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Enzymes
Enzymes
Catalyst: A substance that speeds up the rateof a chemical reaction but is not itselfconsumed.
Most biological catalysts are proteins, enzymes. A few catalysts are RNA: peptide bond
formation is catalyzed by RNA in ribosomes.
Some enzymes require organic coenzymes andor metal ions.
Apoenzyme / Apoprotein = Protein
Holoenzyme = Protein + Coenzyme
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Enzymes
Classification of Enzymes
Add -ase to the activity to obtain the name.
1. Oxidoreductases: transfer e- as H or H-
2. Transferases: transfer groups between molecules
3. Hydrolases: add functional groups to water
4. Lyases: form or add to double bonds.5. Isomerases: isomerize by group transfer
6. Ligases: form C-C, C-S, C-O, C-N bonds,coupled to ATP cleavage
Enzymes
Why are enzymes necessary?
1. Enzymes catalyze chemical reactions. They canacceleratebond formation and breakdown by 106-1012
2. Enzymes are responsible for the majority of all reactionsin living systems.
3. Enzymes are very specific; no side-reactions.4. Enzymes can be regulated.
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Enzymes
Our discussion of enzymes will be organized
into three sections:
1.Thermodynamic background to catalysis2.Mechanisms of catalysis3.Kinetics of catalysis
Enzymes
Fundamental terms and ideas for enzyme catalysis:
a) Substrate (S): the reactant on which the enzyme acts,
to convert it to the product (P)
in many cases, two (or even more) substrates reacttogether; there may be two or more products
b) Enzyme-substrate (E.S) complex: a non-covalent,
reversible association between enzyme and substrate
catalysis occurs in the E.S complexc) Active site: the pocket on the enzyme where the
substrate binds and the reaction is carried out
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Enzymes
(same structure; two representations)
Enzymes
We formulate the uncatalyzed reaction as:
S S P
and the enzyme-catalyzed reaction as:
S + E E.S E.S E.P E + P
Here, the double dagger symbol () refers to an activated
state ofincreased energy (the transition state), which
reactants must pass through on their way to the products. E.S and E.P are the non-covalent complexes
between enzyme and substrate or product.
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Enzymes
Transition state theory is a useful way to analyze what is
needed for catalysis to occur.
The rate of a chemical reaction depends on how muchenergy the reactant (or substrate) must acquire in order to
reach the transition state. In general:
reaction rate = (constant)T e -G /RT
Here, G is the activation energy, the extra energy that
S must acquire to reach the transition state S.
S is of higher energy than S because S mustundergo distortion (bond stretching or bending, locking in a
rare conformation) in order to react
Enzymes
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Enzymes
Since the enzyme catalyzes the reaction (makes it gofaster), it must somehow reduce the activation energy.
The situation for uncatalyzed and enzyme-catalyzed
reactions is shown below:
Enzymes
How does the enzyme do this? A key insight is obtained
from the diagram for the catalyzed reaction, where the
complexes E.S and E.P are intermediates:
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Enzymes
The E.S complex is partway along the reactioncoordinate, the horizontal dimension that measures how
far S has progressed in its conversion to P
that is, in the E.S complex S is partly distorted so it
starts to look like the transition state, S
For the uncatalyzed
reaction, S could not
move this far without
an input of free energy(see red arrow)
Enzymes
When S is bound to enzyme, no extra energy seems to be
needed to reach the distorted form seen in the E.S
complex. How could this be you ask?
1. Evidently, the formation of the E.S complex isenergy-releasing (spontaneous, favourable)
2. Some of this binding energy is used to distort thesubstrate, moving it along the reaction coordinate
3. Thus, even in the ground state E.S complex, theenzyme is already working to catalyze the reaction
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Enzymes
Enzymes
The source of energy which results in the lowering ofG is thebinding ofE to S in which a number of weak interactions areformed: H-bonds, hydrophobic interactions, van der Waalsinteractions.
The energy gained in this way (perhaps ~ 50-100 kJ/mol), wouldaccount for how an enzyme can speed up a reaction >1000x.
Other effects of E.S formation include:
1.A decrease in entropy of S (only one conformation)2.Desolvation (removal of H2O shell around S)3.Induced fit: the enzyme adjusts to the shape of S (the transition
state)
4.Alignment of the groups that must react
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Enzymes
How does the enzyme recognize & bind a substrate?
