Enzyme II Mechanisms of action BSc 2nd sem .pdf

BSc 2nd Sem , Zoology . Enzyme s

1. Enzyme II
Mechanism of enzyme action: ES complex,
transition state, activation energy,
binding energy,
Hypothesis of enzyme action: Key and
Lock theory; Induced fit theory
Classes of enzyme specificity

2. ❖The enzymatic catalysis of reactions is essential to living
systems.
❖Under biologically relevant conditions, uncatalyzedreactions
tend to be slow.
❖Most biological molecules are quite stable in the neutral-pH,
mild temperature, aqueous environment inside cells.
❖Moreover, many common chemical processes are unfavorable
in the cellular environment, such as the transient formation of
unstable charged intermediates or the collision of two or
more molecules in the precise orientationrequired for
reaction.
❖Reactions required to digest food, send nerve signals, or
contract a musclesimply do not occur at a useful rate without
catalysis.
Mechanism of enzyme action

3. ❖Enzyme provides a specific environment within which a given
reaction can occur more rapidly.
❖The distinguishing feature of an enzyme-catalyzed reaction is
that it takes place within the active site.
❖The surface of the active site is lined with amino acid residues
with substituent groups that bind the substrate and catalyze its
chemical transformation.
❖Often, the active site encloses a substrate, sequestering it
completely from solution.
❖In 1880, Charles- Adolphe Wurtz for the first time proposed
existence of enzyme substrate complex which is the key of a
enzyme catalyzed reactions.

4. ❖A simple enzymatic reaction can be written as:
❖where E, S, and P represent the enzyme, substrate, and product; ES and
EP are transient complexes of the enzyme with the substrate and with
the product.
❖The function of a enzyme is to increase the rate of a reaction. It do not
affect reaction equilibria. The direction of equilibrium of any reaction,
can be described by a reaction coordinate diagram.
❖In the coordinate diagram, the free energy of the system is plotted
against the progress of the reaction (the reaction coordinate).
❖The starting point for either the forward or the reverse reaction is called
the ground state.
❖The equilibrium between S and P reflects the difference in the free
energies of their ground states.
❖The free energy of the ground state of P is lower than that of S, so ∆G
’o

for the reaction is negative and the equilibrium favors P.
❖The Position and direction of equilibrium are not affected by any
catalyst.
Enzymes affect reaction rates, not equilibria

5. The free energy of the system is plotted against the progress of
the reaction S → P. Vertical axis represent the energy changes
during the reaction while the horizontal axis (reaction
coordinate) reflects the progressive chemical changes (e.g.,
bond breakage or formation) as S is converted to P.

6. ❖A favorable equilibrium does not mean that the S→P conversion
will occur at a detectable rate
❖The rate of a reaction is dependent on the energy barrier between
the S and P.
❖The energy required for alignment of reacting groups, formation of
transient unstable charges, bond rearrangements, and other
transformations required for the reaction to proceed in either
direction.
❖To undergo reaction, the molecules must overcome this barrier and
therefore must be raised to a higher energy level.
❖At the top of the energy hill is a point at which decay to the S or P
state is equally probable (it is downhill either way). This is called
the transition state.
❖The difference between the energy levels of the ground state and
the transition state is the activation energy, ∆G
‡
. Alternatively
energy required by S to reach at transition state and to convert into
P is called activation energy

7. ❖The rate of a reaction reflects this activation energy: a higher
activation energy corresponds to a slower reaction.
❖Reaction rates can be increased by raising the temperature and/or
pressure, thereby increasing the number of molecules with sufficient
energy to overcome the energy barrier and reach to transition state.
❖Alternatively, the activation energy can be lowered by adding a
catalyst.
❖Enzymes do not affect reaction equilibria, they enhance rate of
reaction by lowering activation energies and providing a lower
energy route for conversion of substrate to product.
❖Enzyme that catalyzes the reaction S → P also catalyzes the reaction
P→ S.
❖The role of enzymes is to accelerate the interconversion of S and P.
The enzyme is not used up in the process, and the equilibrium point
is unaffected.
❖However, the reaction reaches equilibrium much faster when the
appropriate enzyme is present, because the rate of the reaction is
increased.

