Chapter 8 Enzymes: Basic Concepts and Kinetics

1. Chapter 8 Enzymes: Basic Concepts and Kinetics

2. Luminescent jellyfish

3. Enzymes : Basic Concepts and Kinetics • Enzymes : the catalysis of biological systems. • Catalysis takes place at a particular site on the enzyme (= ACTIVE SITE) • Nearly all known enzymes are “proteins”

4. 8.1 Enzymes are powerful and highly specific catalysts • Enzymes accelerate reactions by factors of as much as million or more. • Most reactions in biological systems do not take place in the absence of enzymes. • One of the fastest enzymes known is carbonic anhydrase (hydrate 106 molecules of CO2 per sec.)

5. <Proteolytic enzymes> • In vivo, these enzymes catalyze proteolysis, the hydrolysis of a peptide bond. - In vitro, proteolytic enzymes also catalyze a different but related reaction(=hyrdolysis of an ester bond)

6. Trypsin : digestive enzyme. Quite specific and catalyzes the splitting of peptide bonds only on the carboxyl side of lysine and arginine residues. • Thrombin : participates in blood clotting. More specific then trypsin. Catalyzes the hydrolysis of Agr-Gly bond. Papain!

7. 8.1.1 Many enzymes require cofactors for activity • The catalytic activity of enzymes depends on the small molecules termed cofactors. • Apoenzyme : without its cofactor. • Holoenzyme : complete, catalytically active enzyme. • Cofactors can be divided into two group : metals and small organic molecules. Apoenzyme +cofactor = holoenzyme

8. 8.2 Free energy is a useful thermodynamic function for understanding enzymes • 8.2.1 The free-energy change provides information about the spontaneity but not the rate of a reaction • A reaction can occur spontaneously only if ΔG is negative. • A system is at equilibrium and no net change can take place if ΔG is zero. • A reaction cannot occur spontaneously if ΔG is positive. • ΔG is independent of the path • ΔG provide no information about the rate of a reaction (activation energy ΔGŧ)

9. 8.2.2 The standard free-energy change of a reaction is related to the equilibrium constant A + B ↔C + D ΔG = ΔG0 + RT In[C][D]/[A][B] ΔG0 is the standard free-energy change R is gas constant T is the absolute temperature At equilibrium, ΔG = 0 ΔG0 = -RT In[C][D]/[A][B] The equilibrium constant under standard conditions, Keq = [C][D]/[A][B] ΔG0 = -RT In Keq = -2.303RTlog10Keq Keq = 10- ΔG0 /2.303RT = 10- ΔG0 /1.36 When Keq = 10, ΔG0 = -1.36kcal/mol

11. This reaction takes place in glycolysis. • At equilibrium, Keq = 0.0475 • ΔG0 = -2.303RTlog10Keq • = -1.36Xlog10(0.0475) • = +1.80kcal/mol • → DHAP will not spontaneously convert to GAP ΔG?

12. 8.3 Enzymes accelerate reactions by facilitating the formation of the transition state Enzyme alter only the reaction rate and not the reaction equilibrium The amount of product is the same whether or not the enzyme is present

13. A chemical reaction of substrate S to form product P goes through a transition state Sŧ • Gibbs free energy of activation or activation energy ΔGŧ: between substrate and the transition state • Enzymes accelerate reactions by decreasing ΔGŧ, the free energy of activation. • How?

14. 1 Substrates enter active site; enzyme changes shape so its active site embraces the substrates (induced fit). 2 Substrates held in active site by weak interactions, such as hydrogen bonds and ionic bonds. 3 Active site (and R groups of its amino acids) can lower EA and speed up a reaction by • acting as a template for substrate orientation, • stressing the substrates and stabilizing the transition state, • providing a favorable microenvironment, • participating directly in the catalytic reaction. Substrates Enzyme-substrate complex 6 Active site Is available for two new substrate Mole. Enzyme 5 Products are Released. 4 Substrates are Converted into Products. Figure 8.17 Products Catalysis in the Enzyme’s Active Site • The catalytic cycle of an enzyme

18. 8.3.1 The Formation of an enzyme-substrate complex is the first step in enzymatic catalysis What is the evidence for the existence of an enzyme-substrate complex? 1. The reaction rate increases with increasing substrate concentration until a maximal velocity is reached.