Probably the most important determinants are:
1. Shape consistency or fit (lock and key thesubstrate must fit the active site)
2. Electrostatic consistency - correct matching ofionic and H-bonds within the active site
3. Thermodynamic consistency - can the proteinflex to adopt a substrate or thesubstrate flex to fitinto the active site?
Enzymes
Thus, enzymes provide a special environment inwhich bond formation / breakage is easier.
Example: consider the hydration of carbon dioxide toproduce bicarbonate
H2O + CO
2HCO
3
-H+
+ In the absence of an enzyme, the above reaction is
slow because energy is required to break the O-H
bond of water and stretch one of the C=O bonds.
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Enzymes
As before, we can keep track of the free energy (G)changes by a reaction co-ordinate diagram.
H2O + CO
2HCO
3
-H+
+
P
S
G
Reaction co-ordinate
Lots of G must be added
to stretch the bonds to the
point of breaking in order
to drive the above
reaction.
Enzymes
The reaction catalyzed by Carbonic Anhydrase:
P
S
G
Reaction co-ordinate
E CO2
H2O E +E HCO3
-+ H
+HCO
3
-
is 106 times faster than
the uncatalyzed reaction!
Note that Keq is not affected by
a catalyst. If the enzyme
increases the forward rate k1,
then the back rate k2 will also
be increased since
G P S = G Gp
is lower as well.
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Enzymes
Binding Energy is also used for:1. Entropy Reduction: hold the substrates close
together in proper orientation for reaction.
C
C
O
O
OR
O-
C
C
O
O
O
A
+ OR-
B
CH3C OR
O
CH3C O
-
O
+
CH3
C
O
O
O
CCH3
+ OR-
Fore.g. reaction
A is 105 times
faster than
reaction B.
Enzymes
Binding Energy is also used for:
2. Desolvation: Substrate molecules are surroundedby a water hydration shell that must be removed forreactions to occur.
3. Strain Reduction: Steric and/or electronic strainmust be overcome.
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Enzymes
Enzyme Specificity: Generally, the enzyme is large compared to the substrate and
the substrate binds at a unique location, the active site.
Also, generally, each enzyme catalyzes one reaction using alimited number of substrates.
This enzyme will recognize only D-amino acids, and aseparate enzyme is needed to recognize L-amino acids.
e.g. 1) optical (chiral) specificity
C NH3+
H
COO-
CH2
OH
C
COO-
O
CH2OH
D-serine hydroxypyruvate
D-aminoacid
oxidase
Enzymes
Enzyme Specificity:
eg. 2) geometric specificity
CHOOC
C
COOH-
--
-
CHOOC
H
C
COOH
HO
+ H2O
Fumarate Malate
Fumarase converts fumarate (trans isomer) to malate,
but cannot use maleate (cis isomer) substrate
recognition is very specific.
-
-
C
H COO
C
COOH
Maleate
Fumarase
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Enzymes
Enzyme Specificity:
eg. 3) diastereomeric specificity
COOH
CH2
C COOHHO
CH2
COOH
COOH
CH2
C COOHH
C
COOH
HHO
Citrate Isocitrate
Aconitase
Aconitase can distinguish between the two ends of
citrate even though there is no chiral C - how??
Enzymes
Enzyme Specificity:
eg. 3) diastereomeric specificity
The enzyme provides a binding site that is complementary to the
steric and electronic features of the substrate: hand-in-glove
It can, because the
enzyme is a 3-
dimensional molecule
with 3 sites of
interaction:
O-
O
CCH2
CC
OHH
C
H O
O-
CO
O-
A B
C
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Enzymes
Mechanisms of enzyme catalysisEnzymes use their AA side-chains to participatein the chemical transformation. Here is a classicexample:
General Acid-Base Catalysis
Recall that chymotrypsin is a proteolytic enzymethat cleaves peptide bonds at Trp, Tyr, Phe.
There two important types of catalytic amino acidsin the active site:Acidic residues: donate H+ & accept electrons
Basic residues: accept H+ & donate electrons
Enzymes Mechanism of chymotrypsin
3D Structure of chymotrypsin highlighting
the active site of the enzyme.
catalytic triad
aromatic side
chain pocket
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Enzymes Mechanism of chymotrypsin
Oxyanion hole
Aromatic side
chain
Enzymes Mechanism of chymotrypsin
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Enzymes
The side chains ofHis57, Asp102, Ser195 in
chymotrypsin form a Catalytic Triad.
