Enzymology

This course provides fundamental knowledge of formal and structural enzymology.

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

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

Definition of an enzyme, catalyst. Enzyme nomenclature (E.C)

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

The different types of inhibition (competitive, incompetent, non-competitive, mixed) and their graphical representation are also studied.

Determining inhibition constancy. Irreversible inhibitors.

Reaction speed. Arrhenius' law.

- The third section describes multi-substrate enzymatic kinetics from a formal point of view. With ternary complex. Random or ordered mechanism.

No ternary complex. Ping-Pong, Theorell-Chance mechanism. Cleland representation.

Graphical determination.

- The fourth part deals with equilibrium bonding and allostery.

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

Scatchard demonstration and graphical representation. 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. Cooperative models, concerted (Monod-Wyman-Changeux), sequential (Koshland-Nemethy-Filmer). Role of effectors: activator or inhibitor. Example of hemoglobin and oxygen uptake.

- 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, students 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 various kinetic parameters and put them into perspective within a more general metabolism. He/she will be able to analyze and link the structure and function of enzymes and detail the molecular stages of a catalytic mechanism.