• ΔG = -7 kCal/mol

Without enzymes, chemical traffic through the pathways of metabolism would become terribly congested because
many chemical reactions would take such a long time.
 ENZYMES
• Enzymes – biocatalysts – produced by cells (animal, plant,
microbe) – intracellular /extracellular

• An enzyme is a macromolecule that acts as a catalyst, a chemical

agent that speeds up a reaction without being consumed by the
reaction.

• Usually proteins of high mol. Wt. ; Ribozymes

• Enzymes are specific, resulting in much higher reaction rates as

compared to chemically catalyzed reactions under ambient
conditions.

• Holoenzyme - An enzyme containing a nonprotein group. The

protein part of this enzyme is the apoenzyme (holoenzyme =
apoenzyme + cofactor).

• Isozyme - Enzymes that occur in several different molecular

forms, but catalyze the same reaction

• Cofactor (metal ions-Mg, Zn, Mn, Fe) and coenzyme (NAD, FAD,
CoA etc.)
Most of the coenzymes are derived from vitamins. They act as a transient carrier of groups and
transfer these groups in a biochemical reaction.

• Coenzyme A (CoA) – It is derived from compounds such as pantothenic acid, etc. and carries
acyl groups.
• Flavin adenine dinucleotide (FAD) – It is derived from vitamin B2 (Riboflavin) and carries
electrons.
• Nicotinamide adenine dinucleotide (NAD) – Derived from nicotinic acid (Niacin) and carries
hydride ions.

Cofactor is a prosthetic group bound to the apoenzyme very tightly, often bound covalently.

• Fe2+ or Fe3+ – Catalase, peroxidase, cytochrome oxidase

• K+ – Pyruvate kinase
• Zn2+ – Carbonic anhydrase

The catalytically active apoenzyme-cofactor complex is known as the holoenzyme or conjugate

enzyme.
• All enzymes are described by a four-part Enzyme
Commission (EC) number.

• For example, the enzyme with the trivial name lactate

dehydrogenase has the EC number 1.1.1.27, and is
more correctly called l–lactate: NAD+ oxidoreductase.

• The first part of the EC number refers to the reaction

that the enzyme catalyses.

• The remaining digits have different meanings

according to the nature of the reaction identified by
the first digit.

• For example, within the oxidoreductase category, the

second digit denotes the hydrogen donor and the third
digit denotes the hydrogen acceptor

• Thus, lactate dehydrogenase with the EC number

1.1.1.27 is an oxidoreductase, indicated by the first
digit) with the alcohol group of the lactate molecule as
the hydrogen donor (second digit) and NAD+ as the
hydrogen acceptor (third digit), and is the 27th
enzyme to be categorized within this group.
 Ligase vs Lyase

Lyases are a group of enzymes that catalyzes

the breaking of various chemical bonds by
Ligase is an enzyme that brings about ligation
means other than hydrolysis and oxidation,
of DNA or another substance.
often forming a new double bond or a new ring
structure.

Chemical Bonds
Ligases cause bond formation. Lyases cause bond cleavages.
Reaction Type

Ligases act through hydrolysis reactions. Lyases act through elimination reactions.

Reactants

Ligases act on two reactants at a time. Lyases act on one reactant at a time.

Image from https://www.differencebetween.com/

Application of Enzymes:

• Animal feed enzymes (Improved digestibility - supplementing feed with

enzymes result in faster growth of the animal, improved feed conversion ratio
(i.e. better feed utilisation), more uniform production and better overall health.

• Phytase – Pigs/poultry to digest soy beans and other legumes

• Detergent enzymes – Proteases, lipases, amylases and cellulases (stain

removal

• Wine, brewing and baking (Invertase)

• Fruits and vegetables – Pectinase

 Activation energy barrier

• Chemical reaction – bond breakage/formation

• Changing one molecule into another generally

involves contorting the starting molecule into a highly
unstable state before the reaction can proceed.

• To reach the contorted state where bonds can change,

reactant molecules must absorb energy from their
surroundings. When the new bonds of the product
molecules form, energy is released as heat, and the
molecules return to stable shapes with lower energy
than the contorted state.

• The initial investment of energy for starting a

reaction—the energy required to contort the reactant
molecules so the bonds can break—is known as
activation energy, (EA)

• EA supplied by heat in the form of thermal energy

• EA – determines the rate of the reaction The formation of new bonds releases more
energy than was invested in the breaking of old bonds
Enzymes speed up metabolic reactions by lowering energy barriers
 How enzymes work
- The substrate binds to a specific site on the enzyme known as the
active site.
- Lock & key model
- Proximity effect - Enzymes can hold substrates such that reactive
regions of substrates are close to each other and to the enzyme’s
active site.
- Orientation effect - Enzymes may hold the substrates at certain
positions and angles to improve the reaction rate
- In some enzymes, the formation of an enzyme–substrate complex
causes slight changes in the three-dimensional shape of the enzyme.
This induced fit of the substrate to the enzyme molecule may
contribute to the catalytic activity of the enzyme, too. It can also
stress or bend the critical chemical bonds
- The active site may also provide a microenvironment conducive to a
particular type of reaction than the solution itself.
- Lysozyme and Carboxypeptidase A.
- Orotidine 5'-phosphate decarboxylase (OMP decarboxylase)
It is the shape and charge properties of the active site that enable it to bind to a
single type of substrate molecule, so that the enzyme is able to demonstrate
considerable specificity in its catalytic activity.
• A single molecule of carbonic anhydrase can catalyse the conversion of over half a million
molecules of its substrates, carbon dioxide (CO2) and water (H2O), into the product,
bicarbonate (HCO3−), every second
Effects of Local Conditions on Enzyme Activity

• Each enzyme has an optimal temperature at which its reaction rate is greatest.
• That favor the most active shape of the protein
 Enzyme Immobilization

• Avoid denaturation of sensitive enzymes to reaction conditions; synthetic and economic

consequences
• Inert matrices – similar to native environment
• The interactions between the immobilization matrix and the immobilized enzyme should not
affect the enzyme’s catalytic efficiency and stability
• Advantage – increased functional efficiency and enhanced reproducibility

https://doi.org/10.1016/B978-0-12-819820-9.00012-0
Selection Criteria - Matrix, Method and Conditions of Immobilization

1. Inexpensive, easily available, environmentally friendly, thoroughly inert, stable, and improving
the specificity and reactivity of the immobilized enzyme.
2. Be tolerant to temperature, pH value, mechanical stress, and organic solvents, thus yielding
stable immobilized biocatalyst, under various situations.
3. Be regenerative after the period of life of the immobilized biocatalyst.
4. To load relatively elevated amounts of the enzyme.
5. To deliver disinfectant properties.

https://doi.org/10.1016/B978-0-12-819820-9.00012-0
 Applications

• FOOD, DAIRIES, JUICES, COSMETICS, PHARMACEUTICALS, AND OTHERS