Mechanism of chymotrypsin
1
Enzymes
ES complex
The next step involves
electron flow (arrows)
from the General Base
Catalytic Triad into S.
This creates the 1st
tetrahedral intermediate
(next slide).
Mechanism of chymotrypsin
Substrate binding
compresses the bondbetween Asp102 and
His57, altering the pKa
of His57 to >12.
2
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Enzymes
1st Tetrahedral
Intermediate
The oxyanion isstabilized by H-bonding
to groups in the protein
known as the oxyanion
hole. Notice that a
covalent bond has formed
between the E (Ser195)
and S.
Next, electrons flow from thesubstrate to the General Acid
Catalytic Triad (arrows).
Mechanism of chymotrypsin
3
Enzymes
Acyl-enzyme
intermediate
The C-N bond has now
been cleaved and the C-
terminal peptide
(stabilized by the
donated proton) is
released:
Note: Acyl group:
Mechanism of chymotrypsin
4
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Enzymes
Acyl-enzyme
intermediate
Mechanism of chymotrypsin
The N-terminus is now
going to be released by
hydrolysis:
An incoming water molecule
is activated by His57 (acting
as ageneral base) to create a
hydroxyl group that attacks
the carbonyl carbon of the
enzyme-substrateintermediate
5
Enzymes
2nd tetrahedral
intermediate
Mechanism of chymotrypsin
Next, His57 acts as a general
acid, protonating Ser195,
causing collapse of the 2nd
tetrahedral intermediate.
6
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EP complex
Enzymes Mechanism of chymotrypsin
The N-terminal product is
now free to depart from
the active site and thecatalytic triad is ready to
receive another substrate
molecule.
7
Enzymes Mechanism of chymotrypsin
NOTE: The latter half of the
reaction is the same as in the
first half: General base followed
by general acid catalysis.
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Enzymes
Why on earth study protease mechanism (and many
other enzymes) to such a degree?
Answer: These mechanistic studies provide
fundamental knowledge for understanding the molecular
basis of life, modern drug development, medicine and
agriculture!
Enzymes and HIV
HIV RNA genome
Translation
HIV polyprotein
HIV proteinase
Virus assembly
Individual (now active)
HIV enzymes and
proteins
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Enzymes
X-ray crystal structure of HIVproteinase bound to the
inhibitor Viracept.
Kaldor S.W. et al. (1997)J. Med. Chem. 40, 3979-3985.
HIV proteinase inhibitors are
designed using a combination of
structure based drug design
and enzymology research
Enzymes
Mechanisms of enzyme catalysis
About 1/3 of all enzymes use metal cofactors.1. Weak interactions between metals and the substrate help
stabilize charged transition states and may help orient andbind the substrate.
2. Metals accept & donate electrons in Redox reactions
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Enzymes
Mechanisms of enzyme catalysis
Carboxypeptidase uses zinc
in its active site as an
alternative to using amino
acids to form an oxianion
hole (chymotrypsin).
Note the proximity of
reactive sites and the
importance of binding
interactions
Enzymes
3. Enzyme kineticsBy way of review, for the reaction S P:
Every enzyme will have an optimum set of conditions including
temperature, [salt], pH etc. which must be determined experimentally.
V =d[P]
dt=
d[S]
dt
In moles per L per sec.
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Enzymes
Once the optimal conditions are determined, thesestandard conditions are employed in the main goal of
enzyme kinetics: The determination of v [S]or
the influence of[S]on the velocity which is related tothe enzyme-substrate affinity.
