This document discusses enzyme kinetics and the mechanisms of enzyme catalysis. It begins with definitions of key terms like enzyme and catalysis. It then describes classification of enzymes and the structure of active sites. The mechanisms of enzyme catalysis include acid-base, covalent, and metal ion catalysis. It also discusses models of enzyme-substrate binding like lock-and-key and induced fit. Kinetics concepts covered include Michaelis-Menten kinetics, Lineweaver-Burk plots, and factors affecting reaction rates like temperature and pH. Finally, it describes reversible and irreversible inhibition and different types of inhibitors.

1. Devangi Vanra
Zoology final
Roll no. - 8

2.  Introduction
 Definition
 Classification
 Mechanism of catalytic reaction
 Enzyme kinetics
 Michaelis – Menten equation
 Lineweaver- Burk equation
 Inhibition of Enzyme activity

3.  Frederick W. Kühne coin the term enzyme.
 Enzyme (Greek word - en = in, zyme = yeast), Which means in yeast
 Because of most recognisable reaction popularity known as alcohol fermentation
by zymase enzyme in yeast.
 Enzymes are biological catalysts that speed up the rate of the biochemical
reaction.
 Most enzymes are three dimensional globular proteins (tertiary and quaternary
structure).
 Some special RNA species also act as enzymes and are called Ribozymes
e.g.Hammerhead ribozyme.

4.  With the exception of a few classes of catalytic RNA molecules
enzymes are proteins. Their catalytic activity depends on the
integrity of their native protein conformation.
 If an enzyme is denatured or dissociated into its subunits, catalytic
activity is usually lost. The catalytic activity of each enzyme is
intimately linked to its primary, secondary, tertiary, and quaternary
protein structure.

5.  The active site of an enzyme is the region
that binds substrates, co-factors and
prosthetic groups and contains residue
that helps to hold the substrate.
 Active sites generally occupy less than 5%
of the total surface area of enzyme.
 Active site has a specific shape due to
tertiary structure of protein.
 A change in the shape of protein affects
the shape of active site and function of the
enzyme.

6. Co factor :
 It is an additional chemical ( non protein ) component which is
required for enzyme activity.
 Two types : Co enzyme and prosthetic group
Co enzyme
 Co enzyme is a small , organic, non protein molecules that carry
chemical groups between enzymes.
 Transfer the chemical groups
 Loosely bound, easily removed from the enzyme
 Ex – biotin, which transfers CO2 (Biocytin enzyme)

7.  Prosthetic group :
 Tightly or covalently bound with the enzymes or protein.
 Hard to remove.
 Either metal ion or small organic molecules
 Ex – Mg+² in hexokinase
 Holo enzyme :
 A complete, catalytically active enzyme together with its bound
coenzyme and/or metal ions is called a holoenzyme.
 Apoenzyme / Apoprotein
 The protein part of holo enzyme is called the apoenzyme or
apoprotein.

8. Classification is based on the type of reactions which is
catalyzed by enzymes.
Total 7 classes:

10.  Reaction takes place within the confines of a pocket on the enzyme
called the active site. The active site provides a specific
environment, customized by evolution, in which a given reaction can
occur more rapidly.
 The molecule that is bound in the active site and acted upon by the
enzyme is called the substrate.
 The surface of the active site is lined with amino acid residues with
substituent groups that bind the substrate and catalyze its chemical
transformation.
 The enzyme-substrate complex is central to the action of enzymes.

11.  A simple enzymatic reaction might be written
E + S ⇌ ES ⇌ EP ⇌ E + P
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.

12. The catalytic activity of enzymes can be explained by two
perspective
1) Thermodynamic changes
2) Processes at the active site

13. THERMODYNAMIC CHANGES
 All chemical reactions have energy barrier between reactant and
products.
 The starting point for either the forward reaction or the reverse
reaction is called the ground state,
 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 level of the ground state and the
energy level of the transition state is the activation energy, ΔG‡

15.  Only a few substances cross the activation barrier and change into
products.
 That is why rate of uncatalyzed reactions is much slow.
 Enzymes provide an alternate pathway for conversion of substrate
into products.
 Enzymes accelerate reaction rates by forming transitional state
having low activational energy.
 Hence, the reaction rate is increased many folds in the presence of
enzymes.
 The total energy of the system remains the same and equilibrium
state is not disturbed.

