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1 Note: Graphic 24 has been modified from the original posting: (“Most spherical” was replaced by “least spherical”). Columbia Biological Society First meeting: Tuesday Sep 25 at 9pm in 702 Hamilton. Some prosthetic groups 2 Particular small molecules so tightly bound that they are always found associated with the protein Tetrahydrofolic acid ~ vitamin B9 Pyridoxal phosphate ~ vitamin B6 Riboflavin ~ vitamin B2 Heme 3 Membrane proteins Could be size selective Could be size and charge selective Anion: an ion that would migrate to the anode in an electric field Small molecules bind with great specificity to pockets on protein surfaces 4 Too far Estrogen receptor binding estrogen, a steroid hormone detail estrogen estrogen 5 Protein separation methods Ultracentrifugation Mixture of proteins 6 7 Causing sedimentation: centrifugal force = m(omega)2r m = mass omega = angular velocity r = distance from the center of rotation Opposing sedimentation = friction = foV. Constant velocity is soon reached: centrifugal force = frictional force So: m(omega)2r = foV fo = frictional coefficient (depends on shape) And: V = m(omega)2r/fo, Or: V = [(omega)2r] x [m / fo] Note: formulas wil be provided on exams, as will formulae V proportional to mass (MW) V inversely proportional to fo (shape) V inversely proportional to non-sphericity (Spherical shape moves fastest) Ultracentrifuge 8 + 9 Glass plates + + Large, +++ high positive charge + Large, + low positive charge ++ + + + ++ + Small, +++ High positive charge + ++ + + Small, + Low positive charge + + + + + + + + + + Molecules shown after several hours of electrophoresis + 10 Glass plates + + ++ + + + + + + Winner: Small, +++ High positive charge + ++ + + + + + ++ + + + + + Loser: Large, + low positive charge Intermediate: Large, +++ high positive charge Intermediate: + Small, + Low positive charge Molecules shown after several hours of electrophoresis 11 12 Electrode connection Power supply 13 Electrodes Tracking dyes 14 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 All the polypeptides are denatured and behave as random coils All the polypeptides have the same charge per unit length All are 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) 15 16 Summary of SDS PAGE Separates on MW only, no shape no charge High resolution. Can measure the MW of a protein (subunit MW) by comparig mobiltiy to that of standards. Must first reduce any disulfides to get true subunit MW (e.g., with mercaptoethanol). Molecular sieve chromatography (=gel filtration, =Sephadex chromatography) Sephadex bead 17 Molecular sieve chromatography Sephadex bead 18 Molecular sieve chromatography Sephadex bead 19 Molecular sieve chromatography Sephadex bead 20 Molecular sieve chromatography Sephadex bead 21 22 Plain column of Sephadex Fancy column of Sephadex Handout 4-3: protein separations Handout 23 24 Winners: Largest and most spherical Lowest MW Largest and least spherical Similar to handout, but Winners & Native PAGE added Most charged and smallest 25 Enzymes = protein catalysts 26 Flow of glucose in E. coli Macromolecules Polysaccharides Lipids Nucleic Acids Proteins yn th e tic pa t hw ay monomers bi os intermediates glucose Each arrow = an ENZYME Each arrow = a specific chemical reaction Chemical reaction between 2 reactants H2 + I2 2 HI H2 + I2 2 HI + energy “Spontaneous” reaction: Energy released Goes to the right H-I is more stable than H-H or I-I here That’s why it “goes’ to the right, i.e., it will end up with more products than reactants 27 28 say, 100 kcal/mole say, 103 kcal/mole H2 + I2 2 HI { Change in Energy (Free Energy) 2H + 2I Atom pulled completely apart (thought experiment) -3 kcal/mole Reaction goes spontaneously to the right Energy change is negative: spontaneously to the right = exergonic: energy-releasing Energy change is positive: spontaneously to the left = endergonic: energy-requiring 29 H2 + I2 2 HI H2 + I2 2 HI H2 + I2 2 HI 30 But: it is not necessary to break molecule down to its atoms in order to rearrange them say, 100 kcal/mole say, 103 kcal/mole H2 + I2 2 HI { Change in Energy (Free Energy) 2H + 2I -3 kcal/mole 31 I I + H H I I + H H I I H H I I H H Transition state (TS) (H2 + I2) I H + I H (2 HI) 32 Change in Energy 2H + 2I ~100 kcal/mole H-H | | I-I (TS) Say, ~20 kcal/mole 2 HI { H2 + I2 -3 kcal/mole Activation Energy 33 Allows it to happen Energy needed to bring molecules together to form a TS complex determines speed = VELOCITY = rate of a reaction H2 + I2 2 HI { Change in Energy (new scale) HHII (TS) 3 kcal/mole Net energy change: Which way it will end up DIRECTION of the reaction, independent of the rate Activation energy Biosynthesis of a fatty acid 3 glucose 34 18-carbon fatty acid Free energy change: ~ 300 kcal per mole of glucose is REQUIRED 3 glucose 18-carbon fatty acid So getting a reaction to go in the direction you want is a problem (to be discussed next time) 35 Concerns about the cell’s chemical reactions • 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 – Catalysts deal with this second problem, which we will now consider 36 The velocity problem is solved by catalysts The catalyzed reaction The catalyst takes part in the reaction, but it itself emerges unchanged Change in Energy HHII (TS) 37 Activation energy without catalyst TS complex with catalyst H2 + I2 2 HI Activation energy WITH the catalyst 38 Reactants in an enzyme-catalyzed reaction = “substrates” 39 Reactants (substrates) Not a substrate Active site or substrate binding site (not exactly synonymous, could be part of the active site) 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 40 41 + Enzymes work as catalysts for two reasons: 1. They bind the substrates putting them in close proximity. 2. They participate in the reaction, weakening the covalent bonds of a substrate by its interaction with the enzyme’s amino acid side groups (e.g., stretching). Chemical kinetics Substrate Product SP Velocity = V = ΔP/ Δ t (reactants in enzyme catalyzed reactions are called substrates) So V also = -ΔS/ Δt (disappearance) From the laws of mass action: ΔP/ Δt = - ΔS/ Δt = k1[S] – k2[P] back reaction For the INITIAL reaction, [P] is small and can be neglected: ΔP/ Δt = - ΔS/ Δt = k1[S] So the INITIAL velocity Vo = k1[S] 42 43 44 Vo = the slope in each case Effect of different initial substrate concentrations 0.6 [S4] [S3] P 0.4 [S2] 0.2 [S1] 0.0 t 45 Considering Vo as a function of [S] (which wil be our usual useful consideration)