The hyperbolic relationship
between v and [S] is typical
of an enzyme that exhibits
what are called Michaelis-Mentenkinetics
Enzymes
Deriving the M-M equation is relatively easy andillustrates some of the principles underlying enzyme
kinetics. Consider the following mechanism:
Assumptions include:
1. k3>>k4 so k4 is not a factor (which is the caseearly in the reaction when [P] = 0).2. k1>k3 which makes E + P formation the slow orrate determining part of the reaction.
i.e. v = k3[ES] defines the overall velocity
E + S E.S E + P
k1
k2
k3
k4
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Enzymes
Enzyme can exist free and substrate bound:[Etotal] = [E] + [ES] (eqn A)
The maximum velocity VMAX will occur when allE isbound to S or,
[Etotal] = [ES] or VMAX = k3[Etotal]
E + S E.S E + P
k1
k2
k3
k4
Enzymes
Derivation of the Michaelis-Menten equation
Rate of formation of E.S = k1[E][S]
Rate of breakdown of E.S = k2[E.S] + k3[E.S]
In steady state (when [E.S] remains constant)k1[E][S] = (k2+k3)[E.S] or,
[ES] =k1[E][S]
(k2 +k3)=
[E][S]
(k2 +k3
k1)
E + S E.S E + P
k1
k2
k3
k4
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Enzymes
Derivation of the Michaelis-Menten equation[ES] =
k1[E][S]
(k2
+k3)
=[E][S]
(k
2+k
3
k1
)
we can define:
KM=
k2+ k
3
k1
(the Michaelis constant)
[ES] =[E][S]
KM
So, (eqn B)
Using [Etotal] = [E] + [E.S] we get: [E] = [Etotal] - [E.S]
Substitute this into eqn B: [ES] =([E
total] [ES])[S]
KM
From
Enzymes
Derivation of the Michaelis-Menten equation
[ES]=([E
total] [ES])[S]
KM
is now rearranged:
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Enzymes
Derivation of the Michaelis-Menten equation
Remember also thatVMAX = k3[Etotal] or,
From above,v = k3[ES] andwe can insert eqn C to give:
V =k3[E
total][S]
KM+ [S]
V =VMAX
[S]
KM + [S]
Michaelis-Menten Equation
Enzymes
The equation is straightforward but the significance and meaning of the KM is
very important:
1. KM is a measure of the affinity of E for SK
M=
k2+ k
3
k1
And since k2>>k3 very often,
KM k2
k1Keq
1 for
Thus, a high KM reflects fast dissociation or a low affinity. A low KM reflects slow
or limited dissociation or a high affinity.
E + S E.S
k1
k2
E + S E.S E + P
k1
k2
k3
k4
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Enzymes
2. KM = [S] at 1/2 VMAX For example,
-Thus, a high KM reflects the need for high [S] for areaction (ie an unfavorable reaction).
-A low KM reflects the need for a low [S] for a reaction(ie a favorable reaction).
3. It follows then when KM = [S], 1/2 of E is
bound to S or [E.S] = [E]
Enzymes
KM values are typically in the M to mM region
Determination of KMAttempts to calculate KM from a v vs. [S] graph is
complicated by the necessity of estimating VMAX
The Lineweaver-Burk plot
can give a more accurate
determination (not used
anymore in practice, butprovides a visual means to
determine KM and VMAX)
Vo
Vmax
[S]
Vmax
2
Km
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Enzymes
The Lineweaver-Burk plot equation:
OR
This is an equation for a straight line (y=mx+c)!
Simplifies to:
Enzymes
The Lineweaver-Burk plot: Note: Because you neverreach [S] = , VMAX is
never reached, but
extrapolation of a straight
line is possible
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Enzymes
Other kinetic constants
kcat (catalytic rate constant) = k3Reflects the slow step and the turnover rate of the enzyme.
That is, v = k3[ES]
and remember VMAX = k3[Etotal]
or VMAX = kcat[Etotal]
Therefore,kcat =
VMAX
[E total]
with units t-1(1/time)
That is, how many times per seconddoes a reaction take
place. Kcat values range from 5x10-1 to 4x107 per sec.
E + S E.S E + P
k1
k2
k3
k4
Enzymes
Other kinetic constants
kcat
KM
The specificity constant
(Reflects the catalytic efficiency of an enzyme)
v VMAX
[S]
KM
(when KM >> [S])
v =
kcat
KM
[Etotal
][S]
It is used to compare catalytic efficiencies of different
enzymes or turnover of different substrates. It is much more
accurate than using one of the parameters on its own.
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Enzymes
Enzyme inhibitors - are categorized on the basis ofhow they affect enzyme kinetic parameters.
An inhibitor is a compound which when added to a
solution containing enzyme and substrate reduces the
rate of conversion of S P.
