Protein Structure & Enzyme Kinetics

1. Last updated: 9/22/11 12:44 PM Lec. 6 Intro Bio 2011 Protein structure cont.  Prosthetics groups Membrane proteins Protein purification methodsUltracentrifugation Native gel electrophoresis SDS gel electrophoresis Gel filtration Chemical kinetics Enzyme kinetics Michaelis-Menton equation turnover number, Km

2. Hemoglobin α2β2 tetramer Riboflavin~ vitamin B2 Heme Most prosthetic groups are bound tightly via ONLY weak bonds. Tetrahydrofolic acid ~ vitamin B9 heme

3. Metal ions can be prosthetic groups A “zinc finger” protein domain with two histidines and two cysteines shown Zinc (Zn++) (Voet and Voet, Biochemistry)

4. Membrane proteins Hydrophobic side chains on the protein exterior for the portion in contact with the interior of the phospholipid bilayer. Anions are negatively charged. Cations are positively charged

5. Small molecules bind with great specificity to pockets on protein surfaces Too far

6. A ligand nestled in a tight-binding pocket

7. Estrogen receptor binding estrogen, a steroid hormone detail estrogen estrogen Estrogen receptor is specific, does not bind testosterone

8. Protein binding can be very specific Testosterone Estrogen Estrogen (green) binding to amino acid side chains in the estrogen receptor

9. Protein separation methods Ultracentrifugation Mixture of proteins

10. Ultracentrifuge

11. Causing sedimentation: centrifugal force = mω2r m = mass ω = angular velocity r = distance from the center of rotation Opposing sedimentation = friction = foV. fo = frictional coefficient (shape) V = velocity Constant velocity is soon reached; then, no net force So now: centrifugal force = frictional force (balanced each other out)   V = mω2r/fo, And so: mω2r =  foV Or:  V = [ω2r] x [m / fo]

12. 12 Sample loaded here Glass plates Winner: Small, +++ High positive charge + + + Loser: Large, + low positive charge +++ +++ +++ poly- acrylamide fibers + Intermediate: Large, +++ high positive charge + + Intermediate: Small, + Low positive charge +++ +++ +++ Molecules shown after several hours of electrophoresis

13. Upper resevoir Upper resevoir Cut out for contactof buffer with gel Cut out for contactof buffer with gel

14. Cut out of glass platefor contact of buffer with gel Clamped glass sandwich Electrodeconnection(~ 150 V) Reservoir for buffer Platinum wire electrode

15. Power supply Happy post-doc Tracking dyes

17. SDS PAGE = SDS polyacrylamide gel electrophoresis sodium dodecyl sulfate, SDS (or SLS): CH3-(CH2)11- SO4-- CH3-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-SO4-- SDS Polypeptides denatured, act as random coils All have same charge per unit length All subject to the same electromotive force in the electric field Separation based on the sieving effect of the polyacrylamide gel Separation is by molecular weight only SDS does not break covalent bonds (i.e., disulfides) (but treat with mercaptoethanol for that, boil for a bit for good measure)

18. Reduction of protein disulfide bonds : Protein-CH2-S-S-CH2-Protein  + 2 HO-CH2CH2-SH) (mercaptoethanol) [ a reducing agent ] Protein-CH2-SH+ HO-CH2CH2-S-S-CH2CH2-OH +HS-CH2-Protein Protein gets reduced; mercaptoethanol gets oxidized

19. e.g., “p53” Molecular weight markers (proteins of known molecular weight) P.A.G.E. 12 18 48 80 110 130 160 140 Kd

20. Molecular sieve chromatography (= gel filtration, Sephadex chromatography) Sephadex bead Mild conditions (pH, temperature), proteins in their native state

21. Molecular sieve chromatography Sephadex bead

22. Molecular sieve chromatography Sephadex bead

23. Molecular sieve chromatography Sephadex bead

24. Molecular sieve chromatography Sephadex bead

25. Plain Fancy 4oC (cold room)

26. Larger molecules get to the bottom faster, and …. Non-spherical molecules get to the bottom faster ~infrequent orientation Non-spherical molecules get to the bottom faster

27. Largest and most spherical Lowest MW Winners: Largest and least spherical Similar to handout 4-3, but Winners &native PAGE added Most chargedand smallest Winners:

28. Enzymes = protein catalysts

29. Each arrow = an ENZYME Each arrow = an ENZYME

30. H2 + I2 H2 + I2 2 HI 2 HI + energy Chemical reaction between 2 reactants “Spontaneous” reaction: Energy released Goes to the right H-I is more stable than H-H or I-I here i.e., the H-I bond is stronger, takes more energy to break it That’s why it “goes” to the right, i.e., it will end up with more products than reactants i.e., less tendency to go to the left, since the products are more stable

