Enzyme-221-Allosteric-Enzyme for health sciences

Detailed description of allosteric enzymes

1. Allosteric
Enzyme
Dr. Samina Hyder
H
aq
Dept of Biochemistry King Saud
University

2. Enzyme regulation
•
M
etabolism is the right integration of
varous
processes. There are four
principles ways in which this is
achieved: •
A
llosteric
control
•
M
ultiple forms of enzymes
•
R
eversible covalent modification
•
P
roteolytic
a
ctivation

3. Isozymes
Isozymes
(isoenzymes) are enzymes that differ in
sequence but catalyze the same reaction
•
T
hey usually display different kinetic behavior,
have differing substrate affinities or are regulated in different manners •
T
he existence of isozymes
allows the fine-
tuning of processes (e.g. metabolism) by using different amounts of each isozyme

4. Isozymes: Lactate
Dehydrogenase
•
H
umans have two forms of lactate
dehydrogenase
H
form found in heart
•
M
form found in skeletal muscle
•
T
he two forms are 75% identical and both exist
as homotetramers
(
H4 and M4)
•
T
he H4 form has a higher affinity for substrate
Combinations are possible (e.g. H3M, H2M2) allowing for different affinities

5. Isozymes: Lactate
Dehydrogenase

6. Isoenzyme
in Heart attack
•
T
he pattern of isoenzymes
found in the
plasma serve as a means of identifying the site of tissue damage. For example, the plasma levels of creatine
kinase
(
CK)
are commonly determined in the diagnosis of myocardial infarction.

8. Regulation via Covalent
Modification
•
T
he covalent attachment of a molecule to an
enzyme (or other protein) can alter its activity
•
M
ost such covalent modifications are
reversible
e.g. phosphorylation, acetylation •
S
ome are
irreversible
e.g. attachment of a lipid
group that localizes the protein to the membrane

9. Phosphorylation
•
M
any proteins regulated via
phosphorylation
-
addition of
phosphoryl
group to hydroxyl oxygen of
serine, threonine
or tyrosine
•
T
erminal (
γ
) phosphoryl
group from ATP
transferred to
specific
serine, threonine
and tyrosine residues Catalyzed by
Protein kinases

10. Phosphorylation
•
U
nder physiological condition,
phosphorylation
(and dephosphorylation)
is essentially irreversible
•
-
kinases
and phosphatases
are
required
State of phosphorylation
is then dependant
upon the relative activities of kinases
and
phosphatases

11. Allosteric
Regulation
•
A
llosteric
m
odulators bind at a site other
than the active site and cause activation or inhibition
•
C
an include the substrate itself
•
P
rotein has quaternary structure
•
N
on-Michaelis-Menten
kinetics

12. •
A
llosteric
E
nzyme Kinetics: Sigmoid Curve
instead of Hyperbola.

13. Why Sigmoid Curve.
Affinity for substrate increases with increasing substrate concentration. A plot of product formation as a function of substrate concentration produces a sigmoidal
c
urve because
the binding of substrate to one active site favors
the
conversion of the entire enzyme into the R state, increasing the activity at the other active sites. Thus, the active sites show cooperativity
.

14. Allosteric
Enzyme
•
A
llosteric
enzymes have two conformations:
active (R-state) and less active (T-state)
•
1
. T-state: less active, stabilized by inhibitors
•
2
. R-state: more active, stabilized by substrate
and activators
•
3
. Allosteric
enzymes have multiple subunits.
Cooperativity
results from the R to T transition of
subunits and the interaction of these subunits (quaternary structure)

15. Concerted models:
•
A
ll subunits are either
•
R
or T (explains positive cooperativity)

16. Sequential model
•
s
ubunits convert from R
to T individually (pos. or neg. coop.)
•
P
ositive cooperativity
means activity increases as substrate concentration increases.
•
B
. Negative cooperativity
means activity decreases as substrate concentration increases.

18. Heterotropic
e
ffectors:
•
T
he effector
may be different from the substrate, in which
case the effect is said to be heterotropic. For example, the feedback inhibition . The enzyme that converts D to E has an allosteric
site that binds the endproduct, G.
•
I
f the concentration of G increases (for example, because it
is not used as rapidly as it is synthesized), the first irreversible step unique to the pathway is typically inhibited. Feedback inhibition provides the cell with a product it needs by regulating the flow of substrate molecules through the pathway that synthesizes that product. Heterotropic effectors are commonly encountered, for example, the glycolytic
e
nzyme phosphofructokinase-1 is allosterically
inhibited by citrate, which is not a substrate for the enzyme

19. Feed back inhibition

21. R
egu
l
a
ti
on v
i
a
P
ro
t
eo
l
y
ti
c
Cleavage
Many enzymes are active as soon as they are synthesized and have folded Others are synthesized somewhere you don’t want the
m
to be active -
t
hey are activated after being
transported to the appropriate place
These enzymes can be synthesized as
zymogens
–as
inactive precursors
•
Z
ymogens are activated by
proteolytic
c
leavage
,
which can occur outside the cell

22. Examples of Zymogens
1.
Digestive enzymes: pepsin, chymotrypsin,
trypsin, elastase, carboxypeptidase
2.
Blood clotting -
a
ctivated by a cascade of
proteolytic
a
ctivations
3.
Some hormones: insulin
4.
Collagen -Collagenase
-
enzyme that
breaks down collagen
5.
Caspases
-
p
roteolytic
e
nzymes involved in
apoptosis (programmed cell death

23. Zymogen
A
ctive site
•
Trypsinogen
•
C
hymotrypsinogen
•
Pepsinogen
•
Prothrombin
•
Z
ymogen
p
rovides protection to

the body. As the active E may
destroy body substances if acti
vated in absence of S.e.g.
if
thormbinis
formed in the body, it w
ill convert Fibrinogen to Fibrin.
This will form clot in bl
ood causing heart attack and Stroke.
TRypsin
+
peptide
Enterokinas
Chymotrypsin
+ peptide
Pepsin + Peptide
•
HCl
Thrombin + Peptide
Clotting Factor
Trypsin