Welcome to class of Basic enzymology

1. Welcome to class ofBasic enzymology Dr. Meera Kaur

2. Learning Objectives • To understand • Enzyme and how they differ from nonbiological catalyst • Properties of enzymes and principles underlying their catalytic power • The classifications of enzymes • The factors determining the rate of a reaction (activation energy) • Enzyme’s role in lowering the activation energy in a reaction • The lock and key model to explain the binding and specificity in enzyme action

3. What is an Enzyme? • Enzymes - Are the most remarkable and highly specialized protein - Catalyze hundreds of stepwise reactions in biological systems - Regulate many different metabolic activities necessary to sustain life In short, Living systems use catalyst called enzymes to increase the rate of chemical reactions

4. Why to study enzymology The study of enzymes has immense practical importance: - In medical science: to know the epidemiology, to diagnose, and to treat diseases ( inheritable genetic disorders) - In chemical industries - In food Processing - In agriculture - In everyday activities in the home ( food preparation, cleaning, beauty care etc.)

6. A note on nomenclature… • The International Enzyme Commission (EC) has recommended a systematic nomenclature for enzymes. • This commission assigns names and numbers to enzymes according to the reaction they catalyze. An example of systematic enzyme name is EC 3.5.1.5 urea aminohydrolases for the enzyme that catalyzes the hydrolysis of urea. • The name of an enzyme frequently provides a clue to its function. In some cases, an enzyme is named by incorporating the suffix -ase into the name of its substrate, e.g., pyruvate decarboxylases catalyzes the removal of a CO2 group from pyruvate

7. A note on nomenclature • Certain protein cutting digestive enzymes are exception to this general rule of enzyme nomenclature, e.g., pepsin, trypsin, chymotrypsin and thrombin. Isozyme: Within an organism more than one enzyme may catalyze a given reaction. Multiple enzymes catalyzing the same reaction is called isozymes. - isozymes differ in their catalytic properties. Consequently, the various isozymes that are present in different tissues or at different developmental stages can carry out slightly different metabolic functions.

8. Pyruvate decarboxylase

9. Alanine aminotransferase

10. Enzyme classification Class of Enzyme Type of reaction catalyzed • Oxidoreductases Oxidation-reduction reactions • Transferases Transfer of functional groups • Hydrolases Hydrolysis reactions • Lyases Group elimination to form double bond • Isomerases Isomerization reactions • Ligases Bond formation couples with ATP hydrolysis

11. Enzymes vs. Nonbiological catalysts • Enzymes have their reaction specificity • The activities of many enzymes are regulated so that the organism can respond to the changing condition or follow genetically determined developmental programs • Enzyme activity is affected by the temperature and pH of the medium

12. Properties of enzymes… • All enzymes are proteins or protein in nature (except a small group of catalytic molecules) • Enzymes have molecular weight ranging form 20,000 - 1 million • Some enzymes require no chemical group other than their amino acid residue for activity. • Some enzymes may require an additional chemical compound called cofactors (Zn2+, Mg2+, Fe2+, Mn2+etc.) • Some enzymes may require a complex orgainc or metelloorganic molecule called coenzymes • Sometimes they need both cofactors and coenzymes

13. Properties of enzymes • A coenzyme or metal ion that is covalently bound to the enzyme protein is called a prosthetic group • A complete catalytically active enzymes together with its coenzymes and or metal ions is called a holoenzyme • The protein part of such an enzyme is called the apoenzyme or apoprotein • Enzymes are classified by the reactions they catalyze. Many enzymes have been named by adding the suffix “-ase” to the name of their substrate or to a word or phrase describing their activities. Thus urease catalyzes hydrolysis of urea.

14. How do enzymes work ? • An enzyme circumvents many problems providing a specific environment within which a given reaction is energetically more favorable. • An enzyme-catalyzed reaction occurs within the confinement of a pocket on the enzymes called the active site • The molecule that is bound by the active site and acted upon by the enzyme is called the substrate • The enzyme-substrate complex is central to the action of enzymes.

15. Enzyme specificity Enzymes are specific and efficient catalyst In nonenzymatic reactions, substrates are transformed into product by bond-making and bond-breaking processes. In an enzymatic reaction, these processes occur through interactions of the substrates with appropriate side chains of the amino acid residues of the enzyme or with the coenzyme of the enzyme. - One property of many enzymes is specificity: catalyzing one chemical reaction with only one substrate An Example isCarbonic anhydrase. This enzyme catalyzes only one reversible reaction, the breakdown of carbonic acid (H2CO3) to water and carbon dioxide. At ideal conditions, a single molecule of Carbonic anhydrase is capable of catalyzing the breakdown of about 36 million molecules of carbonic acid in one minute.

16. How do enzymes work? • In a biological reaction, the reacting species must come together and undergo electronic rearrangement that result in the formation of a product. Let us consider an idealized transfer reaction in which compound A—B reacts with compound C to form two new products, A and B—C. • In order for the first two compounds to react, they must approach closely enough for their constituent atoms to interact. • Normally, atoms that approach too closely repel each other; but if the groups have sufficient free energy, they can pass this point and react with each other to form products. • The energy requiring step of the reaction is known as an energy barrier, called as free energy of activation or activation energy (G‡)

17. Idealized transfer reaction

18. Reaction coordinate diagram for the reaction A—B + C A + B—C The progress of the reaction(reaction coordinate) is shown on the horizontal axis, and the free energy(G) is shown on the vertical axis. The transition state of the reaction, represented as a..b..c, is the point of highest free energy.The free energy difference between the reactant and the transition state is the free energy of activation (G‡). .