16. Covalent interaction:
1. General acid base catalysis
2. Covalent catalysis
3. Metal ion catalysis
Non covalent interaction
1) Lock and key hypothesis
2) Induced fit model

17. ACID BASE CATALYSIS
 Transfer of a proton from one molecule to another is the single most
common reaction in biochemistry.
 Catalysis of the type that uses only the H+(H3O+)or OH- ions
present in water is referred to as specific acid-base catalysis.
 The term general acid-base catalysis refers to proton transfers
mediated by weak acids and bases other than water.
 Several amino acid side chains can and do take on the role of proton
donors and acceptors. These groups can be precisely positioned in an
enzyme active site to allow proton transfers, providing rate
enhancements of the order of 10² to 10⁵

18. COVALENT CATALYSIS
 A transient covalent bond is formed between the enzyme and the
substrate.
 Consider the hydrolysis of a bond between groups A and B:
 A—B A + B
 In the presence of a covalent catalyst (an enzyme with a nucleophilic
group X: the reaction becomes
 A—B + X:→ A—X + B → A + X:+B
 Formation and breakdown of a covalent intermediate creates a new
pathway for the reaction, but catalysis results only when the new
pathway has a lower activation energy than the uncatalyzed
pathway.
 These covalent complexes always undergo further reaction to
regenerate the free enzyme.
H2O

19. METAL ION CATALYSIS
 Ionic interactions between an enzyme-bound metal and a substrate
can help orient the substrate for reaction or stabilize charged
reaction transition states.
 Metals can also mediate oxidation-reduction reactions by reversible
changes in the metal ion’s oxidation state
 Nearly a third of all known enzymes require one or more metal ions
for catalytic activity.

20. LOCK AND KEY MODEL
 Proposed by Emil Fischer in 1894.
 Lock and key hypothesis assumed that the enzymes were structurally
complementary to their substrate, so that they fit like a lock and key.
 There is no change in the active site before and after a chemical reaction.

21. INDUCED FIT MODEL
 Proposed by DANIAL KOSH LAND in 1958.
 According to this exposure of an enzyme to substrate cause a change in enzyme,
which causes the active site to change it’s shape to allow enzyme and substrate to
bind..
 It brings specific functional group of the enzyme in proper position.

22.  Introduction :
 “It is a branch of biochemistry in which we study the rate of enzyme
catalyzed reactions.”
 Studying an enzyme’s kinetics in this way can reveal the catalytic
mechanism of that enzyme, its role in metabolism, how its activity is
controlled, and how a drug or an agonist might inhibit the enzyme.

23. RATE OF REACTION AND THEIR
DEPENDENCE ON ACTIVATION ENERGY
 Activation Energy (Ea):
 “The least amount of energy needed for a chemical reaction to take place.”
 Enzyme (as a catalyst) acts on substrate in such a way that they
lower the activation energy by changing the route of the reaction.
 The reduction of activation energy (Ea) increases the amount of
reactant molecules that achieve a sufficient level of energy, so that
they reach the activation energy and form the product
 Example: Carbonic anhydrase catalyses the hydration of 10 CO₂
molecules per second which is 10’x faster than spontaneous
hydration.

24. Enzymes catalysis:
 “It is an increase in the rate of reaction with the help of enzyme(as
catalyst).”
 Catalysis by enzymes that proceed via unique reaction mechanism,
typically occurs when the transition state intermediate forms a
covalent bond with the enzyme(covalent catalysis).
 During the process of catalysis enzymes always emerge unchanged
at the completion of the reaction.

