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Enzymes Regulation
17
17.1. Introduction
Interesting Fact
As we learnt in previous chapters that enzymes are pivotal to biochemical
reactions, their roles in driving the metabolism is indispensable. However, a cell has
Arslan et al. introduced intramolecular crosslinks
enormously complicated network of pathways and therefore it is in order to keep
between two domains of the Escherichia coli
the metabolic flux balanced, the enzymes which are catalysing those reaction must
helicase Rep, which unwinds DNA. By inserting
be regulated. (Otherwise condition would be like driverless cars running without
linkers of different lengths, the domains can
control causing chaos). This chapter on enzymology provides a comprehensive
be held either open or closed. The closed
information on regulation of enzymes. Understanding regulation of enzyme has
conformation activates the helicase, but it
become clinically important domain of science. As most of the enzymes are prime
can also generate super-helicases capable of
targets for developing drugs which either inhibit or augment the enzymatic activities.
unzipping long stretches of DNA at high speed
and with considerable force. Comstock et al. used
It is therefore very important to focus on fundamentals of enzyme regulation and to
optical tweezers and fluorescence microscopy to
understand alterations in their kinetics during such regulation. The cell has several
simultaneously measure the structure and function
mechanisms to regulate enzyme activity, first and the generalised mode (global
of the bacterial helicase UvrD. They monitored its
regulation) of regulation is compartmentalization of metabolism (discussed in
DNA winding and unwinding activity and its shape
chapter 13), usually the anabolic and catabolic compartments are separate and
during these activities. The motor domain also has
enzymes have isoforms with different kinetics abilities in various compartments.
a closed conformation during DNA unwinding and
The second mode of regulation of enzyme activity is availability of accessory
switches to a reversed open conformation during
the zipping-up interaction (Sciecne, 2015 (348)
factors (co-factors) for the enzymes, which are either obtained exogenously
(vitamins) or produced endogenously. The Third mode of regulation is via regulation
of substrate concentration, if there is no substrate, no product will be formed.
However, the above modes do show enzyme specificity. There are some specific routes of enzyme regulation which specifically modulate
enzyme thereby affecting its activity. In the following section we will consider such specific regulatory mechanisms.
Trick to Remember
Regulation of enzymes is like controlling electrical devices in household or industry. For safety purpose there are some general
cut-outs (like MCBs) and centralised switches which can switch off the whole power connection of a building (analogous to
global regulation), while for specific regulation of an electrical appliance we have individual switches (analogous to specific
regulation). Sometimes those devices which are frequently used and need to be operated from multiple places may have
multiple switches like a bulb on staircase may have more than one switch (on each floor) or room light may have additional
switch close to bed, similar to those enzymes which are regulated (analogous to allosteric regulation) .
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Allosteric
Regulation
Homotrophic
Regulation by
Covalent Modification
Reversible
Heterotrophic
K-Type
Regulation by
Peptidyl Cleavage
Regulation by
Peptidyl Cleavage
V-Type
Competitive
Irreversible
Mixed
Uncompetitive
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Fig. 17.1. Classification of major types of enzyme regulation processes (*Noncompetitive is a special case of Mixed)
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In general appearance, the active site is usually a groove or pocket of the enzyme which can be located in a deep tunnel within
the enzyme or between the interfaces of multimeric enzymes. There are two proposed models of how enzymes fit to their specific
substrate: the lock and key model and the induced fit model (Fig 17.2).
Key and lock hypothesis: Emil Fischers proposed the lock and key model, this assumes that the active site is a perfect fit
for a specific substrate and that once the substrate binds to the enzyme no further modification occurs. However, this hypothesis
could not explain the extra-stability of transition state.
Induced fit hypothesis: This was a modified version of key and lock hypothesis given by Daniel Koshland. The induced fit
model is a development of the lock-and-key model and assumes that an active site is flexible and it changes shape until the
substrate is completely bound. The substrate is thought to induce a change in the shape of the active site. The hypothesis also
predicts that the presence of certain residues (amino acids) in the active site will encourage the enzyme to locate the correct
substrate. Conformational changes may then occur as the substrate is bound.
Lock and Key Model (Emil Fischer)
Enzyme
ES complex
Substrate
ES complex
Substrate
Transition state
structure
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Fig. 17.2. Difference between Key and Lock hypothesis and induced fit hypothesis
Native protein
Isolated
and Purified
Electrophoresis
C3
C3
Catalytic Unit
(Dimer of Trimers)
R2
R2
R2
Regulatory Unit
(Trimer of Dimers)
Fig. 17.3. Basic experiment to illustrate the salient features of allosteric enzymes using ATCase as model
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Clinical Note
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Trick to Remember
Similar to the allosteric modulation, covalent modifications are also widespread in proteins which are not enzymes. As
covalent modification is also the common mode to activate or deactivate a non-enzyme protein such as transporter,
receptor or immune effector. Therefore examples here may include some non-enzyme proteins.
