1. Aulani " Biokimia" Presentation 11 Introduction: Protein Aulanni’am Laboratorium Biokimia Jurusan Kimia FMIPA Universitas Brawijaya

2. Aulani " Biokimia" Presentation 11 DNA (Genotype) Protein Introduction:

3. Aulani " Biokimia" Presentation 11 Structure… Structure…

4. Aulani " Biokimia" Presentation 11 Structure cont…

5. Aulani " Biokimia" Presentation 11 COOH NH 2 H + COO - R - C - HR - C - H NH 2 H + R - C - HR - C - H COO - NH 2 R - C - HR - C - H Acidic environmentNeutral environmentAlkaline environment +1 0 pK 1 ~ 2 pK 2 ~ 9 Isoelectric point 5.5

6. Aulani " Biokimia" Presentation 11 Environment pH vs Protein Charge + Net Charge of a Protein Buffer pH Isoelectric point, pI - 3 4 5 6 7 8 9 10 0 -

7. Aulani " Biokimia" Presentation 11 Amino acids -COOH-NH 2 -R GlyG2.349.60 AlaA2.349.69 ValV2.329.62 LeuL2.369.68 IleI2.369.68 SerS2.219.15 ThrT2.6310.4 MetM2.289.21 PheF1.839.13 TrpW2.389.39 AsnN2.028.80 GlnQ2.179.13 ProP1.9910.6 AspD2.099.823.86 GluE2.199.674.25 HisH1.829.176.0 CysC1.7110.88.33 TyrY2.209.1110.07 LysK2.188.9510.53 ArgR2.179.0412.48 pK1pK1 pK1pK1 pK2pK2 pK2pK2 pK3pK3 [OH - ] pH pI pI ? pK 1 + pK 2 2 three pKa two pKa ? ? p K a of Amino Acids

8. Aulani " Biokimia" Presentation 11 HOOC-CH 2 -C-COOH NH3+NH3+ H HOOC-CH 2 -C-COO - NH3+NH3+ H - OOC-CH 2 -C-COO - NH3+NH3+ H NH 2 H +1 0 -2 pK 1 = 2.1 pK 2 = 3.9 pK 3 = 9.8 2.1 + 3.9 2 = 3.0 first second third Isoelectric point Isoelectric point is the average of the two pKa flanking the zero net-charged form pK1pK1 pK2pK2 pK3pK3 Aspartic acid +1 0 -2 [OH]

9. Aulani " Biokimia" Presentation 11 Protein? Protein are linear heteropolymers: one or more polypeptide chains Protein are linear heteropolymers: one or more polypeptide chains Building blocks: 20(?) amino acid residues. Building blocks: 20(?) amino acid residues. Range from a few 10s-1000s Range from a few 10s-1000s Three-dimensional shapes (“fold”) adopted vary enormously. Three-dimensional shapes (“fold”) adopted vary enormously.

10. Aulani " Biokimia" Presentation 11 Primary structure of a protein It is the sequence of amino acids that makes each protein different from the next It is the sequence of amino acids that makes each protein different from the next Dipeptide = 2 amino acids Dipeptide = 2 amino acids Tripeptide = 3 amino acids Tripeptide = 3 amino acids Polypeptide = many amino acids Polypeptide = many amino acids Most proteins have many 100 amino acids Most proteins have many 100 amino acids Peptide Bonds

11. Aulani " Biokimia" Presentation 11 NH 2 COOH 1 NH2NH2 2 NH 2 C NCOOH O H 21 Amino acids are connected head to tail Dehydration -H 2 O

12. Aulani " Biokimia" Presentation 11 HighLow H+H+ lone pair electrons H H H+H+ N H H N Amino H+H+ Ampholyte Ampholyte contains both positive and negative groups on its molecule Carboxylic C O O H C O O pKapKa LowHigh pKapKa

13. Aulani " Biokimia" Presentation 11 Levels of Structure… 1 - Primary structure 2 - Secondary structure 3 - Tertiary structure 4 - Quaternary structure

