1. It is necessary to actively participate during practical lessons, perform all experiments and write lab reports. It is allowed to miss three practical lessons in a semester, out of these, one practical may be missed without compensation and students are obliged to compensate the remaining two practicals with oral presentations of the topics they missed.

2. To pass successfully 2 written tests (with minimal evaluation of mark E).

3. Average mark of tests (written in the begining of every lesson) increases the percentage gain of students.

Student is able to explain the basic principles of enzyme catalysed reaction, of bioenergetics, of metabolic regulation at level of cell and animal organism, respectively. He is able te read metabolic map from saccharide, lipid and steroid metabolism point of view.

Enzymology, kinetics of enzyme reactions, coenzymes,

bioenergetic metabolism, principles of thermodynamics, respiratory chain, oxidative phosphorylation,

metabolic regulation, hormonal control,

biological oxidation, tricarboxylic acid cycle,

metabolism of saccharides (glycolysis, pentose phosphate pathway, gluconeogenesis, glycogenolysis, glycogenesis),

lipid metabolism (fatty acid oxidation, fatty acid synthesis), biosynthesis of triacylglycerols and phospholipids, metabolism of eicosanoids,

cholesterol metabolism (synthesis and its conversion),

lipoprotein metabolism, synthesis and utilization of ketone bodies.

CURRICULUM OF THE LECTURES

1. Enzymology.

Introduction, basic characteristics of biochemistry and its importance for medicine.

Enzymes, enzymatic activity, reaction and substrate specificity, enzyme classes.

Catalytic function of enzymes and active sites; activation energy.

Enzyme catalysis, uncatalyzed reaction, enzyme-catalyzed reaction, principles of enzyme catalysis (serine proteinases as example).

Localization of enzymes in cell (cytoplasm, membrane, mitochondrion)

2. Enzyme kinetics.

Reaction kinetics (equilibrium constants, Guldberg-Waage law, reaction rate, reaction order), catalysis.

Michaelis-Menten kinetics, isosteric and allosteric enzymes.

Kinetic constants Vmax, Km, kcat and C.

Activation and inhibition of enzyme activity (types of inhibitions: reversible and irreversible; competitive, uncompetitive and non-competitive, allosteric).

Isoenzymes (lactate dehydrogenase as an example).

3. Coenzymes.

Redox coenzymes: NAD+, NADP+, FMN, FAD, ubiquinone, ascorbic acid, lipoamide, iron-sulfur cluster, heme.

Group-transferring coenzymes: nucleoside phosphates, coenzyme A, TPP, PLP, biotin, THF, cobalamin.

Activated metabolites (ATP, UDPglucose, CDPcholine, PAPS, SAM).

4. Metabolic regulation.

Intermediary metabolism, metabolic (anabolic, catabolic) pathways.

Regulatory mechanisms.

Allosteric regulation.

Transcription control.

5. Hormonal regulation.

Cell communication modes.

Signal molecules.

Mechanism of action of hydrophilic hormones.

Mechanism of action of lipophilic hormones.

6. Bioenergetic metabolism

Endergonic and exergonic reactions.

Energetic coupling.

ATP as a general energy transporter (structure, types of ATP formation)

Respiratory chain, its localization and enzyme systems.

Electrochemical gradient and proton motive power.

ATP synthesis (oxidative phosphorylation).

7. Biological oxidation.

Tricarboxylic acid cycle (reactions, functions, regulation).

8. Carbohydrate metabolism.

Carbohydrates (monosaccharides, disaccharides, polysaccharides, glycosaminoglycans and proteoglycans).

Glycolysis (reactions, energy profile, aerobic and anaerobic oxidation of glucose).

Oxoacid dehydrogenases – PDH reaction.

9. Carbohydrate metabolism.

Gluconeogenesis.

Pentose phosphate pathway of glucose degradation.

10. Carbohydrate metabolism.

Glycogen metabolism (glycogenesis, glycogenolysis).

Regulation of carbohydrate metabolism.

11. Lipid metabolism.

Fatty acids, fats, phospholipids, glycolipids, isoprenoids, steroids.

Fatty acid degradation (fatty acid transport, β-oxidation).

Degradation of unsaturated fatty acids and oddnumbered fatty acids.

α-oxidation, ω-oxidation, degradation of unusually long fatty acids.

12. Lipid metabolism.

Fatty acid synthesis.

Biosynthesis of triacylgylcerols and phospholipids.

Metabolism of eicosanoids.

13. Lipid metabolism.

Biosynthesis of cholesterol and its interconversions.

Lipoprotein metabolism.

Metabolism of ketone bodies.

SCHEME OF THE PRACTICAL LESSONS

1. Safety rules for working in biochemical laboratory on practical lessons.

Organization of the course in Biochemistry.

