Enzymology

This course provides the fundamental knowledge in formal and structural enzymology.

- The first part of this course deals with formal kinetics (study of reaction rates, determination of the order of a reaction, equilibrium and kinetics, reversible and balanced reaction). Experimental aspects are presented in parallel (determination of kinetic constants by spectrophotometry, fluorescence, radioactivity, immunoassays,...).

- The second part of the course concerns the study of single-substrate enzymatic kinetics.

Definition of an enzyme, catalyst. Nomenclature of enzymes (E.C)

Michaelian kinetics. Michaelis-Menten equation. Definition of the enzymatic parameters,_KM, maximum speed, catalytic constant, turn-over. Different graphical representations (Lineweaer-Burk, Eadie-Hofstee). 

The different types of inhibition are also studied (competitive, incompetent, non-competitive, mixed) as well as their graphic representation.

Determination of the inhibition constancy. Irreversible inhibitors.

Speed of the reaction. Arrhenius law.

- The third part focuses on describing multi-substrate enzymatic kinetics from a formal point of view. With ternary complex. Random or ordered mechanism.

Without ternary complex. Ping-Pong mechanism, Theorell-Chance. Cleland's representation.

Graphical determination.

- The fourth part concerns equilibrium bonds and allostery.

Receptor-Ligand / Enzyme-Substrate binding. Determination of the dissociation (or association) constant. Specific and non-specific binding.

Demonstration and graphical representation of Scatchard. Allosteric receptors (or enzymes). Non-Michaelian enzyme. Notion of cooperativity. Positive and negative cooperativity. Hill number, Hill graph.

Models of allosteric regulation are presented. Allostery. Cooperativity models, concerted (Monod-Wyman-Changeux), sequential (Koshland-Nemethy-Filmer). Role of effectors, activator or inhibitor. Example of hemoglobin and oxygen fixation.

- The fifth part of the course relates enzyme structures to their function using several examples. Description of the 3D structures and catalytic mechanisms of acetylcholine esterase, proteases and nucleoside diphosphate kinase. Notion of catalytic triad, binding pocket, etc... 

At the end of this course, the student should be able to analyze and interpret

enzymatic experimental data and be able to describe all the mechanisms with or without effectors. He/she must be able to calculate the different kinetic parameters and to put them in perspective in a more general metabolism. He/she will be able to analyze and relate the structure and function of enzymes and detail the molecular steps of a catalytic mechanism.