14. Aulani " Biokimia" Presentation 11 Primary structure… This is simply the amino acid sequences of polypeptide chains This is simply the amino acid sequences of polypeptide chains

15. Aulani " Biokimia" Presentation 11 Secondary structure Local organization of protein backbone:  -helix,  -strand (which assemble into  -sheet), turn and interconnecting loop. Local organization of protein backbone:  -helix,  -strand (which assemble into  -sheet), turn and interconnecting loop. Alignment of polypeptides as a right-hand alpha helix Alignment of polypeptides as a right-hand alpha helix Stabilized by hydrogen bonds between carboxyl (C=O) and imido (NH) groups Stabilized by hydrogen bonds between carboxyl (C=O) and imido (NH) groups

16. Aulani " Biokimia" Presentation 11 The  -helix One of the most closely packed arrangement of residues. One of the most closely packed arrangement of residues. Turn: 3.6 residues Turn: 3.6 residues Pitch: 5.4 Å/turn Pitch: 5.4 Å/turn

17. Aulani " Biokimia" Presentation 11 The  -sheet Backbone almost fully extended, loosely packed arrangement of residues. Backbone almost fully extended, loosely packed arrangement of residues.

18. Aulani " Biokimia" Presentation 11 Tertiary structure Three dimensional folding and coiling of polypeptide into globular 3-D structure Three dimensional folding and coiling of polypeptide into globular 3-D structure Caused by additional chemical interactions among side chains Caused by additional chemical interactions among side chains Disulfide bonds Disulfide bonds

19. Aulani " Biokimia" Presentation 11 Quaternary structure… Assembly of homo or heteromeric protein chains. Assembly of homo or heteromeric protein chains. Usually the functional unit of a protein, especially for enzymes Usually the functional unit of a protein, especially for enzymes Interactive folding of several polypeptide chains together to form a “single” functional protein Interactive folding of several polypeptide chains together to form a “single” functional protein Functional proteins also might incorporate minerals or other nonprotein components Functional proteins also might incorporate minerals or other nonprotein components

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21. Enzymes Proteins that catalyze (speed up) chemical reactions without being used up or destroyed in the process. Proteins that catalyze (speed up) chemical reactions without being used up or destroyed in the process. Anabolic (putting things together) and catabolic (breaking things down) functions. Anabolic (putting things together) and catabolic (breaking things down) functions.

22. Aulani " Biokimia" Presentation 11

24. Immune function (antibodies) Antibodies are proteins that attack and inactivate bacteria and viruses that cause infection. Antibodies are proteins that attack and inactivate bacteria and viruses that cause infection.

25. Aulani " Biokimia" Presentation 11

26. Substrate If enzyme just binds substrate then there will be no further reaction Transition stateProduct Enzyme not only recognizes substrate, but also induces the formation of transition state X

27. Aulani " Biokimia" Presentation 11 The Nature of Enzyme Catalysis ● ● Enzyme provides a catalytic surface ● ● This surface stabilizes transition state ● ● Transformed transition state to product B B A Catalytic surface A

28. Aulani " Biokimia" Presentation 11 Active Site Is a Deep Buried Pocket Why energy required to reach transition state is lower in the active site? It is a magic pocket (1) Stabilizes transition (2) Expels water (3) Reactive groups (4) Coenzyme helps (2) (3) (4) (1) CoE + -

29. Aulani " Biokimia" Presentation 11 Enzyme Active Site Is Deeper than Ab Binding Instead, active site on enzyme also recognizes substrate, but actually complementally fits the transition state and stabilized it. Ag binding site on Ab binds to Ag complementally, no further reaction occurs. X

30. Aulani " Biokimia" Presentation 11 Active Site Avoids the Influence of Water Preventing the influence of water sustains the formation of stable ionic bonds - +

31. Aulani " Biokimia" Presentation 11 Essential of Enzyme Kinetics E S + P + Steady State Theory In steady state, the production and consumption of the transition state proceed at the same rate. So the concentration of transition state keeps a constant. S E E

32. Aulani " Biokimia" Presentation 11 Enzyme Kinetics Increase the substrate concentration, observe the change of enzyme activity Substrate concentrationExam Chapters Score Enzyme activity Student A Student B Student C 0 1 2 3 4

