regions are named for the helices they
connect, ex. AB region
Fe in Mb
ferrous iron (Fe2+) is the form that binds oxygen
ferric iron ((Fe3+) in metmyoglobin
y g
does not bind oxygen
yg
3+
Fe :protoporphyrin IX is referred to as hematin. ()
47
48
Myoglobin Structure
Affinity of Heme changed in Mb

F
Free heme:
h
CO affinity
ffi it 25000 X greater
t th
than O2
Mb: CO affinity 250 X greater than O2
Because
B
His
Hi E7 fforce th
the CO tto tilt away!!
The functions of Mb polypeptide:

cradles
dl the
th heme
h
group
protects the heme iron atom from oxidation
provides
id a pocket
k t iinto
t which
hi h th
the O2 can fit
The six liganding positions of an iron ion.

Four ligands lie in the same plane
His F8 is the 5th ligand; in oxymyoglobin
O2 becomes the 6th.
49
50
What Interactions Exist Between the

Subunits of Hemoglobin?
Structures of Hb & Mb
-chains contact with chains more!

1. 11 and 22 ---helices
BG
B
G H
H and GH corner
2. 21 and 12 ---helices CG
C G and
and FG
corner (sliding contacts
Mb: 153 a.a.

Hb:
chain 146 a.a. H helix is shorter
chain 141 a.a. H helix is shorter & Lack D helix
51
There are fewer

-

interactions and -
interactions
52
How Does the Structure of Hemoglobin

Change Upon Binding Oxygen?
When deoxy-Hb crystals are exposed
to oxygen,
yg , theyy shatter
One alpha-beta
p
pair moves relative to
p
the other by 15 degrees upon oxygen
binding
This massive change is induced by
movement of Fe by 0.039 nm when
oxygen binds
bi d
( Mb is 0.029 nm )
The structure of dexoyHb is stablized

Deoxy Hb (T form) is stabilized
by specific hydrogen bonds and 8
salt bridges (ion-pair
(ion pair bonds)
bonds),
which will break in OxyHb (R
form)
H bonds:
In deoxyHb Phenolic -OH
OH groups of
Tyr 140 and Tyr 145 form
intrachain H bonds to Val FG5. ((Val
FG5 is 93 and 98, respectively.)
chain
chain
54
The Bohr Effect
Why Blood Becomes Acid?

CO2 hydration by the enzyme carbonic
y
.
anhydrase
Saturation curve of Hb for O2 is displaced to the right as

acidity increases . This phenomenon is called the Bohr
effect (by Christian Bohr)
DeoxyHb has a higher affinity for proton than oxyHb
HbO2 + H +
HbH + +O2
Other effectors: Protons ,carbon dioxide , chloride ions and

metabolite 2,3- bisphosphoglycerate (BPG)
Many of the are picked up by Hb as O2

dissociates and HCO3- are transported with
the blood back to the lungs.
Hb becomes
b
oxygenated
t d iin th
the llungs, H+ is
i
released and reacts with HCO3 to re-form
H2CO3, from which CO2 is liberated.
56
Hb and CO2 Transportation?
Effect of 2,3-BPG
2 3 BPG
CO2 is directly transported by Hb in the form of

carbamate
b
t (NHCOO
( NHCOO-)
RNH2 + CO2 RNHCOO- + H+
Thi
This reaction
ti to
t right:
i ht in
i tissues
ti
by
b the
th high
hi h CO2
concentration; to left in the lungs where [CO2] is low.
Note: Carbamylation of N-terminus converts it to a
negatively charged functional group that forms a salt
bridge to Arg
Arg-141
141 ( chain) stabilizing deoxyhemoglobin
(T-State)
Binding of 2,3-BPG
2 3-BPG
The strongly
g y negative
g
BPG molecule is
electrostatically bound via interactions with the
positively
p
y charged
g functional g
groups
p of each Lys
y
82, His 2, His 143, and the NH3+-terminal
group
g
p of each
-chain.
The binding of 2
2,3
3 -bisphosphoglycerate
bisphosphoglycerate (2,3(2 3
BPG) to Hb promotes the release of O2
Erythrocytes (red blood cells) normally contain
about 4.5
5 mM BPG
G
4.5 mM BPG equivalent to that of tetrameric
hemoglobin molecules
molecules.
Tetrameric Hb molecule has but one binding
site for BPG. This site is situated within the
central cavity
y formed byy the association of the
four subunits.
57
58
Oxygen binding curves of blood