1.Competitive2.Noncompetitive orMixed3.Uncompetitive
There are three main types of reversible inhibition:
Enzymes
1.Competitive Inhibitors - resemble the substrate and bindin the active site, blocking access of the natural substrate. e.g
Lipitor, Viagra, Protease inhibitors, AZT
E + S + I ES + EI
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Enzymes
1.Competitive Inhibitorse.gmalonic acid, is an inhibitor of succinate dehydrogenase:
COOH
CH2
CH2
COOH
COOH
C
C
HOOC H
H2H
Succinic Acid Fumaric Acid
NOTE: At high [S] the I is displaced from E so Vmax is unchanged.
Enzymes
1.Competitive InhibitorsBut Km is apparently increased because it takes much more S
to reach Vmax. Recall that Km is the apparent affinity of E for S.
Vo
Vmax
[S]
Vmax
2
Km KmI
I
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Enzymes
1.Competitive InhibitorsBut Km is apparently increased because it takes much more S to
reach Vmax. Recall that Km is the apparent affinity of E for S.
1Vo
1 / [S]
I
-1/Km -1/KmI
1
Vmax
Enzymes
2. Non-Competitive Inhibitors
I binds at a site distinctfrom the substrate site, usually
an allosteric site. Allos - Greek - other Stereos -
shape
S
EI
It may bind tofree Eor to E.S. Once bound it will prevent
P formation.
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Enzymes
2. Non-Competitive Inhibitors
S
EI
ESE + S E + P
+ +
I I
EI + S EIS X
KIKI
If the binding affinities ofI to E and ES are identical, therewill be no effect on Km.
But since the I decreases active [Etot] then Vmax must
decrease (Vmax = kcat [Etot] ).
Enzymes
2. Non-Competitive Inhibitors
If the binding affinities ofI to E and ES are identical, therewill be no effect on Km.
But since the I decreases active [Etot] then Vmax must
decrease (= kcat [Etot] ).
[S]Km
Vmax
I
Vo
VImax
VImax
2
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Enzymes
2. Non-Competitive Inhibitors
1 / [S]
1Vo
-1/Km
I
1/Vmax
1/VmaxI
(If the affinity ofI for E and ES are different, Mixed
Inhibition is obtained and both Vmax and Km are changed.)
Enzymes
3. Uncompetitive Inhibitors eg. Roundup
Again, I binds at an allosteric site, but only to the ES complex.
The slopes of 1/Vovs. 1/[S] are unchanged but Vmax is lower, and
so is the apparent [S] needed to reach 1/2 Vmax = Km.
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Enzymes
3. Uncompetitive Inhibitors
Again, I binds at an allosteric site, but only to the ES complex.
The slopes of 1/Vovs. 1/[S] are unchanged but Vmax is lower, and
so is the apparent [S] needed to reach 1/2 Vmax = Km.
VImaxVo I
Vmax
Km [S]KIm
Enzymes
3. Uncompetitive Inhibitors
Again, I binds at an allosteric site, but only to the ES complex.
The slopes of 1/Vovs. 1/[S] are unchanged but Vmax is lower,
and so is the apparent [S] needed to reach 1/2 Vmax = Km.
Vmax
1
-1/KmI -1/Km
I
1 / [S]
1Vo
1
VmaxI
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Enzymes
Allosteric Enzymes vovs [S] plot is Sigmoidal whereas M-M plots are
Hyperbolic.
[S]
Vo
Sigmoidal
Hyperbolic
The enzyme will be very sensitive to [S] over a
narrow range and behaves like an on-off switch.
Enzymes
Allosteric Enzymes The non-MM behaviour arises in multisubunit
enzymes where the occupancy of an active site on
one subunit has an effect on the other subunits.
This is called cooperativity.
The binding of S to one active site of the enzymemakes the binding of subsequent S easier. How?
First, each subunit can exist in 2 conformations:
High affinity - R-state (relaxed)
Low affinity - T-state (taut)
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Enzymes
Allosteric Enzymes Without S, the equilibrium favours T and weak binding.
Binding of S stabilizes the R-state pulling the equilibrium
to the high affinity form simply by the Law of Mass
Action:
+ SS
S
T R
Lots Little
S
S
S
Increasing the amount ofR-state creates
more high-affinity sites and results in more Sbinding, until the enzyme is saturated.
