Enzymes catalyze chemical reactions by reducing the activation energy needed for the reaction to occur. They do this using several mechanisms including acid-base catalysis, covalent bond formation, and metal ion catalysis. Enzymes are also able to increase reaction rates by properly orienting substrates. Enzyme activity can be inhibited through various reversible and irreversible mechanisms such as competitive inhibition where an inhibitor binds to the active site, and suicide inhibition where the inhibitor is converted by the enzyme into a tightly-binding form. The Michaelis-Menten model and Lineweaver-Burk plots are commonly used to study enzyme kinetics and inhibition types.

1. ENZYME CATALYSIS
Department of Pharmacy
University of Peshawar

2. Contents
 Enzyme catalysis
 Mechanisms of enzyme catalysis
 Enzyme inhibition
 Types and mechanisms of enzyme inhibition

3. Definition
Catalysis
“An increase in the rate of reaction due to participation of a catalyst”.
Enzyme Catalysis
It is a sub-type of catalysis and is defined as “an increase in the rate of a chemical
reaction due to the participation of an enzyme”.

4. Characteristics of Enzyme Catalysis
The main characteristics of enzymatic catalysis include:
Efficiency
Enzymes are highly efficient, one molecule of an enzyme can transform more than one
million molecules of substrate a minute.
Specificity
Enzymes are highly selective and produce only one type of product
Activity
Enzymes are highly active under optimum temperature and pH

5. Classification of Enzyme
Based on their function enzymes are classified in to six classes
Oxidoreductases
Catalyzes oxidation-reduction reactions by the transfer of electrons as addition or removal of hydrogen atoms e.g. Cytochrome P450
Transferases
Catalyzes transfer of group usually attached to a co-factor e.g. Hexokinase, Methyl transferase
Hydrolases
Catalyzes hydrolysis reactions i.e. transfer of functional groups of water e.g. Chymotrypsin.
Lyases
Catalyzes the addition of a functional group by breaking a double bond or vice versa e.g. Deaminase
Isomerases
Catalyzes the transfer of functional groups within a molecule forming isomers e.g. Phosphoglucose isomerase.
Ligases
Catalyzes the formation of a C-C, C-S, C-N, C-O bonds due to condensation reaction e.g. DNA ligase

6. Process of Enzyme Catalysis
Enzymes achieve their enormous reaction rate accelerations by reducing the activation
energy via the same catalytic mechanisms used by chemical catalysts.

7. Mechanisms of Enzyme Catalysis
Enzymes carryout their function by the following mechanisms:
 Acid-Base Catalysis
 Covalent Bond Catalysis
 Metal-Ion Catalysis
 Proximity & Orientation Based Catalysis

8. Acid-Base Catalysis
 A proton transfer from an acid or hydroxyl from a base lowers the activation energy
of a reaction
 Ionizable functional groups of amino acyl side chains and of prosthetic groups
contribute to catalysis by acting as acid or base
 The catalytic activity is pH sensitive
 Functional groups of amino acids such as glutamic acid, aspartic acid, lysine,
histidine act as an acid or base.
Example
 Bovine pancreatic RNase A is an acid-base catalyst, it hydrolyzes RNA to its
component nucleotides in the intestine.

9. Covalent Bond Catalysis
 Catalysis is achieved by the transient formation of a covalent bond between the
enzyme and substrate.
 Usually the covalent bond is formed by the reaction of a nucleophilic group on the
catalyst with an electrophilic group on the substrate. Therefore, it is also known as
nucleophilic catalysis.
 The following functional group may form a covalent bond.
• Amino group of Lysine
• Imidazole group of Histidine
• Carboxyl group of Aspartic acid
• Hydroxyl group of Serine
Examples
• Acetylcholine esterase, Chymotrypsin, Trypsin, elastase and alkaline phosphatase

10. Mechanism

11. Metal-Ion Catalysis
 More than 30% of all enzymes require a metal ion for its catalysis.
 Such enzymes are also known as metalloenzymes.
 Most common metal ions include Fe2+, Fe3+, Cu2+, Mn2+, Co2+.
 Catalysis by metalloenzymes takes place by the following three mechanism
• Binding to substrate and orient them properly for reaction.
• Mediating oxidation-reduction reaction through reversible changes in metal ion
oxidation state.
• Electrostatically stabilizing or shielding negative charges.
Examples: Carbonic anhydrase (Zn), Cytochrome P450 (Fe)

12. Mechanism

13. Proximity & Orientation Based Catalysis
 Enzyme substrate interaction align
the reactive chemical groups and hold
them close together in optimal
geometry thus increasing the rate of
reaction.
 This reduces the entropy of the
reactants thus making addition or
transfer favorable.
Example: Nucleotide monophosphate
kinase brings 2 nucleotides close
together and facilitate the transfer of a
phosphoryl group.

14. Proximity & Orientation Based Catalysis

15. Enzyme Inhibition
Enzyme inhibition is process of reducing or abolishing the catalytic activity of an
enzyme due to the interaction of an enzyme inhibitor.
Enzyme Inhibitor
They are low molecular weight compounds that bind to an enzyme and inhibit or reduce
its activity by either preventing the formation or breakdown of enzyme substrate
complex.

