HL Chemistry - Option B: Human Biochemistry

1. HL Chemistry - Option B: Human Biochemistry Enzymes

2. Part 1 Overview of Enzymes

3. Enzyme Fundamentals • Enzymes are protein complexes that speed up biochemical reactions by lowering the activation energy • Enzymes accelerate reactions by facilitating the formation of the transition state • The position of the equilibrium, enthalpy of reaction, and free energy of the reaction are unchanged by an enzyme • The enzymes themselves are the same after the reaction as they was before • Enzymes are powerful and highly specific catalysts • Free energy is a useful thermodynamic function for understanding enzymes • The Michaelis-Menten model accounts for the kinetic properties of many enzymes • Enzymes can be inhibited by specific molecules • Vitamins are often precursors to coenzymes

4. Some Enzyme Terminology • Enzyme – a biomolecule that catalyzes biochemical reaction by lowering activation energy • Substrate – the substance that undergoes a chemical change by an enzyme • Absolute Specificity – the characteristic that an enzyme acts on only one substrate • Relative Specificity – the characteristic that an enzyme acts on several structurally related substrates • Stereochemical Specificity – an enzyme's ability to distinguish between stereoisomers • Cofactor – a nonprotein molecule or ion required by an enzyme for catalytic activity • Coenzyme – an organic molecule required by an enzyme for catalytic activity

5. More Enzyme Terminology • Apoenzyme – a catalytically inactive protein formed by removal of the cofactor from an active enzyme • Active Site – the location on an enzyme where a substrate is bound and catalysis occurs • Enzyme Activity – the rate at which an enzyme catalyzes a reaction • Turnover Number – the number of molecules of substrate acted upon by one molecule of enzyme per minute • Enzyme International Unit (IU) – a quantity of enzyme that catalyzes the conversion of 1 micromole of substrate per minute under specified conditions • Optimum Temperature – the temperature at which enzyme activity is highest

6. And More Enzyme Terminology • Optimum pH - the pH at which enzyme activity is highest • Extremozyme – an enzyme that thrive in extreme environments • Enzyme Inhibitor – a substance that decreases the activity of an enzyme • Competitive Inhibitor – an inhibitor that binds to the active site of an enzyme • Noncompetitive Inhibitor – an inhibitor that binds at a location other than the enzyme’s active site • Zymogen (proenzyme) – the inactive enzyme precursor • Modulator – a substance that binds to an enzyme at a location other than the active site that alters the enzyme's catalytic activity

7. And Yet More Enzyme Terminology • Allosteric Enzyme – an enzyme with a quaternary structure whose activity is changes by the binding of a modulator • Activator – a substance that binds to the allosteric enzyme and increases its activity • Feedback Inhibition – a process in which the end product of a sequence of enzyme catalyzed reaction inhibits an earlier step in the process • Enzyme Induction – the synthesis of enzyme in response to a cellular need • Isoenzyme – a slightly different form of the same enzyme produced by different tissues • Holoenzyme – apoenzyme + cofactor

8. Examples of Enzyme Cofactors • Apoenzyme + • cofactor = • holoenzyme • Cofactors often • derived from • vitamins • Many enzymes • use same • cofactor • When tightly • bound to enzyme, • cofactor = • prosthetic group

9. Cofactor Function and Co-Enzymes!

10. Enzymes Cofactors may be Metal Ions Metal ions are present in trace amounts (e.g. Mg+2, Ca+2, Zn+2)

11. Enzyme Cofactors Coenzyme: a non-protein organic (may be a vitamin)

12. Example of Enzymatic Catalysis: Hydration of CO2 • This reaction is catalyzed by carbonic anhydrase (106 molecules of CO2per sec: 107 times faster than without enzyme!) • Speeds up transfer of CO2 from tissue to blood to alveolar air No wasteful by-products! Product Substrates

13. Selected Enzyme Reaction Rates

14. Example of Enzyme Substrate Specificity: proteolysis • Enzymatic hydrolysis of a specific peptide bond in vivo Substrates Products

