Content

Theoretical content.

Unit 1. Introduction to biocatalysis.

Concept of biocatalysis. Market and use of biocatalysts. Prejudices in the use of enzymes. Historical perspective. Waves of innovation in biocatalysis. Advantages and disadvantages of biocatalysts. Different types of biocatalysis processes. Cellular and enzymatic systems: properties. Factors to consider in a biocatalytic process: source of the biocatalyst and optimization of the process.

Unit 2. Properties, classification and nomenclature of enzymes.

General properties of enzymes: Concept and biological, chemical and practical significance. Definitions. Enzyme-substrate complex. Decreased activation energy. Transition state. Enzymatic cofactors. Nomenclature and classification of enzymes. Databases with enzyme information.

Unit 3. Methods of determination of enzymatic activity and of obtaining enzymes.

Production and characterization of enzymes. Sources of enzymes. Techniques for the extraction of enzymes. Methods of determination of enzymatic activity. Dirct and indirect, continous and discontinous assays. Initial rate: concept, determination, representation. Units of enzymatic activity. Effect of enzyme concentration.

Unit 4. Analysis of enzyme kinetics.

Enzyme kinetics. Reactions with one substrate. Effect of substrate concentration: Michaelis-Menten equation. Pre-steady and steady-states: concepts. Steady state hypothesis: treatment of Briggs-Haldane. Enzymatic reactions with more than one enzyme-substrate intermediate complex. Significance of the parameters kcat,  KM and kcat/KM. Determination of kinetic parameters. Methods with linear representations: Lineweaver-Burk, Eadie-Hofstee and Hanes-Woolf. Other methods. Michaelis-Menten equation for reversible reactions: Haldane relationship.

Unit 5. Inhibition of enzyme catalysis.

Inhibition of enzymatic catalysis: types of inhibitors. Reversible inhibitors: competitive inhibition, acompetitive and mixed inhibition (includes non-competitive inhibition). General model. Graphic analysis of the different types of inhibition. Determination of the inhibition constants. Concept of IC50 and its relation with the inhibition constants. Inhibition by excess substrate. Discrimination between competing substrates. Pseudo-irreversible inhibitors and irreversible inhibitors. Affinity labels. Suicide inhibitors. Use of enzyme inhibitors as drugs.

Unit 6. Analysis of enzyme kinetics in reactions with more than one substrate.

Reactions with more than one substrate: Cleland notation. Sequential ordered mechanism, statistical sequential mechanism, double displacement mechanism (ping-pong). Mathematical treatment and graphical analysis. Methods for determining the type of mechanism. Isotopic exchange and isotopic effect.

Unit 7. Kinetics of transient states.

Characteristics of rapid kinetic methods. Mixing methods: continuous flow, stopped flow and quenched-flow. Relaxation methods: temperature jump (T-jump), pressure jump ( P-jump). "Bursts" and "lags". Analysis of the "Burst" of a reaction: determination of the concentration of active centers. Application of the fast reaction kinetics to the nitrogen assimilation process.

Unit 8. Effect of pH and temperature on enzymatic reactions.

Action of the temperature on enzyme kinetics. Representation of Arrhenius. Enzymes of extremophile organisms. Effects of pH on enzyme kinetics. Ionization of essential residues. Influence of pH on the kinetic parameters. Evaluation of ionization constants. Identification of the ionizable groups involved in the processes of binding and catalysis. Effects of the micro environment on the pK. Exemples.

Unit 9. Cooperativity and Allosterism.

Ligand binding to proteins. Concept and types of cooperativity. Analysis of cooperativity. Union of oxygen to hemoglobin. Cooperativity models. Model of Monod, Wyman and Changeux. Explanation of the homotropic cooperative effects by the MWC model. Allosteric enzymes. K-systems and V-systems. Koshland, Nemethy and Filmer model. Determination of the cooperative model that follows a certain enzyme. Example of enzyme with allosteric regulation: aspartate carbamyl transferase.

Unit 10. Enzymatic specificity.

The active center, specificity and three-dimensional structure. Definition of active center. Characteristics of the active center. Theories about the coupling between the enzyme and the substrate. Fisher's theory (key-lock). Koshland theory (induced-fit). Hexokinase as an example of induced coupling. Hypothesis of three-point union. Hypotheses involving tension. Stabilization of the transition state. Evidence supporting the theory of the transition state. Catalytic antibodies and their applications.

Unit 11. Study of the active center.

The active center. Identification of the binding and catalytic centers. Labelling with a part of the substrate. Use of artificial substrates. Chemical modification with specific irreversible inhibitors. Affinity labels. Suicide inhibitors, examples with pharmacological interest. Directed mutagenesis. Serine proteases: subtilisin. Comparison of mutagenesis and chemical labeling. Investigation of the three-dimensional structure of proteins: X-rays, NMR, molecular modeling. Restriction endonucleases. "Editorial" and error correction mechanisms: aminoacyl-tRNA synthetases.

Unit 12. Mechanisms of enzymatic catalysis.

Mechanisms of catalysis. Introduction to the mechanisms of enzymatic action. Acid-basic catalysis. Covalent catalysis. Pyridoxal phosphate. Catalysis with metal ions. Mechanisms of alcohol dehydrogenase and carbonic anhydrase. Environmental effect: electrostatic catalysis. The lysozyme Mechanism of subtilisin. Superoxide dismutase. Effects of proximity and orientation.Channeling intermediaries. Multifunctional enzymes. Enzymeswith additional non-enzymatic functions "moonlighting enzymes".

Unit 13. Cofactors and ribozymes.

Cofactors and ribozymes. Catalytic activity of RNA. Type of ribozymes. The ribosome is a ribozyme. Biological meaning of ribozymes. Applications of ribozymes.

Unit 14. Regulation of enzymatic activity.

Regulation of enzyme activity. Modification of the enzyme concentration. Regulation of the synthesis and degradation of enzymes. Degradation mechanisms. Variation of the enzymatic speed in function of the concentration of substrate, product and cofactors. Activation by precursor and retro inhibition. Functional meaning of cooperativity and allosterism. Hormonal control. Isozymes. Polymerization-depolymerization. Binding to other proteins. Irreversible covalent modification. Reversible covalent modification. Enzymatic cascade systems.

Unit 15. Biomedical and biotechnological applications of enzymes.

Enzymes in clinical biochemistry and biotechnology. Enzymes as therapeutic agents. Enzyme indicators of pathologies. Plasma enzymes. Factors that affect the levels of plasma enzymes. Examples of enzymes with diagnostic interest. Aminotransferases. Creatine kinase. Lactate dehydrogenase. Indicators of myocardial infarction. Enzymes as reagents in clinical biochemistry. Enzymes and inborn errors of metabolism, examples. Enzymes in the industry. Large scale production of enzymes. Applications: drugs, food industry, detergents, textile industry. Immobilized enzymes. Enzymes as biosensors.

Unit 16. Directed evolution.

Methods to improve biocatalysts. Design and synthesis of new catalysts. Directed evolution. Generation of mutants. Selection and screening of the desired enzymatic activity. Re-design of enzymes to modify their thermostability and enantioselectivity. Adaptive evolution in the laboratory.

Problems.

The problems that are proposed refer to the analysisof enzyme activity and determination and interpretation of kinetic parameters. The statements of the problems will be delivered through the Virtual Campus. 

Delivery of works through the tool of the "Virtual Campus":

Two works will be proposed through the Virtual Campus, which must be worked out by the teams (of three/four people) of students established at the beginning of the course. The works must be delivered before a specific date through the Virtual Campus.