Biochemical engineering and biotechnology
- S. Kendereški, Osnovi enzimologije, TMF, Beograd, 1986.
- Ž. Petronijević, Opšta i primenjena enzimologija, Tehnološki fakultet, Leskovac, 2000.
- Đ. Petrović, Osnovi enzimologije, Zavod za udžbenike i nastavna sredstva, Beograd, 1998.
- T. Palmer, “Understanding enzymes”, Prentice Hall/Ellis Horwood, 1995.
- A.G. Marangoni, Enyzme kinetics: a modern approach, John Wiley & Sons, Inc., Hoboken, New Jersey, 2003.
- Z. Knežević, internal material
The course informs the students about general properties of enzymes and mechanisms of their action. It is intended to provide an introduction to enzyme engineering, and to give a balanced, reasonably detailed account of all the theoretical and applied aspects of the enzyme catalysis. In particular, the course covers the structure and classification of enzymes, mechanisms and nature of enzyme catalysis, specificity of enzyme action, kinetics of enzyme-catalyzed reactions, enzyme inhibition and activation as well as general rules for handling enzymes, plus aspects of their practical applications.
The course is designed to introduce students to the theoretical and practical aspects of enzyme catalysis and general aspects of enzyme regulation. The aim of the course is to give the students a thorough understanding of general characteristics of enzymes such as classification and nomenclature of enzymes, structure and function of enzymes, mechanism of action, kinetics, and regulatory aspects of enzymes. In addition, students gain knowledge about the most important representatives of all enzyme classes.
I An introduction to enzymology
- The subjects of enzymology. A brief history of enzymes
- Enzymology and other sciences
- Basic definitions in enzymology
II General properties of enzymes
Chemical nature of enzymes. Simple and conjugated enzymes.
The structure of enzymes. Monomeric enzymes. Oligomeric enzymes. Multienzyme complexes and multifunctional enzymes. Membrane-bound enzymes. Isoenzymes.
o Enzyme deactivation. Thermal inactivation. Deactivation under the influence of salts and solvents. Correlation between stability and structure.
Coenzymes and other low-molecular-mass cofactors. Mechanisms of cofactors action. The structure and classification of cofactors. Coenzymes of oxidoreductases. Coenzymes as carrier of groups. Coenzymes of lyases, isomerases and ligases. Prosthetic groups.
Metal-activated enzymes and metalloenzymes.
Enzyme as catalyst. Theory of enzyme catalysis. The investigation of active site structure and mechanisms of the binding of substrates to enzymes.
Specificity of enzyme action. Types of specificity. Lock and key and induced fit hypotheses
III Enzyme classification and nomenclature
- Systematic name of enzymes and a code number. An example of enzyme nomenclature. The six main classes of enzymes. The system of enzyme classification. Practical examples.
- Oxidoreductase. Transferases. Hydrolases. Lyases. Isomerases. Ligases.
IV Kinetics and mechanismis of enzyme reactions
An introduction to bioenergetics, catalysis and kinetics
Mechanisms of reactions catalyzed by enzymes
Enzyme activity. Definition of Enzyme Units. Specific and molecular activity.
Methods used for investigating the kinetics of enzyme-catalyzed reactions
Kinetics of single-substrate enzyme-catalyzed reactions. Factors affecting the rates of enzyme reactions. Effect of enzyme concentration. Effect of substrate concentration.
The Michaelis-Menten equation. General strategy and methods for determination of the kinetic constants. The significance of the kinetic constants.
The Briggs-Haldane modification of the Michaelis-Menten equation
Effects of pH and temperature on enzyme activity
Enzyme inhibition and types of inhibitors. Irreversible inhibition. Reversible inhibition. Competitive inhibition. Non-competitive inhibition. Uncompetitive inhibition. Substrate inhibition. Product inhibition. Methods for determination of inhibitor constant.
Activators and stabilizers. Mechanisms of enzyme activation. Metal ion activation. Cofactor activation. Activation of proenzymes.
Allosteric enzymes and sigmoidal kinetics. The Hill equation. Mechanisms of regulation of enzyme activity.
V Applied enzymology
- Some examples of enzymes with industrial application.
- Oxidoreductase, Amylases, proteases, pectinases, isomerases, lipases
Factors affecting the rates of enzyme reactions. The effect of enzyme concentration on reaction rate of sucrose hydrolysis catalyzed by invertase. The effect of substrate concentration. Experimental determination of enzyme catalytic parameters from the progress curve. Effect of temperature on activity of invertase. Effect of pH on enzyme activity. Determination of types of inhibitors. Calculation of enzyme activity. Invertase activity assays. Lipase activity assays. Determination of enzyme content in commercial preparation by Lowry method. Effect of type of organic solvent on reaction rate of olive oil hydrolysis catalyzed by lipase.
Students will demonstrate an ability to use fundamental and advanced concepts to solve biochemical problems dealing with enzyme structure, kinetics and mechanism. Furthermore, the course may serve as a groundwork to more advanced courses such as industrial enzymatic processes providing the students to proceed further in this area of biotechnology.
Prepared by dr Zorica Knežević