Enzyme catalysis

1. Enzyme catalysis a.a. 2015/2016

2. Programme • Introduction • Basic principles of chemical kinetics • Enzyme kinetics • Simple inhibition systems • Reactions of more than one substrate • Effect of pH and temperature on enzyme activity • Multisite and allosteric enzymes

3. Textbooks A. Cornish-Bowden Fundamentals of Enzyme Kinetics Portland Press Ltd, London 2004 I. Segel Enzyme Kinetics Wiley, N. Y. 1975

4. INTRODUCTION The earliest known references to enzymes are from ancient texts dealing with the manufacture of cheeses, breads, and alcoholic beverages, and for the tenderizing of meats. These processes were known as fermentation. Kühne, studying catalysis in yeast extracts, first coined the term ‘‘enzyme’’ (1878). The word derives from the medieval Greek word enzymos, which relates to the process of leavening bread.

5. Today enzymes continue to play key roles in many food and beverage manufacturing processes and are ingredients in numerous consumer products, such as laundry detergents (which dissolve protein-based stains with the help of proteolytic enzymes). Enzymes are also of fundamental interest in the health sciences, since many disease processes can be linked to the aberrant activities of one or a few enzymes.

6. While the ancients made much practical use of enzymatic activity, these early applications were based purely on empirical observations, rather than any systematic studies or appreciation for the chemical basis of the processes being utilized. In the eighteenth and nineteenth centuries scientists began to study the actions of enzymes in a more systematic fashion.

7. The famous French scientist Réaumur performed some of the earliest studies on the digestion of buzzards. Réaumur designed a metal tube with a wire mesh at one end that would hold a small piece of meat immobilized, to protect it from the physical action of the stomach tissue. He found that when a tube containing meat was inserted into the stomach of a buzzard, the meat was digested within 24 hours. René-Antoine Ferchault de Réaumur (1638-1757)

8. Spallanzani showed that the digestion of meat encased in a metal tube took place in the stomachs of a wide variety of animals, including humans. Using his own gastric juices, Spallanzani was able to perform digestion experiments on pieces of meat in vitro. Lazzaro Spallanzani 1729-1799 Gastric juices contain an active ingredient: a yeast

9. The vitalist debate: what is it a yeast? The controversy was focused on alcoholic fermentation The fermentation is catalyzed by a vital force contained within the yeast cells called ferments which function only within living organisms (~1858). Louis Pasteur

10. Friedrich Wöhler Justus von Liebig Yeast is not a living organism: alcoholic fermentation is just a chemical process

11. 1897: Eduard Buchner using the ‘‘pressed juice” from rehydrated dried yeast demonstrated that alcoholic fermentation could be performed in the absence of living yeast cells.

12. What are the enzymes? 1926: crystallization of urease James Sumner 1930: crystallization of pepsin, trypsin, chymotrypsin, and carboxypeptidase. John Howard Northrop Enzymes are (almost always) proteins

13. 1965: the X-ray structure of lysozyme David Chilton Phillips, Baron Phillips of Ellesmere

14. Convert substrates into products Present in all living cells Made of protein Enzymes Biological catalysts Increase the rate of chemical reactions Remain unchanged by chemical reaction

15. The substance acted on by an enzyme is called asubstrate. The part of an enzyme where substrates bind and undergo a chemical reaction is the active site

16. A catalyst increases the rate or velocity of a chemical reaction without itself being changed in the overall process. Therefore, they can continue to catalyze subsequent reactions. Enzymes speed up reactions by many orders of magnitude. For example, the enzyme catalase speeds up the conversion of hydrogen peroxide to water and oxygen by a factor of a billion. Catalysts change the rates of reactions, but do not affect the equilibrium of a reaction. That is, you cannot make more product from an enzyme-catalyzed reaction than you can from the same reaction without it. The enzyme simply helps to reach the equilibrium state faster than if it were not present.

18. Gli enzimi si combinano transientemente col substrato e ne abbassano l’energia di attivazione: stabilizzano lo stato di transizione

20. The lock and key model Fischer, 1894: The lock and key model assumes that the active site is a perfect fit for a specific substrate and that once the substrate binds to the enzyme no further modification is necessary.

21. The induced fit model Koshland, 1958: This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding.

22. “I think that enzymes are molecules that are complementary in structure to the activated complexes of the reactions that they catalyze, that is, to the molecular configuration that is intermediate between the reacting substances and the products of reaction for these catalyzed processes. The attraction of the enzyme molecule for the activated complex would thus lead to a decrease in its energy and hence to a decrease in the energy of activation of the reaction and to an increase in the rate of reaction.” - Linus Pauling Nature 161(1948):707

23. X-ray diffraction studies of the enzyme hexokinase both without and with glucose bound. The binding of glucose causes two domains of the enzyme to fold toward each other.

24. Enzymes are divided into six major classes 1. Oxidoreductases catalyze oxidation - reduction reactions. 2. Transferasescatalyze transfer of functional groups from one molecule to another. 3. Hydrolases catalyze hydrolytic cleavage. 4.Lyases catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement. 5. Isomerases catalyze intramolecular rearrangement. 6. Ligases catalyze reactions in which two molecules are joined.