Lecture 8 Enzyme

1. Lecture 8 Enzyme

2. Outline • Composition, structure and properties of enzyme • Enzyme kinetics • Catalytic mechanisms of enzyme • Regulation of enzyme activities

3. 1. Introduction to enzymes (1). Much of the early history of biochemistry is the history of enzyme research (2). Biological catalysts were first recognized in studying animal food digestion and sugar fermentation with yeast (brewing and wine making) (3). Ferments (i.e., enzymes, meaning in “in yeast”) were thought (wrongly) to be inseparable from living yeast cells for quite some time (Louis Pasteur)

4. (4). Yeast extracts were found to be able to ferment sugar to alcohol (Eduard Buchner, 1897, who won the Nobel Prize in Chemistry in 1907 for this discovery) (5). Enzymes were found to be proteins (1920s to 1930s, James Sumner on urease and catalase ,“all enzymes are proteins”, John Northrop on pepsin and trypsin, both shared the 1946 Nobel Prize in Chemistry) (6). Catalytic RNA (also called ribozyme ---from ribonucleic acid enzyme, or RNA enzyme)were found in the 1980s (Thomas Cech, Nobel Prize in Chemistry in 1989)

5. 1. Definition of enzyme • Enzymes are biological catalysts. • A Catalyst is defined as "a substance that increases the rate of a chemical reaction without being itself changed in the process.”

6. What is the difference between an enzyme and a protein? Protein RNA Enzymes • All enzymes are proteins except some RNAs • not all proteins are enzymes

7. Enzymes are the most remarkable and specialized biological catalysts • An enzyme catalyzes a chemical reaction at a specifically structured active site, being often a pocket. • Enzymes have extraordinary catalytic power, often far greater than those non-biological catalysts. • Enzymes often have a high degree of specificity for their substrates. • Enzymes are often regulatory. • Enzymes usually work under very mild conditions of temperature and pH. • The substance acted on by an enzyme is called a substrate, which binds to the active site of an enzyme in a complementary manner.

8. 2. How enzymes work (important!) 1) Enzymes lower a reaction’s activation energy • All chemical reactions have an energy barrier, called the activation energy, separating the reactants and the products. • activation energy: amount of energy needed to disrupt stable molecule so that reaction can take place.

9. Enzymes Lower a Reaction’s Activation Energy

10. 2) The active site of the enzyme • Enzymes bind substrates to their active site and stabilize the transition state of the reaction. • The active site of the enzyme is the place where the substrate binds and at which catalysis occurs. • The active site binds the substrate, forming an enzyme-substrate(ES) complex. Binding site Active site Catalytic site

11. Enzymatic reaction steps 1. Substrate approaches active site 2. Enzyme-substrate complex forms 3. Substrate transformed into products 4. Products released 5. Enzyme recycled

12. Characteristics of active sites • The active site takes up a small part of the total volume of the enzyme. • The active site is 3-dimensional and is generally found in a crevice or cleft on the enzyme. • The active site displays highly specific substrate binding.

13. Active center of lysozyme 129 aa, discovered by Fleming in 1922 in tears Active center may include distant residues

15. 3. Properties of enzymes (important!) • Catalytic efficiency– high efficiency, 103 to 1017 faster than the corresponding uncatalyzed reactions • Specificity- high specificity, interacting with one or a few specific substrates and catalyzing only one type of chemical reaction. • Mild reaction conditions-37℃, physiological pH, ambient atmospheric pressure

16. Urease Urease High specificity 1). Absolute specificity:the enzyme will catalyze only one reaction. e.g

17. A — B or A — B e.gα-D-glucosidase 2). Relative specificity (i) Group specificity:the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups.

18. esterase fumarate hydratase (ii) Bond specificity:the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure. 3). Stereospecificity:the enzyme will act on a particular steric or optical isomer. malate

19. 4). Hypotheses of enzyme specificity (i) Lock and Key model Proposed by Fischer in 1894 In this model, the active sites of the unbound enzyme is complementary in shape to the substrate

20. (ii) Induced-fit model Proposed by Koshland in 1958 In this model, the enzyme changes shape on substrate binding

21. An Example: Induced conformational change in hexokinase • Catalyzes phosphorylation of glucose to glucose 6-phosphate during glycolysis • such a large change in a protein’s conformation is not unusual

22. Conclusion • Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate. • The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction. • Not a lock & key but induced fit – the enzyme and/or the substrate distort towards the transition state.

23. 4 Chemical composition of enzymes (1) Simple protein (2) Conjugated protein Holoenzyme= Apoenzyme+ Cofactor Coenzyme: loosely bound to enzyme (non-covalently bound). Cofactor Prosthetic group: very tightly or even covalently bound to enzyme (covalently bound)

24. Cofactors often function as transient carriers of specific (functional) groups during catalysis. • Many vitamins and organic nutrients required in small amounts in the diet, are precursors of cofactors.

