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

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

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

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.
Almost all enzymes are proteins that act as biological catalysts
1. have complex structure (sequence of aa’s) spped up chemical reaction
2. Biomolecules that catalyze, increase the rates of chemical Reactions.
3. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products.
Enzymes lower the activation energy of a reaction

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 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.
Earliest known use of enzymes comes from the Egyptian civilization which used yeast for fermentation and called the product Boza.

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 )
Create larger molecules from smaller ones. (Lehninger et al., 1993, p. 198; Campbell & Reece, 2002, pp. 295, )
Coordinate biological reactions between different systems in an organism. (Lehninger et al., 1993, p. 198; Campbell & Reece, 2002, pp )

10. Importance of Enzymes
They are catalysts so they make reactions easier to increases productivity and yield
As catalysts they are not consumed by the reaction may 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.
Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions.

14. Enzymes and biotechnology

15. Almost all processes in a biological cell need enzymes in order to occur at significant rates. Since enzymes are extremely selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.
Every cell in every plant and animal contains many different types of enzyme. Each enzyme catalyzes a different reaction.

16. Biotechnology – an old art
Can you think of some products that have been made using biotechnology for thousands of years?
bread
cheese and yoghurt
beer and wine

17. What is Fermentation? + +
Yeast cells contain enzymes that converts sugars (such as glucose and sucrose) into alcohol (ethanol) and carbon dioxide. This reaction is called fermentation.
glucose  +
ethanol
carbon dioxide
C6H12O6 (aq)
C2H5OH (l)
CO2 (g)  +
Fermentation usually takes place at 20-30°C. It must take place in anaerobic conditions (without oxygen) otherwise the ethanol would react with oxygen and turn into vinegar.

18. Fermentation and beer-making
Barley grains are warmed with water to germinate. This produces sugar
Barley is boiled with water to release the sugar
Hops are added for flavour
Yeast is added and enzymes in this convert the sugar to alcohol
Beer is usually filtered and the yeast recycled to make more beer

19. Fermentation and yoghurt
Pasteurized or sterilized milk is used to kill unwanted bacteria
The milk is mixed with specially-cultured bacteria and kept warm
The enzyme lactase from the bacteria convert milk sugar (lactose) into lactic acid, which gives a sour taste and makes the product semi-solid

20. Biotechnology – a new science
Can you think of more recent uses of biotechnology?
using enzymes to improve detergents
transferring disease-resistance genes into plants
manufacturing medicines such as penicillin

21. Biological washing powder
Biological washing powders contain enzymes to help remove stains.
Proteases break down proteins in stains such as grass, blood and sweat.
Lipases break down stains containing fat and oil.
Carbohydrases break down stains containing carbohydrates, such as starch.
wax coat
The enzymes are coated with a special wax. This melts in the wash, releasing the enzymes. Once the stains have been broken down, they are easier to remove by the detergent.

22. Modern Biotechnology Enzymes in DNA-technology
DNA is basically a long chain of deoxyribose sugars linked together by phosphodiester bonds. Organic bases, adenine, thymine, guanine and cytosine are linked to the sugars and form the alphabet of genes. The specific order of the organic bases in the chain constitutes the genetic language.
Genetic engineering means reading and modifying this language. Enzymes are crucial tools in this process.

23. Modern Biotechnology Enzymes in DNA-technology
DNA-technology has revolutionized both traditional biotechnology and opened totally new fields for scientific study.
Recombinant DNA-technology allows one to produce new enzymes in traditional overproducing and safe organisms
Protein engineering is used to modify and improve existing enzymes.

24. Enzymes and Industry
Enzymes are becoming more common as catalysts for industrial processes. Why is this the case?
Enzymes work at fairly low temperatures – this saves energy and money, and reduces pollution.
Enzymes work in fairly mild conditions (normal pressure, in water and pH close to 7) – this reduces the need for potentially dangerous chemicals.
Enzyme-reactions can be easily controlled – by slightly changing the temperature or pH.
Enzymes are biodegradable – they reduce pollution and environmental problems.

25. There are thousands of different enzymes in your body.

26. Enzyme action
Enzymes are large molecules that have a small section dedicated to a specific reaction. This section is called the active site.
The active site reacts with the desired substance, called the substrate.
The substrate may need an environment different from the mostly neutral environment of the cell in order to react. Thus, the active site can be more acidic or basic, or provide opportunities for different types of bonding to occur, depending on what type of side chains are present on the amino acids of the active site.

27. Enzyme action Why are there so many different enzymes?
Each enzyme has its own unique protein structure and shape, which is designed to match or COMPLEMENT on its one type of SPECIFIC substrate. Products have a different shape from the substrate.

30. The shape of the active site (binding site) of the enzyme, matches the shape of the substrate. Allowing the two molecules to bind during the chemical reaction.
The part of the enzyme with which the reactant binds is called the active site. This is a very specific shape.

31. Enzyme Action Theories

32. Lock & Key Hypothesis
Fit between the substrate and the active site of the enzyme is exact
The key is analogous to the enzyme and the substrate analogous to the lock.
Temporary structure called the enzyme-substrate complex formed
Once formed, they are released from the active site
Leaving it free to become attached to another substrate
This theory of enzyme action is called the ‘lock-and-key’ hypothesis
Like a key fits into a lock very precisely
Lock and Key: This theory, postulated by Emil Fischer in 1894, proposed that an enzyme is “structurally complementary to their substrates” and thus fit together perfectly like a lock and key. This theory formed the basis of most of the ideas of how enzymes work, but is not completely correct.
Enzymes are very specific, because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model.