25. Temperature
Hydrogen ion concentration (pH)
Substrate concentration

26.  Raising the temperature increases the rate of enzyme catalyzed
reaction by increasing kinetic energy of reacting molecules.
 Enzymes work maximum over a particular temperature known as
optimum temperature. Enzymes for humans generally exhibit
stability temperature up to 35-45 C
 The temperature coefficient is a factor Q, by which the rate of
biological processes increases for a 10° C increase in temperature.
 For most biological processes Q = 2.
 However some times extreme heat can denature the enzyme.

28. Effect of pH
 Rate of almost all
enzymes catalyzed
reactions depends on PH
 Most enzymes exhibit
optimal activity at pH
value between 5 and 9
 High or low pH value
than optimum value will
cause ionization of
enzyme which result in
denaturation of enzyme

29.  Michaelis-Menten Model:
 “According to this model the enzyme reversibly combines with substrate to form an ES
complex that subsequently yields product, regenerating the free enzyme.”
Where, S is the substrate
E is the enzyme
ES-is the enzyme substrate complex
P is the product
K1,K-1 and K2 are rate constants

30.  “It is an equation which describes how reaction velocity varies with
substrate concentration.”
Where
V0 is the initial reaction velocity.
Vmax is the maximum velocity.
Km is the Michaelis - Menten constant
[S] is the substrate concentration.

38.  This form of the Michaelis-
Menten equation is called the
Lineweaver- Burk equation.
 It is like linear graph equation
 Y = mX + b
 Graph is plotted against 1/[S] vs
1/V0

39. LINEWEAVER – BURK GRAPH

40. INHIBITION
 The prevention of an
enzyme process as a
result of interaction of
inhibitors with the
enzyme.
 INHIBITORS:
 Any substance that can
diminish the velocity of
an enzyme catalyzed
reaction is called an
inhibitor.

41. Reversible
 Inhibitor binds to enzyme reversibly
through non covalent interaction.
 The activity of enzymes is fully
restored after removing inhibitor.
 Types :
Competitive
Non competitive
Un competitive
Irreversible
 Inhibitor binds at or near the active
site of the enzyme irreversibly, usually
by covalent bonds, so it can’t dissociate
from the enzyme.
 Irreversible inhibitors occupy or
destroy the active sites of the enzyme
permanently and decrease the reaction
rate.
 Types :
Active site directed
Suicide inhibitor

42.  A competitive inhibitor often has structural features similar to those
of the substrate whose reactions they inhibit.
 This means that a competitive inhibitor and enzyme’s substrate are
in direct competition for the same binding active site on the enzyme.

43.  These are not influenced by the concentration of the substrate. It
inhibits by binding irreversibly to the enzyme but not at the active
site.
 They also bind with the same affinity to the free enzyme and form
the Enzyme-Substrate complex.
 It change the shape of enzyme and active site.

44.  Uncompetitive inhibitors do not bind to the free enzyme. They bind only to the
enzyme-substrate complex to yield an inactive E. S. complex.
 Uncompetitive inhibitors frequently observed in multi substrate reaction.
 Inhibition can’t be reversed by increasing the [S] since I doesn’t compete with S for
the same binding site.

45.  Active site directed inhibitor is also called as affinity label.
It is a chemically reactive compound that is designed to
resemble the substrate of an enzyme so that it binds at the
active site and forms a stable covalent bond with a
susceptible group of the nearby residue in the enzyme
protein.
 Affinity labels are very useful for identifying catalytically
important residues.

46.  A suicide inhibitor is a relatively inert molecule that is transformed
by an enzyme at its active site into a reactive compound that
irreversibly inactivates the enzyme
 They are substrate analogs designed so that via normal catalytic
action of the enzyme, a very reactive group is generated.
 The latter forms a covalent bond with a nearby functional group
within the active site of the enzyme causing irreversible inhibition.

47. Lehninger : Principles of biochemistry (8th edition
https://www.slideshare.net/KamalKishor31/enzyme-
kinetics-57408548)