Chemical catalysis (transition state theory, principles of chemical catalysis, principles of enzyme catalysis, kinetic isotope effect, enzyme cofactors). Selected examples of enzyme catalysis (proteases, metaloenzymes). The basic equations of enzyme kinetics (steady-state kinetics, Michaelis-Menten model, inhibition, multisubstrate systems). Measurements and magnitude of individual rate constants (conventional methods, rapid methods including stopped flow, relaxation and quenching, analysis of pre-steady state and relaxation kinetics). The pH dependence of enzyme catalysis. Practical methods for kinetics and equilibria (fluorescence spectroscopy, kinetic and equilibrium methods for determination of Kd). Detection of intermediates in enzymatic reactions. Irreversible inhibition. Alosteric interactions (positive and negative cooperativity, Hill equation). Enzyme-substrate complementarity and the use of binding energy in catalysis (tyrosyl-tRNA synthetase as a selected example, catalytic antibodies). Selected examples of enzymatic reactions (restiriction endonucleases, DNA polymerases). Catalytic RNA and ribonucleoprotein complexes (RNase P).
1. Explain and demonstrate the ability to distinguish between various principles of enzyme catalysis (acid-base catalysis, covalent catalysis, electrostatic catalysis, utilization of binding energy in catalysis)
2. Explain the requirement for cofactors in biocatalysis, the models of monsubstrate and multisubstrate enzyme reactions, type of enzyme inhibition and the pH dependency of the enzyme reactions.
3. Explain the Michaelis-Menten and Briggs-Haldane models of enzyme catalysis, demonstrate the understanding of kinetic parameters reached under steady-state (the advantages and limitations of steady-state kinetics) and write the basic equations used for data analysis.
4. Explain the transient kinetic approach (single turnover and burst kinetics), demonstrate the understanding of the kinetic parameters reached under pre-steady-state conditions and write the basic equations used for data fitting .
5. Explain the methods for studying protein:ligand interactions, demonstrate the understanding of the evaluated parameters and write the basic equations describing the most frequent, simple, models of protein:ligand interaction.
6. Summarize and explain the methods of fluorescent spectroscopy used in biological systems.
7. Visualize the protein structures, in apo or lignad-bound states, using the coordinates from the protein data bases.
8. Compare and conclude about the similarities and differences between protein and ribonucleoprotein enzymes.
9. Demonstrate the understanding of the basic phenomena in biocatalysis related to the overall protein structure and enzyme modular or oligomeric architecture.
10. Demonstrate the ability to evaluate various kinetic complementary approaches intended to unveil the enzyme mechanisms in the original scientific papers and in the independently developed mini-project.
- 1. A. Fersht, Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding, W. E. Freeman and Company, New York, 1999.
2. Pregledni znanstveni radovi po izboru nastavnika
3. Izvorni znanstveni radovi po izboru nastavnika
- Perry A. Frey and Adrian D. Hegeman, Enzymatic Reaction Mechanisms, Oxford University Press, 2007
I. H. Segel, Enzyme Kinetics : Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, Wiley Classics Library Edition, 1993