8. Reaction coordinate diagram comparing enzyme catalyzed and
uncatalyzed reactions. In the reaction S → P, the ES and EP intermediates
occupy minima in the energy progress curve of the enzyme-catalyzed reaction.
The terms ∆G
‡
uncat and ∆G
‡
cat correspond to the activation energy for the
uncatalyzed reaction and the overall activation energy for the catalyzed
reaction, respectively.
The activation energy is lower when the enzyme catalyzes the reaction.

9. ❖Transition state is a fleeting molecular moment of S in which events
such as bond breakage, bond formation, and charge development
have proceeded to the precise point at which decay to either substrate
or product is equally likely.
❖The transition state is not a chemical species with any significant
stability and should not be confused with a reaction intermediate (such
as ES or EP).
❖This transition state has features of both substrate and product and falls
apart to yield product, which dissociates from the enzyme.
❖Transition state is highly unstable and transient
❖In every chemical transformation the S must reach the transition state
in order to convert into products
Transition State

10. Activation energy
❖The difference between the energy levels of the ground state and the transition
state is the activation energy, ∆G
‡
. Alternatively energy required by S to reach at
transition state and to convert into P is called activation energy
❖Activation energies are the energy barriers to chemical reactions.
❖The physical and thermodynamic factors that contribute to ∆G
‡
, are as follows:
❖Entropy (freedom of motion) of molecules in solution, which reduces the
possibility that they will react together
❖Solvation shell of hydrogen-bonded water that surrounds and helps to
stabilize most biomolecules in aqueous solution;
❖Distortion of substrates that must occur in many reactions
❖Need for proper alignment of catalytic functional groups on the enzyme.
❖The rate at which a molecule undergoes a particular reaction decreases as the
activation barrier for that reaction increases.
❖These barriers are crucial to life itself.
❖Without such energy barriers, complex macromolecules would revert
spontaneously to much simpler molecular forms, and the complex and highly
ordered structures and metabolic processes of cells could not exist.
❖Over the course of evolution, enzymes have developed to lower activation
energies selectively for reactions that are needed for cell survival.

11. ❖Enzymes are extraordinary catalysts. The rate enhancements they
bring about are in the range of 5 to 17 orders of magnitude.
❖ Enzymes are also very specific, readily discriminating between
substrates with quite similar structures.
❖How enzymes are able to enhance enormously the rate of
reaction with very specific substrate(s).
❖ How enzymes lower the activation eneg

Catalytic power and substrate specificity
Enzyme Rate Enhancements
Cyclophilin 10
5
Carbonic anhydrase 10
7
Triose phosphate isomerase 10
9
Carboxypeptidase A 10
11
Phosphoglucomutase 10
12
Succinyl-CoA transferase 10
13
Urease 10
14
Orotidine monophosphate decarboxylase 10
17

12. ❖Energy required to lower activation energies is derived from
weak, noncovalent interactions between substrate and enzyme.
❖Formation of each weak interaction in the ES complex is
accompanied by release of a small amount of free energy that
stabilizes the interaction.
❖The energy derived from enzyme-substrate interaction is called
binding energy, G
B. It is a major source of free energy used
by enzymes to lower the activation energies of reactions.
❖The Binding energy G
B contribute both in catalysis and
specificity of the reaction.
❖Weak interactions are optimized in the reaction transition
state.
❖Enzyme active sites are complementary not to the substrates
per se but to the transition states through which substrates pass
as they are converted to products during an enzymatic reaction.

