Enzymes

Prof. Ganti S. Murthy

Biosciences and Biomedical Engineering
Indian Institute of Technology-Indore
Indore, India
Email: Ganti.Murthy@iiti.ac.in
 Enzymes
α amylase Glucoamylase

Starch
Sugars

Dextrins
Liquefaction Saccharification

Pretreatment

Starch Granule

Ref: http://food.oregonstate.edu/learn/starch.html
 Enzymes
Enzymes act as biological catalysts and help in lowering the activation
energy. They, just like any other catalyst do not change the equilibrium.
• Most enzymes are proteins. Tertiary and quaternary structure of protein
provide the functionality of the enzyme.
• Most enzymes are very specific to
a reaction/functional group.
• Lock and Key; Induced fit model
are models to explain enzyme action
 Enzymes
Enzyme Kinetics: Enzyme reactions are most commonly described using
Michaelis-Menton Equation (Important: assumes quasi steady state).

K1
E+S  ⎯⎯→ E+P
K2
ES
K −1

k −1 + k 2
k [ E ][ S ]
[ ES ] = 1 km =
k −1 + k 2 k1

dP [S ]
= k 2 [ ES ] = Vmax
dt km + [S ]

Under what conditions is Michaelis-Menton equation inadequate to explain

enzyme action? Limited/ restricted mobility of enzymes, two phase
reactions, enzyme is not limiting, allosteric regulation
 Enzymes
Enzyme inhibitors interfere in the enzyme action and reduce its activity.
Irreversible inhibitors: Bind irreversibly to enzyme and inactivate it.
Reversible Inhibitors: Inhibition is reversible.
• Competitive Inhibitors: This type of inhibitor binds to the active site of the
enzyme. Changes km and Vmax remains the same
• Uncompetitive Inhibition: This type of inhibitor binds to Enzyme Substrate
complex and renders it inactive.
• Non competitive inhibition : They do not bind to the active site. Changes
Vmax and km remains the same.
• Mixed inhibitors: Consist of a combination of competitive and non
competitive inhibitors.
 Enzymes
History of Enzymology
• Cheese manufacturing is one of the oldest applications of enzymes
(Rennet).
• 1913 Michaelis-Menten Equation was first published.
•Koshland introduced theory of induce fit to explain enzyme activity in
1957
•Allosteric regulation was first used by Monod in 1963
•First list of enzymes was published by Dixon and Webb in 60 years ago.
• International Union of Biochemistry and Molecular Biology (IUBMB)
classified enzymes based on their function (https://www.enzyme-
database.org/)
 Enzyme Classification
Seven categories of enzymes
1. Oxidoreductases (AH2 + B = A +BH2 or AH2 + B = A +BH + H)
2. Transferases (AX+B= A +BX)
3. Hydrolases (A+B+H2O =AH +BOH)
4. Lyases (A–B→ A+B, Reverse reaction is synthase)
5. Isomerases (A-B-C=A-C-B)
6. Ligases (synthases) (A+B+NTP =A-B + NMP +PP or A+B+NTP =A-B +
NDP +P)
7. Translocases (“assists in the movement of another molecule usually
across a cell membrane”)
 Enzyme Classification
Four Digit Classification system
1. First digit indicates the class of the enzyme.
2. Second digit, the subclass indicating the compound/group involved
3. Third digit indicates the sub-subclass indicating the type of reacton
4. Fourth digit is a serial number to classify the enzyme within the sub-
sub class.

Rules for classification and nomenclature of enzymes

Please see the following website: https://www.ebi.ac.uk/intenz/rules.jsp
 Enzyme Classification
•International Union of Biochemistry and Molecular Biology (IUBMB) classified enzymes based on
their function (https://www.enzyme-database.org/)
 Enzyme Classification
Amylases
• α-amylase (EC 3.2.1.1) 1,4-α-D-glucanohydrolase : This is an endo enzyme.
Breaks down amylose and amylopectin by hydrolyzing α 1→4 bonds and
yields dextrins. Optimum pH is ~6.0 and temperature 90°C.

• β-amylase (EC 3.2.1.2) 1,4-α-D-glucan maltohydrolase: This is an

exoenzyme. It also breaks down amylose and amylopectin by hydrolyzing α
1→4 yielding a disaccharide, maltose. Optimum pH of 5.4

• γ-amylase (EC 3.2.1.3) Glucan 1,4-α-glucosidase/ glucoamylase/

amyloglucosidase: This exoenzyme hydrolyzes α 1→4 and α 1→6 (20 times
slower) and releases glucose. Optimum pH is 4.5 and optimum temperature
60°C
 Enzyme Classification
Pullulanases

• EC 3.2.1.41 is also known as α-dextrin endo-1,6-alpha-glucosidase

(Debranching enzyme). This is an exo enzyme
• Type I act only on α-1→6 whereas type II can also act on α-1→4
• EC 1.11.1.6 Catalase Breaks down hydrogen peroxide into water and
oxygen. This is one of the fastest known enzymes in nature (why? How is
this related to Elephant toothpaste?)
 Enzyme Classification
• What are the limitations of the IUBMB approach to enzyme classification?
•Isoenzymes
•Specificity information
•Structural considerations are absent
•Kinetic information
• Organisms producing the enzyme
•Other Online comprehensive databases is BRENDA: https://www.brenda-
enzymes.org/index.php This database contains the kinetic information.