15. Example of Enzyme Substrate Specificity: (continued) • Example (A): Trypsin cleavage site at Lys or Arg (digestive enzyme) • Example (B): Thrombin cleavage site at Arg only • (blood clotting enzyme) • One particular enzyme, Subtilisin, will cleave any • peptide bond

16. Close Up of Thrombin Cleavage Site The specificity of an enzyme is due to the precise interaction of substrate with the enzyme.This is a result of the unique three-dimensional structure of the enzyme

17. Enzyme Classes Most named for substrates & for reactions, with suffix “ase” (e.g.: ATPase breaks down ATP, ATP synthase makes ATP) • 1964, classification & nomenclature of enzymes was developed by the International Enzyme Commission (IEC): • e.g. Nucleoside Monophosphate (NMP) Kinase = IEC 2.7.4.4 2 = class, 7 = phosphoryl group, 4 = phosphate acceptor, 4 = precise acceptor (NMP)

18. Part 2 Enzyme Kinetics

19. The Enzyme-Substrate Complex • The catalytic power of enzymes is derived from the formation of the transition states in enzyme-substrate (ES) complexes • A substrate must be brought into favorable orientation at a specific region of the enzyme called the active site • Evidence Supporting ES Complex Formation: • An enzyme-catalyzed reaction has a maximal velocity suggesting the formation of a discrete ES complex (at high S concentrations catalytic sites are filled) 2. X-ray crystallography has provided high resolution images of substrates and substrate analogs bound to the active sites of many enzymes 3. Spectroscopic characteristics of many enzymes and substrates change on formation of an ES complex

20. The Active Site of an Enzyme The active site is the region that binds the substrates (& cofactors if any) 2. It contains the residues that directly participate in the making & breaking of bonds (these residues are called catalytic groups) The interaction of the enzyme and substrate at the active site promotes the formation of the transition state 4. The active site is the region that most directly lowers the Free Energy (G‡) of the reaction - resulting in rate enhancement of the reaction

21. Common Features of Active Sites Enzymes differ widely in, structure, specificity, & mode of catalysis, yet, active site have common features: The active site is a 3-dimensional cleft formed by groups that come from different parts of the amino acid sequence 2. The active site takes up a relatively small part of the total volume of an enzyme. Why are enzymes so big? Answer: Scaffolding, regulatory sites, interaction sites for other proteins, & channels 3. Active sites are clefts or crevices – they exclude H2O 4. Substrates are bound to enzymes by multiple weak attractions such as electrostatic interactions, hydrogen bonds, Van der Waals forces, & hydrophobic interactions 5. The specificity of binding depends on the precisely defined arrangement of atoms at the active site

22. Active Sites are Composed of Distant Residues

23. The Enzyme – Substrate Complex Is Usually Stabilized by Hydrogen-Bonds EXAMPLE: Ribonuclease (cleaves RNA)

24. Lock-and-Key (ES) Model This model assumes that a unique substrate binds to the active site. Thus, there must be a 1:1 ratio between substrates and enzymes. This is in fact not true, since there are many more substrates than enzymes. Therefore this model is not currently favored by most biochemists.

25. Induced Fit (ES) Model In this model, the active site can change shape slightly to accommodate substrates with similar shapes and charges. This model is favored by most biochemists.

26. Comparison of Lock & Key vs. Induced Fit Models Diagrammatic representation of the two (ES) binding theories illustrates how the Lock & Key Theory (a) yields a 1:1 ratio of substrate to enzyme, whereas the Induced Fit Model (b) suggests the enzyme can accommodate several types of substrates.