Tables 17.2 represents some of the commonly known protein-modifications their donor, biological roles and also the residues that
are prone to specific modification (some of them are examples of proteins but not enzymes)
Table 17.2. Commonly known covalent modifications and their examples.
Modification
Donor molecule
Protein function
Phosphorylation
ATP
Glycogen phosphorylase
Glucose homeostasis
Acetylation
Acetyl CoA
Histones
DNA packing
Myristoylation
Myristoyl CoA
Src
Signal transduction
ADP-ribosylation
NAD
RNA polymerase
Transcription
Farnesylation
Farnesyl pyrophosphate
Ras
Signal transduction
Thrombin
Blood clotting
g -Carboxylation
HCO3
Sulfation
3PAP*
Fibrinogen
Blood-clot formation
Ubiquitination
Ubiquitin
Cyclin
*39-Phosphoadenosine59-phosphosulfate
Table 17.3 summarises additional modification on proteins (Some of them are non-enzymatic proteins and key residues on which
these modifications occur)
Table 17.3. Key covalent modifications on proteins and amino acids involved.
Name
Phosphorylation
Group added
-PO3
2-
Examples
Regulatory enzymes,
Receptors
Acetylation
-CH2COO-
Histones
Methylation
-CH3
K,R
Histones
Acylation
Palmitic acid
G-protein-coupled receptors
Prenylation
Prenyl group
Ras p21
ADP-ribosylation
ADP-ribose
H,R
AMP
Adenylylation
G protein, eEF
Glutamine Synthetase
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Online Support
Video Lecture Available: You tube channel: video 17.2: Various types of reversible enzyme inhibition.
A. Competitive Inhibition
Competitive inhibition, as the term suggests, indicate the competition between substrate and inhibitor for the same binding site
on the enzyme. Substrate on binding to active site forms product, but inhibitor on binding with same site leads to formation of
non-productive EI complex. Increasing concentration of EI complex would inhibit the product formation more severely due to
a decrease in ES complex. Now the kinetics of EI formation is same as that of ES formation hence, the rate constant for the
formation of EI complex is given by Ki and a term alpha (a ) is introduced which represents the strength of inhibition (a 5 11
[I]/K I), where [I] is inhibitor concentration and Ki is the equilibrium constant for the breakdown of EI complex back to E and I,
hence with increasing inhibitor concentration the value of alpha would increase (Fig 17.7)
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V + [S ]
) (alpha is define in text
Apperant Km Increased by a factor a or Km (apperant) 5 a Km (Equation: Vo = max
a
+
K
S
[
]
m
above)
Inhibitor
Competition
for same site
=2
=1
No Inhbitor
ES
EI
NON-PRODUCTIVE
Vmax unchanged
Km increases
1
[S]
Increasing inhibitor
conc.
EI
=3
Vmax constant
Rate of Reactions
Ki
E+P
1
Vo
E+S
ES
+
1+ [I]
I = Ki
Hyperbolic plot
Increasing inhibitor
conc.
Mechanism of inhibition
Km increases
Substrate concentration
Fig. 17.7. Illustration of competitive inhibition a. Mechanism b. Lineweaver Burk plot c. Michaelis plot
B. Uncompetitive Inhibition
Uncompetitive inhibition, on the other hand, does not involve the competition with substrate, but the inhibitor binds to enzyme
substrate complex leading to an unproductive ESI complex. Infact, this occurs due to a conformational change in the enzyme
after binding of substrate which created a new binding site for inhibitor. So in this type of inhibition one would not observe the
EI complex in the reaction mixture. Similar to competitive inhibition the strength of inhibition in this case is indicated by similar
parameter (a 5 11 [I]/KI), where [I] is inhibitor concentration and Ki is the equilibrium constant for the breakdown of EI
complex back to E and I, hence with increasing inhibitor concentration the value of a would also increase (Fig 17.7).
Salient features of competitive inhibition are:
Inhibitor Binds to a site different than that of the binding site of substrate
It actually binds to ES complex to form non-functional ESI complex
ESI complex don not lead to catalysis
Apperant Vmax Vmax/a [Vmax is reduced] (a 5 11 [I]/KI) where [I] is inhibitor concentration and Ki is the
equilibrium constant for the breakdown of ESI complex
[Km is also reduced]
Apperant Km - Km / a
V + [S ]
Equation: Vo = max
'S
Km +a[ ]
Example: The membrane-bound Na1/K1 ATPase is inhibited by ouabain, a toxic glycoside.