Oxygen-binding
In the absence of 2,3-BPG, oxygen
binding to Hb follows a rectangular
hyperbola
The sigmoid binding curve is only

observed in the presence of 2,3BPG
BPG bind better to deoxyHb than

t oxyHb,
to
Hb causing
i a shift
hift iin
equilibrium in favor of O2 release
Fetal hemoglobin
Abnormal Hb
Fetal hemoglobin exists as an 22 tetramer
Sickle-cell anemia patients have abnormally-shaped

red blood cells
The erythrocytes are crescent-shaped instead of
disc-shaped
The sickle cells pass less freely through the
capillaries, impairing circulation and causing tissue
damage
Gamma has a serine substituted for histidine

at AA 143 (lack two of the positive charges in
the central BPG-binding cavity. )
Decreased BPG binding in fetal hemoglobin
gives it a higher affinity for oxygen than
maternal
61
The Cause of sickle-cell

sickle cell anemia
62
Sickle-Cell Anemia is a Molecular

Genetic Disease
A single amino acid substitution in the

-chains
chains
of Hb causes sickle-cell anemia
Glu at position 6 of the -chains
chains is replaced by
Val
As
A a result,
lt Hb S molecules
l
l aggregate
t iinto
t llong,
chainlike polymeric structures
The polymerization of Hb S via the interactions
between the hydrophobic Val side chains at
position b6 and the hydrophobic pockets in the
EF corners of b-chains in neighboring Hb
molecules.
64
Hemoglobin and Nitric Oxide
Hemoglobin and Nitric Oxide
Nitric oxide (NO)

(NO ) is a simple gaseous molecule
that acts as a neurotransmitter and as a second
messenger in signal transduction (see Chapter
32)
NO is a high-affinity
high affinity ligand for Hb,
Hb binding to the
heme iron 10,000 times more tightly than O2
But
B t why
h iis NO
NO nott bound
b
d iinstantaneously
t t
l tto Hb,
Hb
preventing its physiological effects?
NO reacts with the SH of Cys93, forming an
S-nitroso derivative:
The S-nitroso
S nitroso group is in equilibrium with other
S-nitroso compounds formed by reaction of nitric
oxide with small-molecule
small molecule thiols such as free
Cys or glutathione:
These small-molecule thiols transfer NO from

erythrocytes to endothelial receptors, where it
exerts its physiological effects
65
66
End of Part 3
End of the class
Ask yourself.
You should learned
Whats the similarities and differences of

myoglobin and hemoglobin?
What is Bohr effect?
How many ways the activity of Hb could be
regulated?
Besides oxygen, what else Hb could carry?
Common regulation
g
mechanisms of enzymes
y
Phosphorylation modification
Allosteric modification
MWC model
The
Th example
l off glycogen
l
phosphatase
h
h t
The properties of hemoglobin

Allosteric effects on Hb
Cargo of Hb
67
68

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Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

Before this class, ask your self the following

What are the properties of regulatory enzymes?

How do regulatory enzymes sense the momentary

How signal is delivered?

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

15 1 What Factors Influence Enzymatic

Part 1 Factors that influence enzymatic

1. The availability of substrates and cofactors!

Part 2: The general features of allosteric

Genetic regulation of enzyme synthesis and decay

4. Regulation of Enzyme activity

The mechanisms of allosteric regulation

Part 3: Special focus on hemoglobin and

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

The proteolytic activation of

Zymogens are inactive

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

How to stop bleeding?

Proteolytic Enzymes of the

Ann Ny Acad Sci 2001 Mosesson

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

Two routes to blood clot formation

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

Mammalian lactate dehydrogenase (LDH), which exists

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

activities of enzymes can also be

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

Target residues on target proteins:

Typically recognize specific amino acid

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

Cyclic AMP-dependent protein kinase

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

Phosphorylation is Not the Only

Types of Protein chemical modification > 100

Related to the energy state of cell

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

Regulatory Enzymes Have Certain

Substrate binding is cooperative

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

Mechanism of allosteric enzymes

MWC Concerted Model (1965, Monod,

The enzyme exists in only two

The enzyme exists in only two

KNF model (Koshland, Nemethy, and

Hanjias Biochemistry Lecture

Hanjias Biochemistry Lecture

Understand MWC model: Step 2

Understand MWC model: Step 3

Most of the enzyme oligomers will assume the

The R and the T states have different

The equilibrium constant L is assumed to be