Enzymology

This course provides 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 reactions). The experimental aspects are presented in parallel (determination of kinetic constants by spectrophotometry, fluorescence, radioactivity, immunoassays, etc.).

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

Definition of an enzyme, catalyst. Enzyme nomenclature (EC)

Michaelis kinetics. Michaelis-Menten equation. Definition of enzyme parameters,_KM, maximum velocity, catalytic constant, turnover. Different graphical representations (Lineweaer-Burk, Eadie-Hofstee). 

The different types of inhibition are also studied (competitive, noncompetitive, mixed) as well as their graphical representation.

Determination of inhibition constancy. Irreversible inhibitors.

Reaction rate. Arrhenius law.

- The third section focuses on describing multi-substrate enzyme kinetics from a formal perspective. With ternary complexes. Random or ordered mechanism.

Without ternary complex. Ping-Pong mechanism, Theorell-Chance. Cleland 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. Concept of cooperativity. Positive and negative cooperativity. Hill number, Hill graph.

Allosteric regulation models are presented. Allostery. Cooperative models: concerted (Monod-Wyman-Changeux) and sequential (Koshland-Nemethy-Filmer). Role of effectors, activators, or inhibitors. Example of hemoglobin and oxygen binding.

- The fifth part of the course links enzyme structures and their function using several examples. Description of the 3D structures and catalytic mechanisms of acetylcholinesterase, proteases, and nucleoside diphosphate kinase. Concept of catalytic triad, binding pocket, etc. 

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

experimental enzymatic data and be able to describe all mechanisms with or without effectors. He/she must know how 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 steps of a catalytic mechanism.