00061 - Biochemistry

Learning outcomes

By the end of the course, the student will know: - the biological processes at the molecular level - the structure-function relationships of biomolecules with particular reference to proteins - energy metabolism. They will have acquired an integrated view of cell signaling and metabolism; - the correct procedures for working in a biological laboratory; - the basics of structural biochemistry and enzymology; - the main methods for the determination of proteins by spectrophotometry; - the use of experimental protocols for the calculation of the specific activity of enzymes . Students must also know how to critically evaluate the results obtained by writing a report on the experiments carried out.

Course contents

1st module (6 credits) Prof Romana Fato

1. Stucture and catalysis

• Proteins structure and function:  Hemoglobin and myoglobin. Structure of heme. Saturation curves and cooperativity.
• Enzymes: Classification of enzymes. The enzymatic catalysis. Activation energy. Structure and general properties of enzymes. Cofactors and coenzymes.
• Enzyme kinetics: Km, Vmax, kcat. The Michaelis-Menten equation. Effect of pH and temperature on enzyme activity. Inhibition of enzyme activity: reversible inhibition competitive and non-competitive. Regulation of enzyme activity: allosteric enzymes and cooperativity, positive and negative modulators; reversible, irreversible and covalent modifications.

2. Bioenergetics and biochemical reactions.

• Bioenergetics and Thermodynamics
• Chemical Logic and Common Biochemical Reactions
• Phosphoryl Group Transfers and ATP
• coupled reactions

3. Metabolism.

• Bioenergetics and metabolism: catabolism and anabolism. Free Energy Change and coupled reactions. Transfer of phosphate groups and ATP. The thioesters. The biological oxidation-reduction reactions. Flow regulation in the metabolic pathways.

4. Glucose metabolism.

• Glycolysis. The fate of pyruvate. The glycolysis regulation.
• The pentose phosphate pathway.
• Gluconeogenesis and glycogen metabolism: glycogen degradation and synthesis of and their control. Gluconeogenesis: reactions and adjustment.

5. Tricarboxylic acid cycle.

• Tricarboxylic acid cycle its regulation and interrelationships with other metabolisms (anaplerotic role).

6. Electron transport and oxidative phosphorylation.

• The electron carriers in the mitochondrial respiratory chain. Oxidative phosphorylation and ATP synthesis. Uncoupling regulation of oxidative phosphorylation and inhibition.
  
  

7. Lipid metabolism.

• β-oxidation of fatty acids. Training and use of ketone bodies. polyunsaturated fatty acids.
• Biosynthesis of fatty acids: reactions and adjustment.
• Elongation and desaturation of fatty acids.
• Biosynthesis of triacylglycerols and phospholipids.
• Cholesterol and regulation of metabolic pathway.

8. Degradation of amino acids and urea cycle.

• transamination and oxidative deamination reactions. Protein degradation.

9. Signal transduction.

• cAMP-dependent signaling pathway. The inositol phosphate signaling pathway. Pathway that involves a receptor tyrosine kinase phosphorylation cascade.

2nd Module (2 CFU) Prof.Giovanna Farruggia and Prof. Concettina Cappadone

The Biochemistry Laboratory module consists of a laboratory integrated into the teaching activity of the Biochemistry course. The laboratory is placed at the beginning of the course and introduces students to the basics of the study of enzymes. Students will learn to calculate the specific activity of an enzyme in a cellular protein lysate. They will evaluate the kinetic parameters of a purified enzyme as well as the dependence of enzyme activity on pH and substrate specificity. In particular, the following activities will be carried out:

1. Preparation of a cell lysate in hypotonic buffers.

2. Determination of the protein content of the lysate using the Bradford method.

3. Polyacrylamide gel electrophoresis of the cell lysate and/or standard proteins.

4. Determination of Vmax and Km of the enzyme alkaline phosphatase.

5. Determination of the effect of pH and specificity on enzyme activity.

6. Determination of the molar extinction coefficient of NADPH using the glucose 6-phosphate dehydrogenase reaction.

7. Staining of slides with cells of different histogenesis to highlight the presence of neutral lipids.

At the end of the module, an analysis session of the collected data assessed individually and globally will be held. How to write a scientific report will be collectively discussed based on the results obtained.

In addition to the practical part, there will be a limited theoretical part covering methods of protein extraction from biological samples, quantitative analysis by UV-Vis. absorption spectroscopy and separation by electrophoresis.

 

Readings/Bibliography

1st Module

Berg, Tymoczko, Stryer "Biochemistry" Ed. Zanichelli .

Nelson, Cox "Lehninger Principles of Biochemistry" Ed.Zanichelli.

Garret, Grisham "Biochemistry" 5th edition Ed. Piccinin

Biochemistry

Concepts and Connections . Dean R. Appling - Spencer J. Anthony-Cahill - Christopher K. Mathews Pearson ed.

2nd module
The student will be provided protocols with a detailed description of the procedures to be followed during the workshop.

Teaching methods

1st module: lectures
2nd module: laboratory practice

Assessment methods

The learning assessment at the end of the course aims to evaluate the achievement of the following objectives:

1. Module:

-Knowledge of the structural and functional relationships of key biological macromolecules and the theoretical basis of enzymology.

- Knowledge of cellular bioenergetics, the most important metabolic pathways and the mechanisms underlying their regulation and signal transduction.

- Knowledge of the basic molecular mechanisms of living systems and the molecular logic of their adaptation.

2. Module:

-Knowledge of the most important methods for determining proteins

-Knowledge of the experimental methods for measuring enzyme activity

For the 2nd module, the examination consists of the assessment of a written report on a practical laboratory course, for the 1st module it consists of an oral examination.

Biochemistry module 1: the examination consists of an oral examination lasting 20-30 minutes with three questions aimed at assessing the student's theoretical knowledge of the structure and function of biological macromolecules, metabolism and its regulation.

2. Biochemistry module 2: evidence of assessment consists of a written report on the experience gained in the laboratory. A score between 0 and 30 is awarded for the written report.

The examination is passed if the score achieved is between 18 and 30. The final grade is determined by adding up the weighted grades.

Teaching tools

1st Module: power point slides

2nd module: practical laboratory equipped with spectrophotometers and thermostatic baths. Computer laboratory with single-seat. Cell cultures laboratory.

Office hours

See the website of Romana Fato

See the website of Concettina Cappadone

See the website of Giovanna Farruggia