Kinetics of enzyme-catalyzed reaction. pH dependence of enzyme reactions.

a) The determination of the pH optimum for trypsin-catalyzed (EC 3.4.21.4) hydrolysis of N-α-benzoyl-DL-arginyl-p-nitroanilide (BAPA).

2. Kinetics of enzyme-catalyzed reaction. The influence of enzyme reactions. The influence of temperature and enzyme concentration on reaction rate.

a) Temperature dependence of the trypsin-catalyzed hydrolysis of BAPA.

b) Time course of the trypsin-catalyzed hydrolysis of BAPA.

c) The influence of trypsin concentration on the rate of hydrolysis of BAPA.

3. Kinetics of enzyme-catalyzed reaction. Substrate concentration dependence of reaction rate.

a) The influence of BAPA concentration on the rate of trypsin-catalyzed hydrolysis of BAPA.

b) The determination of kinetic constants: the Michaelis constant Km (Lineweaver-Burk), catalytic constatnt kcat and overall constant C.

4. Inhibition of enzyme activity.

a) The influence of soy bean inhibitor on the rate of the trypsin-catalyzed hydrolysis of BAPA.

b) The influence of blood serum inhibitor on the rate of the trypsin-catalyzed hydrolysis of BAPA.

c) The influence of α1-antitrypsin on the trypsin-catalyzed hydrolysis of α-tosyl-L-arginyl- p-nitroanilide (tutorial).

5. Activation of enzyme activity.

a) The influence of metal cations on the rate of the trypsin-catalyzed hydrolysis of BAPA.

b) The rate of the trypsin-catalyzed hydrolysis of BAPA in relation to the concentration of calcium ions.

6. 1st written credit test: Enzymology and coenzymes.

Coenzymes are derivatives of water-soluble vitamins.

a) Detection of thiamine (vitamin B1).

b) Detection of riboflavin (vitamin B2).

c) Detection of ascorbic acid (vitamin C).

7. Biological oxidation.

a) Detection of the xanthine oxidase (EC 1.1.3.22).

b) Detection of the catalase (EC 1.11.1.6).

c) Detection of the peroxidase (EC 1.11.1.7).

d) Pseudo-peroxidase property of hemoglobin.

8. Carbohydrate metabolism.

a) Reactions of carbohydrates.

b) Determination of glucose concentration in blood of domestic animals.

9. Carbohydrate metabolism.

Enzyme activity units and their calculation.

Isoenzymes. Lactate dehydrogenase (LDH), its diagnostic application.

c) Determination of total lactate dehydrogenase (LDH, EC 1.1.1.27) activity in blood serum of domestic animals.

10. Polysaccharide metabolism.

a) Iodine test for detection of starch.

b) Determination of α-amylase (AMS, EC 3.2.1.1) activity in blood serum and urine of domestic animals.

11. 2nd written credit test: Carbohydrate metabolisms. Bioenergetics.

Lipid metabolism.

a) Determination of total lipid concentration in blood serum of domestic animals.

b) Determination of triacylglycerol concentration in blood serum of domestic animals.

12. Lipid metabolism.

c) Determination of cholesterol concentration in blood serum of domestic animals.

d) Detection of cholesterol in butter, oil, lard and egg yolk.

13. Intermediary metabolism – metabolic pathways.

Student presentation of their seminary works.

Awarding credit points to students.

Harvey, R.A., Ferrier, D.R.: Lippincott´s Illustrated Reviews: Biochemistry. 5th Edition. Baltimore, Lippincott Wiliams and Wilkins, 2011, 521pp.

Koolman, J., Roehm, K. H.: Color Atlas of Biochemistry. 2nd Edition. Stuttgart; New York : Georg Thieme Verlag, 2005, 476 pp.

Nelson, D. L., Cox, M. M.: Lehninger Principles of Biochemistry. 4th Edition. New York : W. H. Freeman and Company, 2005, 1119 pp.

Stryer, L.: Biochemistry. 3rd Edition. New York : W. H. Freeman and Company, 1988, 1089 pp.

Heinová, D. et al.: Practical Course in Biochemistry I, Košice : UVLF, 2010, 105 pp.

Continuous assessment:

continuous tests (on practical lessons from actually tought topics), solution of tasks (in protocol form of performed practical work on laboratory lessons), two credit written tests from explained chapters

Conditions for completion of course:

1. It is necessary to actively participate during practical lessons, perform all experiments and write lab reports. It is allowed to miss three practical lessons in a semester, out of these, one practical may be missed without compensation and students are obliged to compensate the remaining two practicals with oral presentations of the topics they missed.

2. To pass successfully 2 written tests (with minimal evaluation of mark E).

3. Average mark of tests (written in the begining of every lesson) increases the percentage gain of students.

Final assessment:

credit