16. Types of Enzyme Inhibition
Enzyme Inhibitors
Reversible
Competitive
Non-Competitive
Un-Competitive
Irreversible Suicide
Allosteric

17. Reversible inhibition
 Reversible inhibitors bind to enzyme with noncovalent interactions, such as hydrogen
bonds, ionic bonds, and hydrophobic interactions.
 Can be rapidly dissociated
 Can be readily removed by dialysis
 Fully active enzyme can be recovered

18. Competitive Inhibition
 A Competitive inhibitor is any compound which closely resembles the chemical structure
and geometry of the substrate for an enzyme.
 The inhibitor competes for the same active site as the substrate molecule.
 The inhibitor interacts with the active site of the enzyme but no interaction takes place.
 Because of the presence of inhibitor fewer sites are available for substrate. Since the
enzyme's overall structure is unaffected by the inhibitor, it is still able to catalyze the reaction
on substrate molecules that do bind to an active site.
 Since the inhibitor and substrate bind at the same site, competitive inhibition can be
overcome simply by raising the substrate concentration.
 Therefore, the amount of enzyme inhibition depends upon the inhibitor concentration,
substrate concentration, and the relative affinities of the inhibitor and substrate for the active
site.
 Its inhibitory action is proportional to concentration.

19. Mechanism of Competitive Inhibitors
Examples: ACEIs, Statins

20. Non-Competitive Inhibitors
 The inhibitor usually binds non-covalently to a domain on the enzyme other than the
substrate binding site.
 This occurs when the substances do not resemble the geometry of the substrate & do
not exhibit mutual competition.
 They are not influenced by concentration of the substrate.
 Strong affinity for inhibitors prevent catalysis possibly due to distortion in enzyme
conformation.

21. Examples of Non-Competitive Inhibitors
 Cytochrome C oxidase (Fe) inhibition by cyanide, Organophosphates bind with the
serine residue to acetyl choline esterases, MAOIs, .

22. Un-Competitive Inhibitors
 Inhibitor has no affinity for the enzyme free
enzyme. Inhibitor binds to enzyme substrate
complex.
Example: Arsenic inhibition of Glyceraldehyde 3-
phosphate dehydrogenase (Glycolysis), Lithium
inhibition of inositol monophosphate.

23. Irreversible Inhibition
 Destroys a functional group necessary for the catalytic action.
 The catalytic activity of enzyme is completely lost.
 The enzyme can only be restored by synthesizing new molecules.
 Enzyme bonds covalently with the inhibitor.
 It includes:
• Suicide inhibition

24. Mechanism

25. Suicide Inhibition
 It is a specialized type of irreversible inhibition (aka. Mechanism based inhibition).
 The structural analogue is converted into a more effective inhibitor due to enzymatic
action.
 The substrate like compound initially binds with the enzyme and the first few steps
of the pathway are catalyzed.
 This new product irreversibly binds to the enzyme and inhibits further reactions.
 Example: Allopurinol forms alloxathine which permanently inhibits xanthine
oxidase, 5-fluorouracil forms 5-fluorodeoxyuridylate which inhibitis thymidylate
synthase, Organophosphates with Ach.

26. Allosteric Inhibition
 Allosteric means other site.
 When an inhibitor binds to the allosteric site it induces a conformational change in
the structure of the enzyme, which results in distorted shape of the active site, hence
the substrate can’t bind to it.
 The inhibitor is not a substrate analogue.
 It is partially reversible when excess substrate is added.
Example: Citric acid inhibiting blood clotting.

27. Mechanism

28. Michaelis–Menten kinetics
 It is named after German biochemist Leonor Michaelis and Canadian physician
Maud Menten.
 The model takes the form of an equation describing the rate of enzymatic reactions,
by relating reaction rate to the concentration of a substrate.
 The Michaelis-Menten equation has been used to predict the rate of product
formation in enzymatic reactions for more than a century.
Vo= Rate of reaction
Vmax= Maximum reaction rate
[S]= Substrate concentration
KM= Michaelis constant

29. Michaelis–Menten Plot

30. Michaelis–Menten Plot

31. Lineweaver-Burk Kinetics
 The Lineweaver–Burk plot (double reciprocal plot) is a graphical representation of
the Lineweaver–Burk equation of enzyme kinetics.
 It was first described by Hans Lineweaver and Dean Burk in 1934.
 The plot provides a useful graphical method for analysis of the Michaelis–Menten
equation.
 The Lineweaver–Burk plot was widely used to determine important terms in enzyme
kinetics, such as Km and Vmax, before the wide availability of powerful computers
and non-linear regression software.

32. Lineweaver-Burk Kinetics
 The y-intercept of such a graph is equivalent to the inverse of Vmax.
 The x-intercept of the graph represents −1/Km.
 It also gives a quick, visual impression of the different forms of enzyme inhibition.

33. Lineweaver-Burk Kinetics
 When used for determining the type of enzyme inhibition, the Lineweaver–Burk plot
can distinguish competitive, non-competitive and uncompetitive inhibitors.
 Competitive inhibitors have the same y-intercept as uninhibited enzyme (since Vmax
is unaffected by competitive inhibitors the inverse of Vmax also doesn't change) but
there are different slopes and x-intercepts between the two data sets.
 Non-competitive inhibition produces plots with the same x-intercept as uninhibited
enzyme (Km is unaffected) but different slopes and y-intercepts.
 Uncompetitive inhibition causes different intercepts on both the y- and x-axes .

34. Lineweaver-Burk Plot

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