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Introduction: Protein Aulanni’am Laboratorium Biokimia Jurusan Kimia FMIPA Universitas Brawijaya Aulani " Biokimia" Presentation 11 Introduction: DNA (Genotype) Protein Aulani " Biokimia" Presentation 11 Structure… Aulani " Biokimia" Presentation 11 Structure cont… Aulani " Biokimia" Presentation 11 Acidic environment Neutral environment Alkaline environment pK2 ~ 9 NH2 H+ R-C-H COOH NH2 H+ R-C-H COOpK1 ~ 2 +1 NH2 R-C-H COO- 5.5 0 Isoelectric point Aulani " Biokimia" Presentation 11 -1 Buffer pH Environment pH vs Protein Charge 10 9 8 7 Isoelectric point, pI + 6 5 4 3 0 - Net Charge of a Protein Aulani " Biokimia" Presentation 11 - pKa of Amino Acids Amino acids -COOH -NH2 Gly Ala Val Leu Ile Ser Thr Met Phe Trp Asn Gln Pro Asp Glu His Cys Tyr Lys Arg 2.34 2.34 2.32 2.36 2.36 2.21 2.63 2.28 1.83 2.38 2.02 2.17 1.99 2.09 2.19 1.82 1.71 2.20 2.18 2.17 G A V L I S T M F W N Q P D E H C Y K R -R pH two pKa 9.60 pK2 9.69 9.62 9.68 9.68 9.15 pK1 10.4 9.21 9.13 9.39 three pKa 8.80 pK3 9.13 10.6 9.82 3.86 pK2 9.67 4.25 9.17 6.0 ? 10.8 8.33 pK1 9.11 10.07 8.95 10.53 9.04 12.48 Aulani " Biokimia" Presentation 11 pI pK1 + pK2 2 ? pI ? [OH-] H first HOOC-CH2-C-COOH Aspartic acid +1 NH3+ pK1 = 2.1 H second HOOC-CH2-C-COO- Isoelectric point is the average of the two pKa flanking the zero net-charged form 2.1 + 3.9 = 3.0 2 Isoelectric point 0 NH3+ pK2 = 3.9 H -OOC-CH -C-COO2 -1 NH3+ third pK3 = 9.8 H -OOC-CH -C-COO2 NH2 -2 -2 pK3 -1 pK2 pK1 Aulani " Biokimia" Presentation 11 0 +1 [OH] Protein? Protein are linear heteropolymers: one or more polypeptide chains Building blocks: 20(?) amino acid residues. Range from a few 10s-1000s Three-dimensional shapes (“fold”) adopted vary enormously. Aulani " Biokimia" Presentation 11 Primary structure of a protein It is the sequence of amino acids that makes each protein different from the next Dipeptide = 2 amino acids Tripeptide = 3 amino acids Peptide Bonds Polypeptide = many amino acids Most proteins have many 100 amino acids Aulani " Biokimia" Presentation 11 Amino acids are connected head to tail NH2 1 COOH NH2 2 COOH Dehydration -H2O O NH2 1 C N 2 H Aulani " Biokimia" Presentation 11 COOH lone pair electrons Amino High pKa Low N H H+ H+ N H H H Low pKa High Carboxylic C O H O O C O H+ Ampholyte contains both positive and negative groups on its molecule Aulani " Biokimia" Presentation 11 Levels of Structure… 1 - Primary structure 2 - Secondary structure 3 - Tertiary structure 4 - Quaternary structure Aulani " Biokimia" Presentation 11 Primary structure… This is simply the amino acid sequences of polypeptide chains Aulani " Biokimia" Presentation 11 Secondary structure Local organization of protein backbone: -helix, -strand (which assemble into -sheet), turn and interconnecting loop. Alignment of polypeptides as a right-hand alpha helix Stabilized by hydrogen bonds between carboxyl (C=O) and imido (NH) groups Aulani " Biokimia" Presentation 11 The -helix One of the most closely packed arrangement of residues. Turn: 3.6 residues Pitch: 5.4 Å/turn Aulani " Biokimia" Presentation 11 The -sheet Backbone almost fully extended, loosely packed arrangement of residues. Aulani " Biokimia" Presentation 11 Tertiary structure Three dimensional folding and coiling of polypeptide into globular 3-D structure Caused by additional chemical interactions among side chains Disulfide bonds Aulani " Biokimia" Presentation 11 Quaternary structure… Assembly of homo or heteromeric protein chains. Usually the functional unit of a protein, especially for enzymes Interactive folding of several polypeptide chains together to form a “single” functional protein Functional proteins also might incorporate minerals or other nonprotein components Aulani " Biokimia" Presentation 11 Aulani " Biokimia" Presentation 11 Enzymes 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. Aulani " Biokimia" Presentation 11 Aulani " Biokimia" Presentation 11 Aulani " Biokimia" Presentation 11 Immune function (antibodies) Antibodies are proteins that attack and inactivate bacteria and viruses that cause infection. Aulani " Biokimia" Presentation 11 Aulani " Biokimia" Presentation 11 