Allosteric Enzyme

Dr. Samina Hyder Haq

Dept of Biochemistry
King Saud University
 Enzyme regulation
• Metabolism is the right integration of
varous processes. There are four
principles ways in which this is
achieved:
• Allosteric control
• Multiple forms of enzymes
• Reversible covalent modification
• Proteolytic activation
 Isozymes

Isozymes (isoenzymes) are enzymes that differ in

sequence but catalyze the same reaction
• They usually display different kinetic behavior,
have differing substrate affinities or are regulated
in different manners
• The existence of isozymes allows the fine-
tuning of processes (e.g. metabolism) by using
different amounts of each isozyme
 Isozymes: Lactate
Dehydrogenase

• Humans have two forms of lactate

dehydrogenase H form found in heart
• M form found in skeletal muscle
• The two forms are 75% identical and both exist
as homotetramers (H4 and M4)
• The H4 form has a higher affinity for substrate
Combinations are possible (e.g. H3M, H2M2)
allowing for different affinities
Isozymes: Lactate
Dehydrogenase
 Isoenzyme in Heart attack
• The 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.
 Regulation via Covalent
Modification

• The covalent attachment of a molecule to an

enzyme (or other protein) can alter its activity
• Most such covalent modifications are reversible
e.g. phosphorylation, acetylation
• Some are irreversible e.g. attachment of a lipid
group that localizes the protein to the membrane
 Phosphorylation

• Many proteins regulated via

phosphorylation - addition of
phosphoryl group to hydroxyl oxygen of
serine, threonine or tyrosine
• Terminal (γ) phosphoryl group from ATP
transferred to specific serine, threonine
and tyrosine residues Catalyzed by
Protein kinases
 Phosphorylation
• Under 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
 Allosteric Regulation
• Allosteric modulators bind at a site other
than the active site and cause activation or
inhibition
• Can include the substrate itself
• Protein has quaternary structure
• Non-Michaelis-Menten kinetics
• Allosteric Enzyme Kinetics: Sigmoid Curve
instead of Hyperbola.
 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 curve 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.
 Allosteric Enzyme
• Allosteric 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)
 Concerted models:
• All subunits are either
• R or T (explains positive cooperativity)
 Sequential model
• subunits convert from R
to T individually (pos. or
neg. coop.)
• Positive cooperativity
means activity increases
as substrate
concentration increases.
• B. Negative cooperativity
means activity decreases
as substrate
concentration increases.
 Heterotropic effectors:
• The 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.
• If 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 enzyme phosphofructokinase-1 is allosterically
inhibited by citrate, which is not a substrate for the enzyme
Feed back inhibition
 Regulation via Proteolytic
Cleavage
Many enzymes are active as soon as they are
synthesized and have folded

Others are synthesized somewhere you don’t want them

to be active - they are activated after being
transported to the appropriate place
These enzymes can be synthesized as zymogens –as
inactive precursors
• Zymogens are activated by proteolytic cleavage,
which can occur outside the cell
 Examples of Zymogens
1. Digestive enzymes: pepsin, chymotrypsin,
trypsin, elastase, carboxypeptidase
2. Blood clotting - activated by a cascade of
proteolytic activations
3. Some hormones: insulin
4. Collagen -Collagenase - enzyme that
breaks down collagen
5. Caspases - proteolytic enzymes involved in
apoptosis (programmed cell death
 Zymogen Active site

• Trypsinogen Enterokinas TRypsin + peptide

• Chymotrypsinogen Trypsin
Chymotrypsin + peptide
• Pepsinogen
•HCl
Pepsin + Peptide

• Prothrombin Clotting
Thrombin + Peptide
Factor

• Zymogen provides protection to the body. As the active E may

destroy body substances if activated in absence of S.e.g. if
thormbinis formed in the body, it will convert Fibrinogen to Fibrin.
This will form clot in blood causing heart attack and Stroke.