Can you find an enzyme that impacts your daily life in the
BRENDA database? How does this enzyme impact? Where is it
found? What is the reaction it catalyzes? Which organisms
produce it naturally? Industrially?
 Enzymes
Cellulases

Ref: http://en.wikipedia.org/wiki/Cellulase
 Enzymes

Ref:http://www.fao.org/docrep/w7241e/w7241e08.htm
 Enzymes
Cellulases
• Endo Cellulases: Facilitate hydrolysis by exposing cellulose
chains and disrupting the crystalline structure
• Exo cellulases: They further hydrolyze cellulose and yield
cellobiose (a disaccharide)
• Cellobiase: These enzymes hydrolyze cellobiose to glucose.
• Oxidative cellulases: “Depolymerize cellulose by radical
reactions”
• Cellulose phosphorylases: “Depolymerize cellulose using
phosphates instead of water”
• Progressive and non-progressive cellulases
 Enzymes

Ref: Lynd et al. 2002

 Enzymes
Cellulase producing fungi in nature

Fungi Fungi
Acremonium cellulolyticus Talaromyces emersonii
Aspergillus acculeatus Thielavia terrestris
Penicillium funmiculosum Trichoderma koningii
Phanerochaete Trichoderma reesei
chrysosporium Trichoderma viride
Schizophyllum commune Aspergillus fumigatus
Sclerotium rolfsii Aspergillus niger
Sporotrichum cellulophilum Fusarium solani

Ref:http://www.fao.org/docrep/w7241e/w7241e08.htm
 Enzymes
Effect of pH and Temperature
• Effect of pH on activity and stability of α-amylase.

Ref: Ivanova et al. (1993)

 Enzymes
Effect of pH and Temperature
• Effect of temperature on activity and stability of α- amylase.

Ref: Ivanova et al. (1993)

 Enzymes
Kinetic Models for Cellulose Hydrolysis
• Effect of substrate and product inhibition, competitive inhibition by xylose
were and enzyme adsorption were considered.

Ref: Kadam et al. (2004)

 Enzymes
Kinetic Models for Cellulose Hydrolysis
• Effect of substrate and product inhibition, competitive inhibition by xylose
were and enzyme adsorption were considered.

Ref: Kadam et al. (2004)

 Enzymes
Kinetic Models for Cellulose Hydrolysis
• Effect of substrate and product inhibition, competitive inhibition by xylose
were and enzyme adsorption were considered.

Ref: Kadam et al. (2004)

 Enzymes
Kinetic Models for Cellulose Hydrolysis

Ref: Kadam et al. (2004)

 Enzymes
Kinetic Models for Cellulose Hydrolysis

Ref: Kadam et al. (2004)

 Diseases due to Enzymes
Most inherited metabolic disorders are caused due to defective
gene that results in missing enzyme or enzyme deficiency.
•LSD (Lysosomal Storage Disorders): Enzymes that recycle cellular
waste are missing.
•NP (Nieman-Pick) Disease: Lack of a enzyme to metabolize
lipids leading to accumulation of lipids in spleen, liver, lungs,
bone marrow and brain.
•MPS (Mucopolysaccharidoses): Caused by deficiency or
missing enzymes that break down polysaccharides. This
causes accumulation of complex sugar molecules in cells. A
progressive disease that affects all organs.
Ref: https://nationalstemcellfoundation.org/glossary/enzyme-deficiencies/
Ref: https://www.webmd.com/a-to-z-guides/inherited-metabolic-disorder-types-and-treatments#1
 Diseases due to Enzymes
Most inherited metabolic disorders are caused due to defective
gene that results in missing enzyme or enzyme deficiency.
•Mitochondrial Diseases: Cause problems inside mitochondria.
• Glycogen Storage Disorders: Cuases problems with sugar
storage. (what is Glycogen and how is it related to starch?)
•Galactosemia: Impaired breakdown of galactose (what is this?)

Ref: https://nationalstemcellfoundation.org/glossary/enzyme-deficiencies/
Ref: https://www.webmd.com/a-to-z-guides/inherited-metabolic-disorder-types-and-treatments#1
 Application of Enzymes In Daily Life
• Stone-washed jeans:
•Environmentally friendly detergents:
• Clear fruit juices
•Cheese
•Clear beer
•Soft chapatis and breads
 References
• Kadam et al. (2004)
• Lynd et al. 2002
• Ivanova et al. 1994
• http://www.fao.org/docrep/w7241e/w7241e08.htm
 Enzymes

Prof. Ganti S. Murthy

Biosciences and Biomedical Engineering
Indian Institute of Technology-Indore
Indore, India
Email: Ganti.Murthy@iiti.ac.in