33. Aulani " Biokimia" Presentation 11 Increase Substrate Concentration 213456780 0 2 4 6 8 Substrate (mmole) Product 80 60 40 20 0 S+E↓PS+E↓P (in a fixed period of time )

34. Aulani " Biokimia" Presentation 11 An Example for Enzyme Kinetics (Invertase) V max KmKm S vovo 1/S 1vo1vo Double reciprocalDirect plot 1) 1) Use predefined amount of Enzyme → E 2) 2) Add substrate in various concentrations→ S (x 軸 ) 3) 3) Measure Product in fixed Time (P/t)→ v o (y 軸 ) 4) 4) (x, y) plot get hyperbolic curve, estimate→ V max 5) 5) When y = 1/2 V max calculate x ([S]) → K m 1 V max - 1 K m 1/2

35. Aulani " Biokimia" Presentation 11 A Real Example for Enzyme Kinetics Data no 12341234 0.25 0.50 1.0 2.0 0.42 0.72 0.80 0.92 Absorbance v (mmole/min) [S] 0.21 0.36 0.40 0.46 (1) The product was measured by spectroscopy at 600 nm for 0.05 per mmole (2) Reaction time was 10 min VelocitySubstrate ProductDouble reciprocal 1/S1/v 4 2 1 0.5 2.08 1.56 1.35 1.16 →→→→→→→→ 1.0 0.5 0 v Direct plot Double reciprocal 2.0 1.0 0 1/v -4 -2 0 2 4 1/[S] 0 1 2 [S] 1.0 -3.8

36. Aulani " Biokimia" Presentation 11 Enzyme Inhibition (Mechanism) CompetitiveNon-competitive Uncompetitive E E Different site Compete for active site Inhibitor Substrate Cartoon Guide Equation and Description I [ I ] binds to free [E] only, and competes with [S]; increasing [S] overcomes I Inhibition by [ I ]. I [ I ] binds to free [E] or [ES] complex; Increasing [S] can I not overcome [ I ] inhibition. I [ I ] binds to [ES] complex only, increasing [S] favors I the inhibition by [ I ]. E + S → ES → E + P + I ↓ I E I ← ↑ E + S → ES → E + P + + II I I ↓ II E I + S →E I S ← ↑ ↑ E + S → ES → E + P + I ↓ I E I S ← ↑ X

37. Aulani " Biokimia" Presentation 11 KmKm Enzyme Inhibition (Plots) CompetitiveNon-competitive Uncompetitive Direct Plots Double Reciprocal V max KmKm Km’Km’[S], mM vovo vovo II KmKm V max I Km’Km’ V max ’ V max unchanged K m increased V max decreased K m unchanged Both V max & K m decreased I 1/[S]1/K m 1/v o 1/ V max I Two parallel lines I Intersect at X axis 1/v o 1/ V max 1/[S]1/K m 1/[S]1/K m 1/ V max 1/v o Intersect at Y axis = Km’= Km’

38. Aulani " Biokimia" Presentation 11 How to Separate These Objects 1 2 3 9 10 11 12 6 4 8 5 7 4 5 8 wood stone cotton wood wood cotton stone wood stone cotton stone cotton cotton wood stone Shape Size Density Shape Density Size Sieving different sizes Different sedimentation Different rolling speed 4 6 7 8 5 1 3 4 6 7 8 9 10 11 12 2 5

39. Aulani " Biokimia" Presentation 11 Basic Principles of Protein Purification Ammonium sulfate fractionation Cell Organelle Homogenization Macromolecule Nucleic acid Carbohydrate (Lipid) SizeChargePolarityAffinity Small molecule Cell Debris Protein Amino acid, Sugar, Nucleotides, etc Gel filtration, SDS-PAGE, Ultrafiltration Ion exchange, Chromatofocusing, Disc-PAGE, Isoelectric focusing Reverse phase chromatography, Salting-out Affinity chromatography, Hydroxyapatite

40. Aulani " Biokimia" Presentation 11 Thank you