13. How does an enzyme use binding energy to lower the activation energy
for a reaction?Weak Interactions between Enzyme and Substrate Are
Optimized in the Transition State

14. ❖When ES complex is formed, some weak interactions take place
in the ES complex, but the full complement of such interactions
between substrate and enzyme is formed only when the substrate
reaches the transition state.
❖The free energy (binding energy) released by the formation of
these interactions partially offsets the energy required to reach
the top of the energy hill.
❖The summation of the unfavorable (positive) activation energy
∆G
‡
and the favorable (negative) binding energy G
B results in
a lower net activation energy.
❖The transition state is a very brief point of time when substrate
spends atop an energy hill.
❖The enzyme-catalyzed reaction is much faster than the
uncatalyzed process, however, because the hill is much smaller
❖Weak binding interactions between the enzyme and the
substrate provide a substantial driving force for enzymatic
catalysis.

15. Binding energy G
B and Catalysis
❖The rate of any reaction is determined by the concentration of the reactant (or reactants)
and by a rate constant.
V = k[S];
If rate constant is↑ → rate of reaction is ↑
If rate constant is low → reaction will be occurring at slower rate.
❖There is relationship between magnitude of a rate constant and the activation energy.
❖Where k is the Boltzmann constant and h is Planck’s constant. The important point here
is that the relationship between the rate constant k and the activation
energy ∆G‡ is inverse and exponential.
❖In simplified terms, a lower activation energy means a faster reaction rate.
❖Binding energy accounts for the huge rate accelerations brought about by enzymes.
❖By using the above equation, it can be easily explained that decreasing the ∆G
‡
by 5.7
kJ/mol accelerate a first-order reaction by a factor of ten, under conditions commonly
found in cells.
❖The energy available from formation of a single weak interaction is generally estimated to
be 4 to 30 kJ/mol. The overall energy available from a number of such interactions is
therefore sufficient to lower activation energies by the 60 to 100 kJ/mol required to explain
the large rate enhancements observed for many enzymes.

16. ❖The binding energy that provides energy for catalysis
also gives an enzyme its specificity, the ability to
discriminate between a substrate and a competing
molecule.
❖If an enzyme active site has functional groups arranged
optimally to form a variety of weak interactions with a
particular substrate in the transition state, the enzyme
will not be able to interact to the same degree with any
other molecule
❖In general, specificity is derived from the formation of
many weak interactions between the enzyme and its
specific substrate molecule.
Binding energy G
B and Specificity

17. ❖The glycolytic enzyme triose phosphate isomerase catalyzes the
inter-conversion of glyceraldehyde 3-phosphate and dihydroxy-
acetone phosphate.
❖This reaction rearranges the carbonyl and hydroxyl groups on
carbons 1 and 2.
❖More than 80% of the enzymatic rate acceleration has been traced
to enzyme-substrate interactions involving the phosphate group on
carbon 3 of the substrate.
❖This was determined by comparing the enzyme-catalyzed
reactions with glyceraldehyde 3-phosphate and with
glyceraldehyde (no phosphate group at position 3) as substrate

18. reaction?
(1) The entropy (freedom of motion) of molecules in
solution decreases during the reaction
❖Binding energy allows active site to holds the substrates
in the proper orientation and close proximity.
❖Substrates can be precisely aligned on the enzyme, with
many weak interactions between each substrate and
strategically located groups on the enzyme clamping the
substrate molecules into the proper positions.
❖Studies have shown that constraining the motion of two
reactants can produce rate enhancements of many
orders of magnitude
Binding Energy overcome the factors of
activation energy barrier

19. (2)The solvation shell of hydrogen-bonded water that surrounds and
helps to stabilize most biomolecules in aqueous solution;
❖ Formation of weak bonds between substrate and enzyme results in
desolvation of the substrate.
❖ Enzyme-substrate interactions replace most or all of the hydrogen
bonds between the substrate and water.
(3) Many reaction involves the distortion of substrates
❖Binding energy involving weak interactions formed only in the
reaction transition state helps to compensate thermodynamically for
any distortion, primarily electron redistribution, that the substrate
must undergo to react.
(4)Need for proper alignment of catalytic functional groups on the
enzyme
❖Enzyme itself usually undergoes a change in conformation when the
substrate binds, induced by multiple weak interactions with the
substrate. This is called as induced-fit a mechanism postulated by
Daniel Koshland in 1958

20. Hypothesis of Enzyme Mechanism

21. Lock and Key theory
❖In the lock-and-key model, the enzyme is assumed to be the
lock and the substrate the key
❖The enzyme and substrate are made to fit exactly
❖This model fails to explain the activities of allosteric
enzymes.