31. Atom pulled completely apart (a “thought” experiment) 2H + 2I say, 100 kcal/mole say, 103 kcal/mole Change in Energy (Free Energy) H2 + I2 { -3 kcal/mole 2 HI Reaction goes spontaneously to the right If energy change is negative: spontaneously to the right = exergonic: energy-releasing If energy change is positive: spontaneously to the left = endergonic: energy-requiring

32. H2 + I2 2 HI H2 + I2 2 HI H2 + I2 2 HI Different ways of writing chemical reactions H2 + I2 2 HI H2 + I2 2 HI

33. But: it is not necessary to break molecule down to its atoms in order to rearrange them 2H + 2I say, 100 kcal/mole say, 103 kcal/mole Change in Energy (Free Energy) H2 + I2 { -3 kcal/mole 2 HI

34. + I I I I I I I I I I (H2 + I2) H H H H H H H H H H Transition state (TS) + (2 HI) Reactions proceed through a transition state + Products

35. 2H + 2I ~100 kcal/mole Change in Energy H-H | | I-I (TS) Say, ~20 kcal/mole H2 + I2 Activation energy { -3 kcal/mole 2 HI

36. HHII (TS) Allows it to happen Energy needed to bring molecules together to form a TS complex Change in Energy (new scale) Activation energy determines speed = VELOCITY = rate of a reaction H2 + I2 { 3 kcal/mole 2 HI Net energy change: Which way it will end up. the DIRECTION of the reaction, independent of the rate 2 separate concepts

37. Direction We need it to go in the direction we want Speed We need it to go fast enough to have the cell double in one generation time Concerns about the cell’s chemical reactions

38. Example Biosynthesis of a fatty acid 3 glucose’s 18-carbon fatty acid Free energy change: ~ 300 kcal per mole of glucose used is REQUIRED So: 3 glucose 18-carbon fatty acid So getting a reaction to go in the direction you want is a major problem (to be discussed next time)

39. Direction We need it to go in the direction we want Speed We need it to go fast enough to have the cell double in one generation time Catalysts deal with this second problem, which we will now consider Concerns about the cell’s chemical reactions

40. Got this far

41. The velocity problem is solved by catalysts The catalyzed reaction The catalyst takes part in the reaction, but it itself emerges unchanged

42. HHII (TS) Activation energy without catalyst TS complex with catalyst Change in Energy Activation energy WITH the catalyst H2 + I2 2 HI

43. Reactants in an enzyme-catalyzed reaction = “substrates”

44. Reactants (substrates) Active site or substrate binding site (not exactly synonymous, could be just part of the active site) Not a substrate

45. Substrate Binding Unlike inorganic catalysts, enzymes are specific

46. Small molecules bind with great specificity to pockets on ENZYME surfaces Too far

47. Unlike inorganic catalysts, enzymes are specific                                       succinic dehydrogenase HOOC-HC=CH-COOH <-------------------------------> HOOC-CH2-CH2-COOH +2H fumaric acid                                                     succinic acid NOT a substrate for the enzyme: 1-hydroxy-butenoate:    HO-CH=CH-COOH (simple OH instead of one of the carboxyls) Maleic acid Platinum will work with all of these, indiscriminantly

48. + • Enzymes work as catalysts for two reasons: • They bind the substrates putting them in close proximity. • They participate in the reaction, weakening the covalent bonds • of a substrate by its interaction with their amino acid residue side groups (e.g., by stretching bonds).

49. Dihydrofolate reductase, the movie: FH2 + NADPH2  FH4 + NADP or: DHF + NADPH + H+  THF + NADP+ Enzyme-substrate interaction is often dynamic. The enzyme protein changes its 3-D structure upon binding the substrate. http://chem-faculty.ucsd.edu/kraut/dhfr.mpg

50. Substrate  Product (reactants in enzyme catalyzed reactions are called substrates) S  P Velocity = V = ΔP/ Δ t So V also = -ΔS/ Δt (disappearance) From the laws of mass action: ΔP/ Δt = - ΔS/ Δt = k1[S] – k2[P] For the INITIAL reaction, [P] is small and can be neglected: Initial ΔP/ Δt = - ΔS/ Δt = k1[S] So the INITIAL velocity Vo = k1[S] back reaction Chemical kinetics O signifies INITIAL velocity