C. Mixed Inhibition
Competitive inhibition, is the mechanistic combination of competitive and uncompetitive inhibition, in this mode the inhibitor binds
to enzyme and forms EI complex, as well as it also binds to additional site created at non-catalytic site after binding of substrate
to enzyme, thus forming ESI complex. Therefore one can observe both EI and ESI complex in a reaction mixture operating by this
mechanism. The kinetics of this inhibition will also be a combination of above two, Infact the degree of EI formation or ESI formation
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EI
=3
=2
=1
S
E
ES
No Inhbitor
Inhibitor
ESI
NON-PRODUCTIVE
Km decreases
1
[S]
Vmax decreases
Increasing inhibitor
conc.
1+ [I]
Ki
E+P
Rate of Reactions
Ki
ES
+
I
1
Vo
E+S
Hyperbolic plot
Increasing inhibitor
conc.
Mechanism of inhibition
Km decreases
Substrate concentration
Fig. 17.8. Illustration of uncompetitive inhibition a. Mechanism b. Lineweaver Burk plot c. Michaelis plot
Apperant Km a Km / a
[Km is increased when a > a ; Km is decreased when a <a ; and
remains unchanged when a 5a ]
V + [S ]
Equation: Vo = max
'S
aK m + a [ ]
Examples: L Threonine Dehydratase is inhibited by end
product isoleucine
Trick to Remember
As a smart trick to remember whether Km
or Vmax change in which type of inhibition,
an analogy may be created. In case of
competitive inhibition the lines on double
reciprocal plot are intersecting while on
a plot of uncompetitive lines are parallel
(Quite obvious! things that compete must
intersect and those which do not compete
remain parallel). So graph may be plotted
from this point and every new line placed
away from origin indicate increasing inhibitor
conc. Now recall the determination of Vmax
and Km on double reciprocal plot (chapter
16) and determine if Km and Vmax are
increasing or decreasing)
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EI
S
E
ES
No Inhbitor
1+ [I]
Ki
Inhibitor
1+ [I]
Ki
Vmax decreases
EI
ESI
Km increases or decreases
or remains unchanged (depends on and )
NON-PRODUCTIVE
Increasing inhibitor
conc.
EI
Ki
E+P
Vmax decreases
Rate of Reactions
Ki
ES
+
I
1
Vo
E+S
+
I
Hyperbolic plot
Increasing inhibitor
conc.
Mechanism of inhibition
Km decreases
1
[S]
Substrate concentration
No Inhbitor
Vmax decreases
Km decreases with increasing inhibitor
1
[S]
No Inhbitor
Also Known as
NON-COMPETITIVE
INHIBITION
Vmax decreases
Km decreases with increasing inhibitor
1
[S]
Increasing inhibitor
conc.
Increasing inhibitor
conc.
1
Vo
Increasing inhibitor
conc.
Fig. 17.9. Illustration of mixed inhibition a. Mechanism b. Lineweaver Burk plot c. Michaelis plot
No Inhbitor
Vmax decreases
Km Unchanged
1
[S]
Fig. 17.10. Illustration of three conditions during mixed inhibition, the last curve shows non- competitive inhibition
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Concept Map
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Q1. A Protein moved more slowly is an SDS- PAGE. Isoelectric focusing [IEF] showed that here was no change in the
pI Mass spectrometric analysis showed that the modification was on serine The modification that the protein
undergoes is likely to be
[CSIR-NET DEC 2015]
a. Phosphorylation
b. Glycosylation
c. Ubiquitination
d. ADP- ribosylation
Solution: This question may be related to the activity of enzymes [by possible mode of protein modification],
however solving this does not require enzymology, but basic knowledge of proteins, Phosphorylation, Ubiquitination
and ADP ribosylation will cause a change in the ionization state of the protein and also the isoelectric point so the
separation on the pI will alter if there is any such mutation. But if modification is glycosylation the protein will
show a change in SDS PAGE but not in IEF. Therefore option b is correct.
Q2. Reaction products inhibit catalysis in enzymes by [CSIR NET JUNE 2013]
a. Covalently binding to the enzyme.
Solution: Reaction product often interact with the enzyme in many ways as stated in option a, c, and d, so correct
option may be b. Altering the enzyme structure.
Q3. Allosteric enzymes are
[GATE]
Solution: Allosteric enzymes are generally larger and more complex than simple enzymes (refer text for details)
Q4. Which one of the following statements is true about non-competitive inhibition?
a. Km increases
b. Km decreases
c. Vmax increases
d. Vmax decreases
[IISc, 2012]
Solution: In non-competitive inhibition , which a special case of mixed inhibition the lines on double reciprocal
plot converse at one point on X axis which indicates the constant value of Km but the value of Vmax decreases
by increasing inhibitor concentration .
Q5. Abzymes are
[IISc, 2011]
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