19. 2. X-ray crystallography has provided high-resolution images of substrates and substrate analogs bound to the active sites of many enzymes. Time-resolved crystallography; exposure to a pulse of light converts the substrate analogue to substrate

20. 3. The spectroscopic characteristics of many enzymes and substrates change on formation of an ES complex. • Tryptophan synthetase catalyzes the synthesis of L-tryptophan from L-serine and indole-derivative. • Pyridoxal phosphate (PLP) prosthetic group

21. 8.3.2 The active sites of enzymes have some common features • The active site of an enzyme is the region that bonds the substrates. • Generalizations concerning their active site : • The active site is a three-dimensional cleft formed by groups that come from different parts of the amino acid sequence. • The active site takes up a relatively small part of the total volume of an enzyme. What are the roles of the remaining parts?

22. 3. Active sites are unique microenvironments; water is usually excluded. Nonpolar microenvironment enhances the binding of substrates as well as catalysis 4. Substrates are bound to enzymes by multiple weak attractions. (~3-12 Kcal/mol) - Non-covalent interactions : electrostatic interaction, hydrogen bonds, van der Waals forces, and hydrophobic interaction. Shape complementarity is crucial. ribonuclease

23. 5. The specificity of binding depends on the precisely defined arrangement of atoms in an active site. Lock and key model - A substrate must have a matching shape to fit into the site. Induced fit model - Enzyme changes shape on substrate binding. The active site forms a shape complementary to that of the transition stat only after the substrate is bound

24. The binding Energy between enzyme and substrate is important for catalysis The binding energy: The free energy released on binding The maximal binding energy is released when the enzyme is In the transition state. Because the full complement of complex is only formed in Transition state.

25. 8.4 The Michaelis-Menten model accounts for the kinetic properties of many enzymes (판서) Kinetics is the study of reaction rates - The extent of product formation is determined as a function of time for a series of substrate concentrations.

26. Vo : defined as the number of moles of product formed per second. • Km : equal to the substrate concentration at which the reaction rate is half its maximal value. Vmax/2

27. V0 = Vmax[S]/Km+[s] 1/V0 = Km+[s] /Vmax[S] 1/V0 = Km /Vmax[S] + [s] /Vmax[S] = Km /Vmax[S] + 1/Vmax Michaelis-Menten equation Lineweaver-Burk equation

28. 8.4.1 The significance of Km and Vmax values • The Km values of the enzymes range widely. • Km provides approximation of substrate concentration in vivo. • For most enzymes, Km lies between 10-1 and 10-7M. • High Km indicates weak binding. • Low Km indicates strong binding.

29. The maximal rate, Vmax reveals the turnover number of an enzyme. • Turnover number : the number of substrate molecules converted into product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate. Also called kcat. • Most enzymes range from 1 to 104 per sec.

30. 8.4.2 Kinetic perfection in enzymatic catalysis : The kcat/Km criterion Multienzyme complex to overcome diffusion using tunnel ! • Most enzymes are not saturated with substrate • (In physiological condition, [s] << Km) • kcat/Km can be used as a measure of catalytic efficiency. • Diffusion limits the catalytic efficiency. Why? kcat/Km < K1

31. 2-16. Multifunctional Enzymes with Tunnels Class 3. Some bifunctional enzymes shuttle unstable intermediates through a tunnel connecting the active site. ; A physical channelallows the product of one reaction to diffuse through the protein to another active site Figure2-44. The two active sites of the bifunctional enzyme tryptophan synthase are linked by an internal channel

32. 2-16. Multifunctional Enzymes with Tunnels • Carbamoyl phosphate synthetase • The single-chain protein has three separate active sites connected by two tunnels through the interior of the protein • The entire journey from first substrate to final product covers a distance of nearly 100Å Ammonia + carboxyphosphate  carbamate Carbamate + ATP  carbamoyl phosphate + ADP Figure2-45. Three consecutive reactions are catalyzed by the three active sites of the enzyme carbamoyl phosphate synthetase

33. - Chymotrypsin clearly has a preference for cleaving next to bulky, hydrophobic side chains.