26. 5 Classification of enzymes (1). By their composition 1). Monomeric enzyme 2). Oligomeric enzyme 3). Multienzyme complex: such as Fatty acid synthase

27. (2) Nomenclature • Recommended name • Enzymes are usually named according to the reaction they carry out. • To generate the name of an enzyme, the suffix -ase is added to the name of its substrate (e.g., lactase is the enzyme that cleaves lactose) or the type of reaction (e.g., DNA polymerase forms DNA polymers). • Systematic name (International classification) • By the reactions they catalyze (Six classes)

28. Lactate dehydrogenase Transfer electrons (hydride ions or H atoms); play a major role in energy metabolism. e.g., the transfer of a phosphoryl group from ATP to many different acceptors. NMP kinase Chymotrypsin i.e., the transfer of functional groups to water. These are direct bond breaking reactions without being attacked by another reactant such as H2O. Fumarase Triose phosphate isomerase Leading to the formation of C-C, C-S, C-O, C-N bonds. Aminoacyl-tRNA synthetase

29. Each enzyme is given a systematic name and a unique 4-digit identification number for identification by the Enzyme Commission (E.C.) of IUBMB (since 1964) lactate + NAD+ pyruvate + NADH + H+ Lactate dehydrogenase (lactate:NAD+ oxidoreductase) 1 Indicates type of cofactor Indicates type of substrate

30. 6 Catalytic mechanisms of enzymes • Mechanisms - the molecular details of catalyzed reactions • How do enzymes stabilize the transition state of a reaction • General Acid-base catalysis • Covalent catalysis • Catalysis by proximity and orientation • Metal catalysis

31. 1). General acid-base catalysis • The active sites of some enzymes contain amino acid functional groups that can participate in the catalytic process as proton donors or proton acceptors --general acid-base catalysis. • A general acid (BH+) can donate protons • A covalent bond may break more easily if one of its atoms is protonated

32. Amino acids in general acid-base catalysis

33. A-X + E X-E + A X-E + B B-X + E 2). Covalent catalysis • Covalent catalysis involves the substrate forming a transient covalent bond with residues in the active site of the enzyme or with a cofactor. • This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction. • Group X can be transferred from A-X to B in two steps via the covalent ES complex X-E

34. nucleophilic center (X:) • Examples of covalent bond formation between enzyme and substrate. • In each case, a nucleophilic center (X:) on an enzyme attacks an electrophilic center on a substrate.

35. Nucleophilic group (X:) Electrophilic group

36. 3). Catalysis by proximity and orientation • This increases the rate of the reaction as enzyme-substrate interactions align reactive chemical groups and hold them close together.

37. Analogous to an effective increase in concentration of the reactants.

39. Substrate 4).Many enzymes have metal ions in their active centers playing important roles in catalysis. • help activate substrates • Stabilize charged transition states by forming ionic bonds. Enzyme • An enzyme may use a combination of several catalytic strategies to bring about a rate enhancement.

40. 7. Enzyme activity • Enzymes are never expressed in terms of their concentration (as mg or μg etc.), but are expressed only as activities. • Enzyme activity = moles of substrate converted to product per unit time. • The rate of appearance of product or the rate of disappearance of substrate • Test the absorbance: spectrophotometer

41. Units of enzyme activity: • Katal (kat) – 1 kat denotes the conversion of 1 mole substrate per second (mol/sec) • International unit (IU) - amount of enzyme activity that catalyses the conversion of 1 micromol of substrate per minute (μmol/min).

42. 8. Factors affecting enzyme activity • Concentration of substrate • Concentration of enzyme • Temperature • pH • Activators • Inhibitors

43. Enzyme velocity • Enzyme activity is commonly expressed by the intial rate (V0)of the reaction being catalyzed. (why?) • Enzyme activity = moles of substrate converted to product per unit time.

44. Velocity decreases as time increases as: • S may be used up • P may inhibit reaction(E) • Change of pH may occur and decrease enzyme reaction • Cofactor or coenzyme may be used up • Enzyme may loss activity

45. (1). Substrate concentration ([S]) affects the catalytic velocity (rate) • At relatively low concentrations of substrate, Voincreases almost linearly (A) with an increase in [S]. • At higher [S], Vo increases by smaller and smaller amounts (B) in response to the increase in [S]. • Finally, a point (a plateau of maximum velocity, Vmax) (C) is reached. C B A • [S] is a key factor affecting the rate.

46. Quantitative expression of relationship between [S] and V0 • 1913, Leonor Michaelis and Maud Menten deduced the equation, Michaelis-Menten equation, based on the exist of intermediate ([ES]) in the enzyme reaction. Leonor Michaelis (1875-1949) Maud Menten (1879-1960)

47. k1 k3 E + S ES E + P k2 Proposed Model • EScomplex formed when specific substrates fit into the enzyme active site (at the beginning of reaction) • When [S] >> [E],E issaturated with S. • k1,k2 and k3 represent the velocity constants for the respective reactions.

48. [S] V = Vmax [S] + KM Michaelis-Menten equation (very important!) • Michaelis-Menten equation describes how reaction velocity (V) varies with substrate concentration [S]. • The following equation is obtained after suitable algebraic manipulation. Note: V means V0 Km:Michaelis constant Km = (k2 + k3)/k1

49. [S] V = Vmax Zero order reaction [S] + Km First order reaction • The equation fits the observed curve very well. • When [S] is very low (<<Km), then V = (Vmax/ Km)[S] or V is linearly dependent on [S]. • when [S] is very high (>>Km), then V = Vmax; that is, the V is independent of [S].

50. Significances of Km 1) When [S] = Km, Vmax [S] Vmax [S] Vmax V = = = Km+[S] [S] + [S] 2 so, whenV = 1/2 Vmax, Km = [S],the unit of Km is as [S] 2) For a specific substrate, Km is a constant for the enzyme. 3) Km can be a measure of the affinity of E for S. A low Kmvalue indicates a strong affinity between E and S.