22. Induce Fit theory
❖Daneil Koshland (1958) proposed the induce fit theory for
enzyme action.
❖It states that substrate binding to the enzyme induces a
conformation change in the active site which makes it perfectly
complementary to the substrate in the transition state.
❖During the enzyme substrate interactions enzyme forms multiple
weak interactions with the substrate
❖The theory explains the flexible nature of active site.

23. Possible Types of Transition State Changes
❖The enzyme might bring two reactants into close proximity and
maintain proper orientation
3.

24. Possible Types of Transition State Changes
❖The enzyme might put “stress” on a bond facilitating bond
breakage

25. ❖Acid-base catalysis: A molecule other than water
plays the role of a proton donor or acceptor. The
enzyme might modify the pH of the
microenvironment, donating or accepting a H
+
❖Covalent catalysis: The active site contains a
reactive group, usually a powerful nucleophile that
becomes temporarily covalently modified in the
course of catalysis.
❖Metal ion catalysis: Metal ions can serve as
electrophilic catalyst, stabilizing negative charge
on a reaction intermediate.

26. Classes of Enzyme Specificity
Enzymes show varied degree of specificities which are of
four types
❖Absolute: enzyme reacts with only one substrate
❖Group: enzyme catalyzes reaction involving any
molecules with the same functional group
❖Linkage: enzyme catalyzes the formation or break up of
only certain category or type of bond
❖Stereochemical: enzyme recognizes only one of two
enantiomers

27. Absolute Specificity
❖Some enzyme are capable of acting on only one
substrate. For example,
❖urease acts only on urea to produce ammonia and
carbon dioxide.
❖Similarly, carbonic anhydrase brings about the union
of carbon dioxide with water to form carbonic acid.
❖ Carbonic anhydrase
❖ H2O + CO2 → H2CO3

28. Group specificity
❖Some other enzymes are capable of catalyzing the reaction of
a structurally related group of compounds.
❖For example, lactic dehydrogenase (LDH) catalyzes the
interconversion of pyruvic and lactic acids and also of a number
of other structurally-related compounds.

29. Linkage specificity
❖Enzyme catalyzes the formation or break up of only certain
category or type of bond.
❖For example proteolytic enzymes pepsin, trypsin, chymotrypsin
and thrombin

30. Optical specificity
❖The most striking aspect of specificity of enzymes is that a
particular enzyme will react with only one of the two optical
isomers.
❖For example, arginase acts only on L-arginine and not on its
D-isomer. Similarly, D-amino acid oxidase oxidizes the D-
amino acids only to the corresponding keto acids.
❖Although, the enzymes exhibit optical specificity, some
enzymes, however, interconvert the two optical isomers of a
compound. For example, alanine racemase catalyzes the
interconversion between L- and D-alanine.

31. Geometrical specificity
Some enzymes exhibit specificity towards the cis and trans
forms.
❖As an example Fumarase catalyzes a stereospecific trans addition
of a hydrogen atom and a hydroxyl group. The hydroxyl group
adds to only one side of the double bond of fumarate; hence, only
the L isomer of malate is formed.

32. References
❖Nelson DL and Cox MM,
LehningerPrinciplesof Biochemistry,
5thEdition2008,W.H. Freemanand
Company, New-York​​
❖Berg,TymoczkoandStryerBiochemistry5th
Edition, W.H. Freeman andCompany, New-
York​​
❖JL Jain, Sixth edition 2005, S. Chand and
Company Ltd, New Delhi, India