This can be shown to produce sigmoidal Vo
vs. [S] graphs.
Enzymes
Allosteric Enzymes Anything that displaces the equilibrium will affect the
kinetic curveand this includes activators (A) &
inhibitors (I).
Inhibitors bind selectively to T-state, and therefore pullthe equilibrium towards T-state. Activators bind to R-
state (as S does) and pulls the equilibrium towards R-
state:
+I +A AI
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Enzymes
Allosteric EnzymesFor example, the enzyme phosphofructokinase(substrate: fructose-6-P) is inhibited by high levels ofATP and activated by AMP.
Vo+AMP
+ATP
[Fructose-6 phosphate]
Inhibitors and activators do
not bind at the activesite,
but at otherallosteric sites,
from which they can still
influence the R T
equilibrium of the protein.
Enzymes
Allosteric proteins Hemoglobin is not an enzyme, but allostery is an important part
of its function. Hemoglobin binds O2 cooperatively. This allows
it to respond to changes in O2 demand by different tissues.
HMS Cell Biology Visualization website
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Enzymes
Allosteric proteinsO2 binding to hemoglobin is also inhibited by 2,3-bisphosphoglycerate (BPG),
always present in red blood cells. Because fetal hemoglobin (Hb F) has a lower
affinity for BPG than adult hemoglobin (Hb A) does, it has a higher affinity for
O2. This situation permits the fetus to extract O2 from mothers blood.
Enzymes
Advantages of Allosteric enzymes A metabolic pathway is one in which the product of
the 1st Enzyme is a substrate for the 2nd Enzyme etc.
Usually regulation occurs at the beginning of thepath to prevent waste.
In this example, E1 is threonine dehydratase. It isthe key regulatory enzyme in the pathway and is
inhibited by the end product,L-Ile.
E5
E4
E3E2E1
DCBA L-IleL-Thr
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Enzymes
Advantages of Allosteric enzymes
This is known as end-product inhibition orfeedback
inhibition
end-product inhibitors bind at allosteric sites, since
they do not resemble the substrate for the firstenzyme, and cannot bind at the active sites.
E5
E4
E3E2E1
DCBA L-IleL-Thr
Enzymes
Advantages of Allosteric enzymesA more subtle advantage lies in increased sensitivity to
changes in [S] over a narrow [S] range:
Over a physiological substrate range, activity of the
allosteric enzyme is much more sensitive to [S] than the
MM enzyme is, allowing tight regulation of its activity.
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Enzymes
Other types of regulationCovalent Regulation
e.g. Glycogen phosphorylase is activated by phosphorylationof Ser (catalyzed by a protein kinase) a covalent
modification that induces an allosteric conformational
change. This is reversible by a phosphatase that removes
the phosphate from the Ser side chain.
Phosphorylation of enzymes to activate them is an importantmetabolic control mechanism.
A number of other covalent modifications of enzymes arealso known.
Enzymes
Other types of regulation
Zymogens Enzyme activation by proteolytic cleavage. The pancreas produces inactive trypsinogen,
chymotrypsinogen, proelastase, procarboxy-peptidase to
prevent digestion of the pancreas.
A duodenal enzyme enteropeptidase activates trypsin byremoving AA 1-6 of trypsinogen
Trypsin then activates the other proenzymes, whichfunction in digestion of proteins in the duodenum
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Enzymes
Irreversible Enzyme Inhibitors An irreversible inhibitor forms a covalent bondwith an
active site AA.
Penicillin reacts with the active site Ser in transpeptidase,an enzyme involved in bacterial cell wall synthesis.
Aspirin acetylates Ser in the enzyme COX-2 (producesprostaglandin, a hormone that stimulates inflammation),
and relieves inflammation-linked disease like arthritis.
Diisopropylfluorophosphate reacts with Ser-195 inchymotrypsin, inactivating the enzyme.
Enzymes
Summary of enzymes Energetics of catalysis Enzyme specificity Mechanisms of enzyme catalysis Enzyme Kinetics - Michaelis-Menten eqn
KM, kcat, Lineweaver-Burk plot Inhibitors - competitive, uncompetitive,
noncompetitive Allosteric enzymes - equilibrium RT Other types of regulation
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