Enzymology

1. Enzymology Samra Khalid ASAB, National University of Sciences and Technology

2. Course content • Introduction and history of enzymes • Historical aspects • Discovery of enzymes • Chemistry of enzymes • Function and importance • Enzymes in biotechnology • Characteristics and properties • Catalytic power and specificity • Enzyme substrate complex • Catayltic cycle of enzyme • Nomenclature / Classification and Activity Measurements • Oxidoreductase-dehydrogenase • Transferase • Hydrolase • Lyase • Isomerase • Ligase • Activity measurements • Enzyme Purification and Assay • Initial velocity measurements • Assay types • Enzyme units of activity • Turnover number and properties • Purification and assessment • Methods for measurement • Enzyme kinetics • Michaelis-Menten Kinetics • Introduction • Assumptions • Derivation • Description of voversus [S] • Michaelis constant (KM)

3. Course content • Specificity/Substrate constant (SpC) • Graphical Analysis of Kinetic Data, pH and Temp Dependence • Graphical Analysis • Lineweaver-Burk Analysis • Hanes-Woolf Analysis • Eadie-Hofstee Analysis • Direct Linear Plot (Eisenthal/Cornish-Bowden Plot) • Nonlinear Curve Fitting • pH-dependence of Michaelis-Menten Enzymes • Temperature-Dependence of Enzyme Reactions • Single Molecule Enzymology • ATP Synthase • ATP Synthase with Tethered Actin • Myosin-V • Kinesin motor attached to a fluorescent bead • Single Molecule Studies of Cholesterol Oxidase • β-galactosidase: a model Michaelis-Menten enzyme? • Enzyme inhibition and kinetics • Classification of inhibitors • Reversible, Irreversible, Iodoacetamide, DIFP • Classification of Reversible Inhibitors • Competitive, Uncompetitive, Noncompetitive, Substrate • Multi-substrate Reactions and Substrate Binding Analysis • Substrate Binding Analysis • Single Binding Site Model • Binding Data Plots • Direct Plot • Reciprocal Plot • Scatchard Plot • Determination of Enzyme-Substrate Dissociation Constants • Kinetics • Equilibrium Dialysis • Equilibrium Gel Filtration • Ultracentrifugation • Spectroscopic Methods • Mechanism of enzyme catalysis • Engineering More Stable Enzymes • Incorporation of Non-natural Amino Acids into Enzymes • Protein Engineering by Combinatorial Methods • DNA Shuffling

4. Enzymes Biological catalyst… Biomolecules catalyze, increase the rates of chemical reactions Almost all enzymes are proteins. act only upon a specific substrate (or substrate group) do not change the energetics of the reaction Living systems use enzymes to accelerate and control the rates of vitally important biochemical reactions.

5. Historical Background 2100 BC Codex of Hammurabi-description of wine making 700 BC Homer’s Iliad: “As the juice of fig tree curdles milk, and thickens it in a moment though it be liquid, even so instantly did Paeeon cure fierce Mars” 1700s Réaumur - studies on the digestion of buzzards- digestion is a chemical rather than a physical process Late 1800s Kühne - term 'enzyme': Greek "in yeast" Hans &  Eduard Buchner – filtrates of yeast extracts could catalyse fermentation! No need to living cells E. Fischer – “lock and key” hypothesis 1903 Henri – first successful mathematical model 1913 Michaelis and Menten – NZ rate equation.... 1950s-1960s Koshland – “Induced fit” model 1965 Monod, Wyman and Changeux – allosteric regulation

6. History of Enzymes • As early as the late 1700s and early 1800s, the digestion of meat by stomach secretionsand the conversion of starch to sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified.

7. History of Enzymes • In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms. He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells.

8. In 1878 German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme, which comes from Greek ενζυμον "in leaven", to describe this process. The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment used to refer to chemical activity produced by living organisms. • In 1897 Eduard Buchner began to study the ability of yeast extracts that lacked any living yeast cells to ferment sugar. In a series of experiments at the University of Berlin, he found that the sugar was fermented even when there were no living yeast cells in the mixture. • He named the enzyme that brought about the fermentation of sucrose "zymase". In 1907 he received the Nobel Prize in Chemistry“ for his biochemical research and his discovery of cell-free fermentation".

9. Functions of Enzymes • Break down nutrients into useable molecules. (Lehninger et al., 1993, p. 198) • Store and release energy (ATP). (Lehninger et al., 1993, p. 198; Campbell & Reece, 2002, pp. 162-163) • Create larger molecules from smaller ones. (Lehninger et al., 1993, p. 198; Campbell & Reece, 2002, pp. 295, 316-317) • Coordinate biological reactions between different systems in an organism. (Lehninger et al., 1993, p. 198; Campbell & Reece, 2002, pp. 101-102)

10. Importance of Enzymes • They are catalysts so they make reactions easierto increases productivity and yield • As catalysts they are not consumed by the reactionmay be used over and over again • Most enzyme reaction rates are millions of times faster than those of un-catalyzed reactions. • Enzymes show specificity to the reaction they control • Enzymes are sensitive to their environment so they can be controlled by adjusting the temperature, the pH or the substrate concentration • However, enzymes do differ from most other catalysts by being much more specific.

11. Properties of enzymes as catalysts

12. Catalytic Power of Enzyme • Most enzyme reaction rates are millions of times faster than those of comparable uncatalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific. • The ratio of uncatalyzed to catalyzed reaction rate is called the catalytic power. For uncatalyzed hydrolysis of urea the reaction rate is 3x104 and for catalyzed reaction it is 3X10-10 • The Catalytic power is therefore 3x1014 .

13. Specificity • Enzymes are highly specific to their substrate and reaction catalysed • Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. • Most enzymes can be denatured that is, unfolded and inactivated by heating, which destroys the three-dimensional structure of the protein. • Depending on the enzyme, denaturation may be reversible or irreversible.