Enzymology

1. Enzymology Dr. Nasir Jalal 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 • Enzymes as catalysts • Enzyme - substrate interactions • Lock & key model • Induced fit model • Transition state model • Quantum tunnelling model • Enzymes as proteins • Non-protein cofactors • Metal ions • Organic cofactors • 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. My office hours • Thursdays 9:00-11:00 • Fridays2:00-4:00 • Nasir.Jalal@doctor.com

5. Introduction to enzymology • Enzymes are Biomolecules that catalyze, increase the rates of chemical reactions by 1015 to 1017 fold. • Almost all enzymes are proteins. • In enzymic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. • Living systems use enzymes to accelerate and control the rates of vitally important biochemical reactions.

6. Brief history • Earliest known use of enzymes comes from the Egyptian civilization which used yeast for fermentation and called the product Boza. • HISTORY of Enzymes As early as the late 1700s and early 1800s, the digestion of meat by stomach secretions and 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. Fermentation 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. Meaning of “enzyme” (Greek) In 1878 German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme , which comes from Greek ενζυμον (enzymon)"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. History of Biotechnology

10. Stages of Biotech evolution • Ancient • Classical • Modern

11. Ancient Biotechnology

12. Ancient Biotech • Begins with early civilization • Developments in agriculture and food production • Few records exist

13. Ancient Biotech • Archeologists research • Ancient carvings and sketches sources of information

14. Ancient Biotech • Not known when biotech began exactly • Focused on having food and other human needs

15. Ancient Biotech • Useful plants brought from the wild, planted near caves where people lived • As food was available, ability to store and preserve emerged

16. Ancient • Food preservation most likely came from unplanned events such as a fire or freeze.

17. Domestication • 15,000 years ago, large animals were hard to capture • People only had meat when they found a dead animal • Came up with ways of capturing fish and small animals

18. Domestication • Food supplies often seasonal • Winter food supplies may get quite low • Domestication is seen by scientists as the beginning of biotech

19. Domestication • Adaptation of organisms so they can be cultured • Most likely began 11,000 – 12,000 years ago in the middle east

20. Domestication • Involved the collecting of seed from useful plants and growing crude crops from that seed • Involved the knowledge that the seed had to properly mature. • A most recent find in Peru, documents the first civilization at 15000 years.

21. Domestication • Proper planting • Need for water, light and other conditions for plant growth • Earliest plants likely grains and other seeds used for food

22. Domestication • Raising animals in captivity began about the same time in history. • Easier to have an animal close by than to hunt and capture a wild one.

23. Domestication • Learned that animals need food and water. • Learned about simple breeding. • How to raise young. • Cattle, goats and sheep were the first domesticated food animals.

24. Domestication • About 10,000 years ago, people had learned enough about plants and animals to grow their own food • The beginning of farming.

25. Food • Domestication resulted in food supplies being greater in certain times of the year. • Products were gathered and stored.

26. Food • Some foods rotted • Others changed form and continued to be good to eat • Foods stored in a cool cave did not spoil as quickly

27. Food • Foods heated by fire also did not spoil as quickly • Immersing in sour liquids prevented food decay

28. Food preservation • Using processes that prevent or slow spoilage • Heating, cooling, keeps microorganisms (mo’s) from growing

29. Food preservation • Stored in bags of leather or jars of clay • Fermentation occurs if certain mo’s are present • Creates an acid condition that slows or prevents spoilage

30. Classical Biotechnology

31. Classical Biotech • Follows ancient practices. • Makes wide spread use of methods from ancient practices, especially fermentation. • Methods adapted to industrial production e.g., salting, canning.

32. Classical Biotech • Produce large quantities of food products and other materials in short amount of time. • Meet demands of increasing population. • Many methods developed through classical biotech are widely used today.

33. Cheese • One of the first food products made through biotechnology • Began some 4,000 years ago • Nomadic tribes in Asia

34. Cheese • Strains of bacteria were added to milk • Caused acid to form • Resulting in sour milk

35. Cheese • Enzyme called “rennet” was added • Rennet comes from the lining of the stomachs of calves

36. Cheese • Rennet is genetically engineered today. • Not all cheese is made from produced rennet. • Rennet is a complex of enzymes produced in mammalian stomach, and is used in the production of cheese. Rennet contains several enzymes, including the proteolytic enzyme protease that coagulates the milk, causing it to separate into solids (curds) and liquid (whey). They are also very important in the stomach of young mammals as they digest their mothers' milk. The active enzyme in rennet is called chymosin or rennin.

37. Yeast • Long used in food preparation and preservation • Bread baking • Yeast produces a gas in the dough causing the dough to rise

38. Yeast • Fermented products • Vinegar • Require the use of yeast in at least one stage of production. • Sugar (glucose or fructose) → alcohol (ethanol) + carbon dioxide C6H12O6 → 2 CH3CH2OH + 2 CO2

39. Yeast • Species of fungi • Some are useful • Some may cause diseases

40. Vinegar • Ancient product used to preserve food • Juices and extracts from fruits and grains can be fermented

41. Fermentation • Process in which yeast enzymes chemically change compounds into alcohol • In making vinegar the first product of fermentation is alcohol

42. Fermentation • Alcohol is converted to acetic acid by additional microbe activity • Acid gives vinegar a sour taste • Vinegar prevents growth of some bacteria

43. Vinegar • Keeps foods from spoiling • Used in pickling • Biblical references to wine indicate the use of fermentation some 3,000 years ago

44. Fermentation control • In ancient times, likely happened by accident • Advancements occurred in the 1800’s and early 1900’s

45. Fermenters • Used to advance fermentation process • Specially designed chamber that promotes fermentation

46. Fermenters • Allowed better control, especially with vinegar • New products such as glycerol, acetone, and citric acid resulted

47. Development • Of yeasts that were predictable and readily available led to modern baking industry

48. Modern Biotechnology

49. Modern Biotech • Manipulation of genetic material within organisms e.g., pruteen. • Based on genetics and the use of microscopy, biochemical methods, related sciences and technologies.

50. Modern Biotech • Often known as genetic engineering • Roots involved the investigation of genes