Enzymology

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Dr Sitaram Swain

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Core Courses

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Course Name : Enzymology

Code(Credit) : CUTM1706(3-1-2)

Course Objectives

  • • To acquire fundamental knowledge on enzymes and their importance in biological reactions.
    • To understand ability to difference between a chemical catalyst and biocatalyst.
    • Exposure to the nature of non-protein enzymes such as ribozymes.
    • Understanding the role of enzymes in clinical diagnosis and industries.

Learning Outcomes

  • • This course will provide fundamental knowledge on enzymes and their importance in
    biological reactions.
    • Students will understand the difference between a chemical catalyst and biocatalyst and understand activation energy.
    • They will study non-protein enzymes such as ribozymes and will be exposed to the Industrial and biomedical applications of enzymes

Course Syllabus

Module:I Enzymes:Definition, historical perspective, general characteristics, co-factors – coenzymes and metal ions.
Module:II Classification and units of enzymes: Based on IUB with examples. Unit of enzyme activity,definition of IU, enzyme turn over number and nature of non-enzymatic and enzymatic catalysis. Specific activity. Enzyme specificity. Concept of active site, ES complex, specificity.
Module:III Theories of enzyme catalysis: Lock and key model, Koshland’s induced fit theory. Enzyme kinetics: Factors affecting rate of enzyme catalyzed reactions.
Module:IV Characterization: Effect of enzyme concentration, substrate concentration, pH and temperature. Michaelis – Menten equation,Lineweaver – Burk(L-B) plot. Determination of Vmax& Km from L-B plot and their significance.Enzyme inhibition –competitive, non competitive and uncompetitive. Graphical representation by L-B plot. Evaluation of Km, Ki and Vmax in presence of inhibitor.
Module:V Chemical modification of active site groups. Site directed mutagenesis of enzymes. Mechanism of action of chymotrypsin .
Module:VI Allosteric enzymes:Sigmoidal curve, positive and negative modulators, qualitative description of “concerted” & “sequential” models for allosteric enzymes. Half site reactivity, Flipflop mechanism, positive and negative co-operativity with special reference to aspartate transcarbamylase and phosphofructokinase.
Module:VII Isoenzymes:Detection, nature, importance. Lactate dehydrogenase as an example. Multi enzyme complex – Pyruvate dehydrogenase complex. – Composition, subunits, assembly, enzymatic reaction functions.RNA as an enzyme. (Ribozymes).
Applications of Enzymes: Enzymes as reagents, Marker enzymes in diagnostics, Immobilized enzymes, Industrial applications of enzymes.
Laboratory Practices
• Isolation of urease and demonstration of its activity
• Isolation of acid phosphatase and demonstration of its activity
• Determination of specific activity of salivary amylase by DNS
• Purification of urease
• Time course of urease reaction
• Influence of substrate concentration and pH on the rate of enzymatic reaction
• Determination of Km and Vmax of salivary amylase
• Determination of initial velocity [time kinetics] of salivary amylase
• Determination of optimum temperature of salivary amylase

Suggested readings:
Text Books:
Cox, M.M and Nelson, D.L. (2008). Lehninger Principles of Biochemistry, VEdition, W.H. Freeman and Co., New York.
Reference Books:
1.Cox, M.M and Nelson, D.L. (2008).Lehninger’s Principles of Biochemistry, Sixth Edition, W.H. Freeman and Co., New York.
2. Voet D and Voet J(2012) Biochemistry . Fifth edition, Wiley.
3. Murray, R.K., Bender, D.A., Botham, K.M., Kennelly, P.J., Rodwell, V.W. and Well, P.A. (2009). Harper’s Illustrated Biochemistry, XXVIII Edition, International Edition, The McGraw-Hill Companies Inc.

Session Plan

Session 1

Enzymes:Definition, historical perspective

https://www.youtube.com/watch?v=ozdO1mLXBQE

Session 2

Session 3

co-factors – coenzymes and metal ions.

https://www.youtube.com/watch?v=jYR0RlMft8Y

Session 4

Classification and units of enzymes: Based on IUB with examples

https://www.youtube.com/watch?v=L1QV1WDyq7M

Session 5

Unit of enzyme activity,definition of IU,

Session 6

enzyme turn over number and nature of non-enzymatic and enzymatic catalysis.

Session 7

Specific activity. Enzyme specificity

Session 8

Concept of active site

Session 9

ES complex

Session 10

specificity.

Session 11

Theories of enzyme catalysis: Lock and key model

Session 12

Koshland’s induced fit theory

Session 13

Enzyme kinetics: Factors affecting rate of enzyme catalyzed reactions

Session 14

Characterization: Effect of enzyme concentration, substrate concentration, pH and temperature.

Session 15

Characterization: Effect of enzyme concentration, substrate concentration, pH and temperature.

Session 16

Michaelis – Menten equation,Lineweaver – Burk(L-B) plot

Session 17

Determination of Vmax& Km from L-B plot and their significance.

Session 18

Enzyme inhibition –competitive, non competitive and uncompetitive.

Session 19

Graphical representation by L-B plot

Session 20

Evaluation of Km, Ki and Vmax in presence of inhibitor.

Session 21

Chemical modification of active site groups

Session 22

Site directed mutagenesis of enzymes

Session 23

Mechanism of action of chymotrypsin

Session 24

Allosteric enzymes:Sigmoidal curve, positive and negative modulators

Session 25

qualitative description of “concerted” & “sequential” models for allosteric enzymes.

Session 26

Half site reactivity

Session 27

Flipflop mechanism

Session 28

positive and negative co-operativity with special reference to aspartate transcarbamylase and phosphofructokinase

Session 29

Isoenzymes:Detection, nature, importance

Session 31

Multi enzyme complex – Pyruvate dehydrogenase complex.

Session 30

Lactate dehydrogenase as an example.

Session 31

Multi enzyme complex – Pyruvate dehydrogenase complex.

Session 31

Multi enzyme complex – Pyruvate dehydrogenase complex.

Session 31

Multi enzyme complex – Pyruvate dehydrogenase complex.

Case Studies

Case Studies

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    Dr Sitaram Swain has received PhD degree from Utkal University in the year 2017. He is also trained and experienced from TATA Memorial Hospital, Mumbai, India. He has experienced with cell line culture, clinical biochemistry, immuno-fixation, immunoassay, cancer cytogenetic, serum protein electrophoresis and tumour marker analysis. He has qualified NET(ICAR-ASRB) in Animal biotechnology. He has […]