27. Enzyme - Catalyzed Reactions: maximal velocity Under initial conditions the plot is linear and first order (or pseudo-first order). After the product concentration starts to build up, the reverse reaction becomes more important and the reaction velocity asymptotically approaches the maximal velocity (Vmax). Vmax Rate = k[enzyme]1 (when [enzyme] << [substrate])

28. Michaelis-Menten Kinetics V0 = moles of product formed per sec. when [P] is low (close to zero time); V0 varies with [S] E + S  ES  E + P Michaelis-Menten Model V0 = Vmax x [S]/([S] + Km) Michaelis – Menten Equation Km = [S] when V0 = Vmax/2 Km is the “Michaelis Constant” It is a function of the kinetic rate constants

29. Initial velocity V0 (when [P] is low) (Ignore the back reaction!)

30. Steady-State & Pre-Steady-State Conditions At equilibrium, there is no net change of [S] & [P] or [ES] & [E] At pre-steady-state, [P] is low (close to zero time), thus, use V0 for initial reaction velocity At pre-steady state, we ignore the back reactions

31. Michaelis-Menten Kinetics Enzyme kinetics based on the Michaelis-Menten Graph: At a fixed concentration of enzyme, V0 is almost linearly proportional to [S] when [S] is small, but is nearly independent of [S] when [S] is large. k1 k3 Proposed Model: E + S  ES  E + P ES complex is a necessary intermediate! k2 Start with:V0 = k3[ES], and derive, V0 = Vmax x[S]/([S] + Km) This equation accounts for graphical data At low [S]: ([S] < Km), V0 = (Vmax/Km)[S] At high [S]: ([S] > Km), V0 = Vmax When [S] = Km: V0 = Vmax/2 Thus, Km = substrate concentration at which the reaction rate (V0) is half max

32. Range of Km values Km provides approximation of [S] in vivo for many enzymes

33. Lineweaver-Burk plot (double-reciprocal) V0 = Vmax x [S]/([S] + Km) Michaelis – Menten Equation • Due to the asymptotic approach to Vmax given by Michaelis-Menten Kinetics,it is sometimes very difficult to find the various components in the aforementioned equation • Rearrangement of the Michaelis-Menten equation gives the Lineweaver-Burk relationship: • This is of the form y = mx + b, so a plot of 1/[S] vs.1/V0 produces a straight line with values as shown on the next slide 1/V = (Km /Vmaxx 1/[S]) + 1/ Vmax )

34. Lineweaver-Burk Plot (double-reciprocal) 1/V = (Km /Vmaxx 1/[S]) + 1/ Vmax )

35. Allosteric Modulation

36. Allosteric Enzyme Kinetics • Sigmoidal dependence of V0 on [S], means the enzyme kinetics are not Michaelis-Menten! • Enzymes can have multiple subunits • and multiple active sites • Substrate binding may be cooperative!

37. Enzyme Inhibition – Competitive vs. Noncompetitive

38. Kinetics of Competitive Inhibition • Increase [S] to overcome • inhibition • Vmax is then attainable, and Km is increased ← Ki = dissociation constant for inhibitor

39. Competitive Inhibitor Lineweaver-Burk Plot Vmax is unaltered, but Km is increased!

40. Kinetics of Non-Competitive Inhibition Unlike competitive inhibition, increasing [S] can not overcome inhibition in the non-competitive case

41. Non- Competitive Inhibitor Lineweaver-Burk Plot Km is unaltered, but Vmax is decreased!

42. Part 3 A Few Enzyme Applications

43. Vitamins as Enzymes Vitamins can either be water soluble or fat soluble • They play important roles in metabolism • If too many or too few vitamins are present, disease will result

44. Vitamins: Water-Soluble

45. Vitamins: Fat-Soluble

46. Structures of Some Water-Soluble Vitamins A few facts: • B series vitamins are components of coenzymes, • They must be modified before they can serve their functions • Ascorbic acid is a reducing agent (an antioxidant)

47. Structure of Some Fat-Soluble Vitamins

48. Enzyme Denaturation • Enzymes are only functional if they have the proper 3-D structure • Changes in temperature, pH, salt concentration, metal ion content, and solvent polarity can cause the enzyme to change conformation, and thus become inactive

49. Denaturation of an Enzyme with pH or Temperature

50. Enzymes – Effect of pH on Activity