19. Reaction coordinate diagram of a reaction: Here reactants and products have different free energies When the free energy of the reactant is greater than that of the product, the free energy changes for the reaction is negative, so the reaction proceeds spontaneously (a). When the free energy of the product is greater than that of the reactant, the free energy change of the reaction is positive, so the reaction does not proceed spontaneously (however the reverse reaction does proceed).

20. Effect of a catalyst on a chemical reaction Here the reactants proceed through a transition state (X‡) during their conversion to products. A catalyst lowers the free energy of activation ( G‡) for the reaction so that G‡cat < G‡uncat. Loweing the free energy of the transition state (X‡) accelerates the reaction because more reactants are able to achieve the transition state per unit time

21. Enzymes use chemical catalytic mechanism • There are three basic kinds of chemical catalytic mechanisms used by enzymes: - Acid-base catalysis - Covalent catalysis - Metal ion catalysis

22. Acid-base catalysis • In acid-base catalysis a proton is transferred between the enzyme and the substrate • It may be either acid catalysis or base catalysis or both • Many enzymes use both

23. Tautomerization of a ketone Tautomers are interconvertible isomers that differ in the placement of a hydrogen and a double bond. The dotted lines represent bonds in the process of breaking or forming.

24. Acid catalysis If a catalyst (H—A) donates a proton to the to the keton’s oxygen atom, it reduces the unfavorable character of the transition state, thereby lowers its energy. Hence, it also lowers the activation energy barriers for the reaction. This is an example of acid catalyst, since the catalyst acts as an acid by donating a proton.

25. Keto-enol tautomerization(base catalysis) The same keto-enol tautomerization reaction shown in the previous slide can be accelerated by a catalyst that can accept a proton. In this case, it is a base catalyst (:B), where the dots represent unpaired electron. Base catalysis lowers the energy of transition state and thereby accelerates the reaction.

26. Amino acid side chains that can act as acid-base catalysts

27. Covalent catalysis… • In covalent catalysis, a covalent bond is formed between the catalyst and the substrate during formation of the transition state • In covalent catalysis, an electron rich group in the enzyme active site forms a covalent adduct with a substrate. This covalent complex is much more stable than a transition state and can be isolated. • Covalent catalysis is also called nucleophilic catalysis, because the catalyst is a nucleophile (an electron-rich group in search of a electron -poor center ( a compound with an electron deficiency is known as an electrophile) • Enzymes that use covalent catalysis undergo a two-part reaction process. So the reaction coordinate diagram contains two energy barriers with the reaction intermediate between them.

28. Protein groups that can act as covalent catalysts

29. Reaction coordinate diagram for a reaction accelerated by covalent catalysis Two transition states flank the covalent intermediate. The relative heights of the activation energy barriers (the energies of the two transition states, X‡1and X‡2) vary depending on the reactions.

30. Metal ion catalysis… • Metal ions participate in enzymatic reactions by mediating oxidation-reduction reactions, or by promoting the reactivity of other groups in the enzyme’s active site through electrostatic effects • A protein bound metal ion can interact directly with the reacting substrate

31. Metal ion catalysis Example:The conversion of acetaldehyde to ethanol catalyzed by the liver enzyme alcohol dehydrogenase. Here a zinc ion stabilizes the developing negative charge on the oxygen atom during formation of the transition state.

32. The unique properties of enzyme catalyst • Enzyme–substrate complexes: • Enzymes are specific and efficient catalyst • Lock and key fit of enzymes and substrates help produce enzyme specificity

33. Lock-and-key model of enzyme action In this early model, the substrate (key) was believed to bind tightly to a site on the enzyme (lock) that was exactly complementary to it in its size, shape and charge.

34. Enzyme-substrate complexes Lock and key fit of enzymes and substrates helps produce enzyme specificity: A substrate molecule must contact an enzyme molecule before it can be transformed into products. Once the substrate has made contact, it must bind to the enzyme at the active site— a place where the conversion of substrate to product can take effect. The active site is usually a dimple, pocket or crevice formed by fold in the chain conformation of the protein. When the substrate key fits the enzyme lock, an enzyme-substrate complex is formed. The fit between enzyme and substrate in binding helps to produce enzyme specificity. Complementarity between enzyme and and substrate is governed by factors besides shape– e.g., the electrical charge. The substrate may be positively charged and the enzyme’s active site negatively charged and vice versa. Hydrogen bonds and other weak forces may also hold the the enzyme- substrate complex together. Hydrophobic regions of the substrate associate with similar regions on the active site of the enzyme. For most enzymes contributions from shape, charge, and a large number of weak forces are responsible for the formation and strength of the complex.

35. Transition state stabilization • Linus Pauling (1946) formulated the principle that • An enzyme increases the reaction rate not by binding tightly to the substrates, but by binding tightly to the reaction’s transition state, i.e., substrate that have been strained toward the structure of the product. In other word, the tightly bound key of the lock and key model is the ‘transition state’, not the substrate. Pauling’s theory of enzyme’s actions is consistent with the chemical catalytic mechanisms, in which the catalyst stabilizes the transition state of the reaction. In an enzyme, tight binding( stabilization) of the transition state occurs in addition to acid-base, covalent, or metal ion catalysis.

36. Effect of very tight substrate binding on enzyme catalysis The red line indicates the free energy changes as the reactants pass through a transition state to products. The blue line indicates free energy changes for the same reaction when the reactants (substrates) are very tightly bound to the enzyme ( they are at a very low free energy level) before they react. The activation energy barrier (G‡) is therefore increased because the tight binding must be overcome before the substrates can reach the transition state.