Substrate Transition state X Product If enzyme just binds substrate then there will be no further reaction Enzyme not only recognizes substrate, but also induces the formation of transition state 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 A A B Catalytic surface 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 + CoE (1) (4) (3) (1) Stabilizes transition (2) (2) Expels water (3) Reactive groups (4) Coenzyme helps Aulani " Biokimia" Presentation 11 Enzyme Active Site Is Deeper than Ab Binding Ag binding site on Ab binds to Ag complementally, no further reaction occurs. Instead, active site on enzyme also recognizes substrate, but actually complementally fits the transition state and stabilized it. X Aulani " Biokimia" Presentation 11 Active Site Avoids the Influence of Water + - Preventing the influence of water sustains the formation of stable ionic bonds Aulani " Biokimia" Presentation 11 Essential of Enzyme Kinetics Steady State Theory E + S E S E +P 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. Aulani " Biokimia" Presentation 11 Enzyme Kinetics Score 0 Student B Student C 1 2 3 4 Exam Chapters Enzyme activity Student A 0 1 2 3 4 Substrate concentration Increase the substrate concentration, observe the change of enzyme activity Aulani " Biokimia" Presentation 11 1 2 3 4 5 6 7 8 S + E ↓ P 80 60 40 20 0 0 2 4 6 Substrate (mmole) Aulani " Biokimia" Presentation 11 8 (in a fixed period of time) Product Increase Substrate Concentration 0 An Example for Enzyme Kinetics (Invertase) 1) Use predefined amount of Enzyme →E 2) Add substrate in various concentrations → S (x 軸) 3) Measure Product in fixed Time (P/t)→ vo (y 軸) 4) (x, y) plot get hyperbolic curve, estimate → Vmax 5) When y = 1/2 Vmax calculate x ([S]) → Km Vmax 1 vo vo 1/2 -1 Km 1 Vmax 1/S" Biokimia" Presentation 11Km Double reciprocal Aulani Direct plot S A Real Example for Enzyme Kinetics Data Substrate Product Velocity Double reciprocal [S] Absorbance v (mmole/min) 0.25 0.21 → 0.42 0.50 0.36 → 0.72 1.0 0.40 → 0.80 2.0 0.46 → 0.92 no 1 2 3 4 1/S 4 2 1 0.5 1/v 2.08 1.56 1.35 1.16 Double reciprocal Direct plot (1) The product was measured by spectroscopy at 600 nm for 0.05 per mmole (2) Reaction time was 10 min 1.0 v 0.5 0 0 1 2 2.0 1.0 1/v 1.0 -3.8 0 -4 Aulani[S] " Biokimia" Presentation 11 -2 0 2 1/[S] 4 Enzyme Inhibition (Mechanism) Equation and Description Cartoon Guide I Competitive I Non-competitive Substrate E S S E I Compete for Inhibitor active site S I I Uncompetitive S E I I Different site E + S← → ES → E + P + I ↓↑ EI E + S← → ES → E + P + + I I ↓↑ ↓↑ EI + S →EIS [I] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [I]. [I] binds to free [E] or [ES] complex; Increasing [S] can not overcome [I] inhibition. Aulani " Biokimia" Presentation 11 S I E + S← → ES → E + P + I ↓↑ EIS [I] binds to [ES] complex only, increasing [S] favors the inhibition by [I]. Enzyme Inhibition (Plots) I Competitive I Non-competitive Direct Plots Vmax vo vo I Double Reciprocal Km Km’ I [S], mM Km = Km’ I Uncompetitive Vmax Vmax Vmax’ Vmax’ [S], mM I Km’ Km [S], mM Vmax unchanged Km increased Vmax decreased Km unchanged Both Vmax & Km decreased 1/vo 1/vo 1/vo Intersect at Y axis 1/Km I I I Two parallel lines 1/ Vmax 1/[S] Intersect at X axis 1/Km 1/ Vmax 1/[S] Aulani " Biokimia" Presentation 11 1/ Vmax 1/Km 1/[S] 1 How to Separate These Objects Shape 2 3 4 5 6 7 8 9 10 11 12 Size Density wood stone cotton wood wood cotton stone wood stone cotton stone cotton 1 2 3 Shape 6 Size 4 5 6 4 7 8 Density 5 cotton 8 7 9 10 11 4 8 wood stone 5 12 Different sedimentation Sieving different sizes Aulani " Biokimia" Presentation 11 Different rolling speed Basic Principles of Protein Purification Organelle Cell Homogenization Macromolecule Small molecule Amino acid, Sugar, Nucleotides, etc Nucleic acid Protein Ammonium sulfate Size Gel filtration, SDS-PAGE, Ultrafiltration Charge Carbohydrate (Lipid) Cell Debris fractionation Polarity Affinity Ion exchange, Reverse phase Affinity Chromatofocusing, chromatography, chromatography, Disc-PAGE, Salting-out Hydroxyapatite Isoelectric focusing Aulani " Biokimia" Presentation 11 Thank you Aulani " Biokimia" Presentation 11