33. Lock and Key Hypothesis
Enzyme may be used again
Enzyme-substrate complex E S P
Reaction coordinate

34. The Lock and Key Hypothesis
This explains enzyme specificity
This explains the loss of activity when enzymes denature

35. E + S <---> [ES] <---> E + P
Enzyme Action
  E + S <---> [ES] <---> E + P
enzymes catalyze reactions by lowering the energy of activation (Ea)                     

36. Induced Fit Hypothesis
Some proteins can change their shape (conformation)
When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation
The active site is then moulded into a precise conformation
Making the chemical environment suitable for the reaction
The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)
Induced Fit: An enzyme that is perfectly complementary to its substrate would actually not make a good enzyme because the reaction has no room to proceed to the transition state of the reaction. To go to completion, a reaction must go through the transition state. In the lock and key theory, the substrate or the enzyme cannot change conformations to the transition state. Therefore, enzymes must actually be complementary to the transition state so the reaction may proceed. This idea was researched by Haldane in 1930, and Linus Pauling in This idea led the Induced Fit theory, postulated by Daniel Koshland in 1958, where the enzyme itself can change conformations to facilitate the transition state of the substrate. This change in conformation of the enzyme allows the necessary functional groups at the active site to move closer to the substrate, enhancing the efficiency of the reaction.
© 2007 Paul Billiet ODWS

37. Induced Fit Hypothesis
Enzymes can form to the shape of its substrate

38. Enzyme activity and inhibition
The “normal” way an enzyme functions is when the specific substrate binds to the active site and creates the products.
A similar substrate can also bond to the active site covalently and irreversibly. This prevents the enzyme from functioning.
A similar substrate can bind to the active site, not permanently, and prevents the desired substrate from entering the active site. This changes the products and functioning of the enzyme. This is called competitive inhibition.
A molecule can bond to another part of the enzyme and cause a change in conformation. This change causes the active site to change shape as well. This change in shape prevents the desired substrate from entering the active site. This is called non-competitive inhibition.

39. Catalytic cycle of an enzyme

40. Enzyme cofactors
A cofactor is a substance that is not a protein that must bind to the enzyme in order for the enzyme to work.
A cofactor can be of organic origin. An organic cofactor is called a coenzyme.
Cofactors are not permanently bonded. Permanently bonded cofactors are called prosthetic groups.
An enzyme that is bonded to its cofactor is called a holoenzyme.
An enzyme that requires a cofactor, but is not bonded to the cofactor is called an apoenzyme. Apoenzymes are not active until they are complexed with the appropriate cofactor.

41. Factors That Affect Enzyme Activity
Enzyme activity can be affected by other molecules.
Inhibitors are molecules that decrease enzyme activity. Many drugs and poisons are enzyme inhibitors.
activators are molecules that increase activity.
Activity is also affected by temperature, chemical environment (e.g. pH)

42. Enzymes and temperature
Enzymes are mostly affected by changes in temperature and pH. (Campbell & Reece, 2002, pp )
Enzyme-catalyzed reactions, usually 20-45°C.
All enzymes work best at only one particular optimum temperature. Different enzymes have different optimum temperatures.
As the temperature decreases below the optimum the enzyme will eventually become inactive. The reaction will stop.
Too high of a temperature will denature the protein components, rendering the enzyme useless.
As the temperature increases above the optimum, the reaction quickly slows down and stops completely. This is because the temperature irreversibly alters the shape of the enzyme and stops it working. At this point, the enzyme is said to be denatured

43. Enzymes and pH Enzyme reactions occur across a range of pH values.
Like for temperature, each enzyme will work best at only one particular pH.
Some enzymes, for example, those in the stomach, work best in acidic conditions.
Other enzymes work best in alkaline conditions.
pH ranges outside of the optimal range will protonate or deprotonate the side chains of the amino acids involved in the enzyme’s function which may make them incapable of catalyzing a reaction.
If the pH value changes sufficiently, the enzyme’s shape irreversibly changes and it will be denatured.

44. Summary of Enzymes Enzymes are mostly proteins
They are highly specific to a reaction
They catalyze many reactions including breaking down nutrients, storing and releasing energy, creating new molecules, and coordinating biological reactions.
Enzymes use an active site, but can be affected by bonding at other areas of the enzyme.
Some enzymes need special molecules called cofactors to carry out their function.
Cofactors that are organic in nature are called coenzymes.
Coenzymes are usually derived from vitamins.
Coenzymes transfer functional groups for the enzyme they work with.
Enzymes are affected by changes in pH, temperature, the amount of substrate, cofactors and inhibitors, as well as the amount of allosteric inhibitors and activators and concentration of products that control feedback inhibition.

45. Glossary
active site – The part of the enzyme into which the reactant molecule fits.
catalyst – A substance that changes the rate of a reaction without being used up.
denatured – The state of an enzyme when it has been irreversibly damaged and has changed shape.
enzyme – A biological catalyst.
fermentation – The conversion of sugar to ethanol and carbon dioxide by enzymes in yeast.
lock and key – A model of how enzymes work and the importance of their shape.