34. 8.4.3 Most biochemical reactions include multiple substrates • Most reactions in biological systems usually include two substrates and two products. • Multiple substrate reactions can be divided into two classes. • 1. Sequential displacement • 2. Double displacement

35. 1. Sequential displacement - Ordered. Lactate dehydrogenase ① ② ① ② - The coenzyme always binds first and the lactase is always released first.

36. 1. Sequential displacement - Random. Creatine kinase ① ② ① ② - The order of addition of substrates and release of products is random.

37. 2. Double-Displacement(Ping-Pong) Aspartate aminotransferase • One or more products are released before all substrates bind the enzyme. • Substituted enzyme intermediate

38. 8.4.4 Allosteric enzymes do not obey Michaelis-Menten kinetics - These enzymes consist of multiple subunits and multiple active sites. • Allosteric enzymes often display sigmoidal plots. (hyperbolic plots) • In allosteric enzymes, the binding of substrate to one active site can affect the properties of other active sites in the same enzyme molecule. • Cooperative, regulatory molecules

39. 8.5 Enzymes can be inhibited by specific molecules • The activity of many enzymes can by inhibited by the binding of specific small molecules and ions. • Competitive inhibitor • Enzyme can bind substrate • or inhibitor but not both. • Inhibitor resembles substrate. • Uncompetitive inhibitorInhibitor and substrate bind to different binding site. • Noncompetitive inhibitorInhibitor and substrate bind to different binding site.

40. - Irreversible inhibitor : dissociates very slowly from its target enzyme. Tightly bound to the enzyme, either covalently or noncovalently. Important drug.(ex. Penicillin, Aspirin) - Reversible inhibitor : rapid dissociation of the enzyme –inhibitor complex. - Methotrexate : structural analog of tetrahydrofolate. Competitive inhibitor. Used to treat cancer.

41. 8.5.1 Competitive and noncompetitive inhibition are kinetically distinguishable • In competitive inhibition, the inhibitor competes with the substrate for the active site. • Can be overcome by a sufficiently high concentration of substrate. • Inhibitor increase the Km value.

42. 8.5.1 Competitive and noncompetitive inhibition are kinetically distinguishable In uncompetitive inhibition. Inhibitor bind only to the ES complex. But ESI complex can not produce product. Vmax is decreased. Km is reduced.

43. 8.5.1 Competitive and noncompetitive inhibition are kinetically distinguishable In noncompetitive inhibition. Substrate can still bind to the enzyme-inhibitor complex. But complex can not produce product. Vmax is decreased. Km is unchanged.

44. 1/Vmax -1/Km

45. 1/Vmax -1/Km

46. 1/Vmax -1/Km

47. 8.5.2 Irreversible inhibitors can be used to map the active site • Irreversible inhibitors can be divided into three categories • Group-specific reagents • Affinity labels or reactive substrate analogs • Suicide inhibitors

48. Group-specific reagents -react with specific R groups of amino acids. -DIPF modifies only 1 of the 27 serine residues in the chymotrypsin and also modifies reactive serine residue in acetylcholinesterase.

49. Group-specific reagents -react with specific R groups of amino acids. - Iodoacetamide modifies reactive cystein of enzyme.

50. 2. Reactive substrate (Affinity labels) - Structurally similar to the substrate for the enzyme that covalently modify active site residues. • TPCK is a substrate analog for chymotrypsin. • TPCK irreversible bind at the active site(histidine).