L#3 & L#4: Myoglobin/Hemoglobin/Immunoglobulins and Kinetics
L#3 & L#4: Myoglobin/Hemoglobin/Immunoglobulins and Kinetics 0280
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This 6 page Class Notes was uploaded by Denise Croote on Saturday January 23, 2016. The Class Notes belongs to 0280 at Brown University taught by Arthur Salomon in Spring 2016. Since its upload, it has received 32 views. For similar materials see Introductory Biochemistry in Biology at Brown University.
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Date Created: 01/23/16
Lecture Three: Myoglobin, Hemoglobin, and Immunoglobulins ● some examples of secondary structures are B haripins, Psi loops ● domains are units of compact structure, folding, and function ● most domains are continuous along the structure ● Myoglobin consists of all alpha helices and has a prosthetic group attached (a group that is stably associated with a protein and contributes to its function) ○ heme = prophyrin + iron has a iron atom bound in the Fe2+ state. Fe2+ can simultaneously react with two O2 to make Fe3+ (which is bad because Fe3+ does not bind oxygen) ○ central iron atom has six bonds, 4 to nitrogen, 1 to oxygen, and 1 to histidine ○ when Fe2+ is nestled into the protein a nitrogen from a His residue occupies one of the coordination bonds so that Fe2+ cannot turn into Fe3+ ● free heme binds CO 25,000 times more tightly than 02 while myoglobin binds CO only 200 times more tightly than O2. ● B/c O2 is in a much higher concentration in the body it binds to myoglobin more frequently and only 1% of myoglobin is coordinated with CO ● Theta θ is the fraction of occupied binding sites in a protein = ( occupied sites/ occupied + unoccupied sites) ● a plot of theta vs [L] ligand gives a hyperbolic function (can be defined in partial pressures and concentrations) ● Kd (which is the dissociation constant) is the molar concentration of the ligand at which half of the available ligand binding sites are occupied ● Because of the high concentration of oxygen in the atmosphere, myoglobin is half saturated with oxygen with only 1.2% of atmospheric oxygen ● myoglobin binds too tightly to oxygen to transport it (higher affinity for oxygen and steeper curve than hemoglobin, hemoglobin is better for transport ● Hemoglobin has 2 alpha and 2 beta subunits, can bind 4 oxygens, is found in RBC, and has a similar tertiary structure to myoglobin (even though the aa sequences differ) ● Hb is a heterotetramer(protein containing four noncovalently bound subunits, wherein the subunits are not all identical) ● Have a T state (tense) where the oxygen is not bound, the Fe is pulled back out of the plane by the proximal His ● Have a R state (relaxed) where the O2 is bound to the heme causing it to be centered and pushed forward ● Affinity for oxygen is higher in the R state. Once one O2 binds the conformation of the entire hemoglobin subunit changes to make it bind O2 more tightly in the R state ● Referred to as cooperative binding, binding of first oxygen enhances binding of the remaining three ● During the transition to the R state the iron units in the unoccupied sites are pushed into the central plane and iron has a high affinity for oxygen ● do not want a high affinity R locked state in the tissues because the hemoglobin would not release the oxygen like it is supposed to, would not want too low of a T locked affinity in the lungs because then it would not pick up enough oxygen to bring to the tissues ● myoglobin has no cooperativity b/c it only has one subunit (nH=1). The binding of the last O2 to the Hb is no longer cooperative because all of the remaining few unoccupied sites are on different molecules. ● concerted theory says that all are either in the R state or the T state, sequential theory says the subunits change independently of each other ● Venous blood is more acidic than arterial blood, the more acidic the blood the more the T state is favored and the lower the affinity for oxygen (need a higher concentration of oxygen to achieve the same proportion of binding) ● BGP is a negative allosteric effector. BPG is negatively charged and is attracted to the positively charged His. BPG can only fit with the T tetramer and promotes the T state (reduces O2 affinity) ● When climbing mountains there is less oxygen available, we hyperventilate to get more oxygen, pH of blood goes up, start making more BPG , binds to His, induces T conformation, affinity for oxygen goes down, pick up less oxygen in the lungs, but you are better at dropping it off in the tissues, body’s method maintain efficiency ● Sickle Cell Anemia puts two sticky patches (valines) of the outside of the hemoglobin causing the RBCs to clump ● Heterozygotes for sickle cell has resistance against malaria ● Immunoglobulin IgG has conformation specific antigen binding sites (CDRs) ● SDS Gel Electrophoresis separates a protein into monomers, migration in the gel is a function of molecular weight ● Western Blot coat surface with antigens, incubate with a primary antibody, incubate with a secondary antibodyenzyme complex, add a substrate, look at a color specific antigen Lecture Four: Kinetics ● use kinetics to figure out more about a reaction mechanism, use kinetics to study the effects of substrates and inhibitors on enzymes to gain greater insight into their physiological function, quantitatively measure enzymatic activity to gain information about diseases ● ΔG´0 measures the standard free energy change at a pH of 7 when all concentrations of the solutes are 1M ● ΔG´0 = –RT ln K ´eq where Keq = [P]/[S]. When ΔG´0 is negative, the products have a lower free energy than the reactants and are present in a higher concentration ● a spontaneous (exergonic) reaction tells you the reaction will go but is does not tell you how fast ● The transition state is a short lived unstable state where the the species is equally as likely to go back to the reactants or forward to the products ● The activation energy for the reverse reaction is greater than the activation energy for the forward reaction if ΔG´0 is less than zero. More reactants will reach the transition state than products so P will accumulate ● transition states have high free energy because they have high entropy (substrate has to adopt an ordered conformation to change to product, more ordered means more entropy) and high enthalpy (may have to break covalent bonds, H bonds, and electrostatic interactions in the substrate to reach the product) ● The absolute height to the transition state determines the rate of the reaction whereas the difference in ΔG´0 for the reactants and products determines the thermodynamic outcome of the reaction. ● an enzyme uses an alternative, lower energy pathway and usually operates through a different reaction mechanism than the uncatalyzed pathway. ● an enzyme increases the rate but does NOT change the equilibrium of the reaction ● enzyme active sites are complementary to the transition state conformations of the substrates. They bind to the transition state intermediate and lower the activation energy ● very stable enzyme substrate complexes are not productive because they result in less species reaching the transition state and a slowed reaction rate ● The initial velocity, V0, is the rate of the reaction at the beginning of the steady state, after the equilibrium between E, S, and ES has been reached, but before a significant amount of substrate is consumed or product has been formed. ● V0 = k2[ES] ● the V0 formula is very similar to the formula for ligand binding ● called the MichaelisMenten Equation and it is used to describe the Km, Vmax, and Kcat for each characteristic enzymatic reaction ● Define: Km ≡ (k2 + k–1)/k1 Km is an apparent dissociation constant that correlates with the dissociation constant Kd. Because Kd = k–1/k1 when k2 is really small Km ≈ Kd, a low concentration of substrate is required to achieve the half Vmax if Km is really small ● [ES] =[Et][S] / Km + [S] ● Substitute V0/k2 for [ES] ● V0/k2 = [Et][S]/ Km + [S] ● rearrange to get V0 = k2[Et][S] / Km + [S] ● Km = [S] when V0 = ½ Vmax (derivation shown in text) ● When substrate concentration is much greater than Km, V0 = Vmax ● When substrate concentration is much smaller than Km, there is a linear dependence ● V max is the maximum velocity that can be achieved with a given total enzyme concentration. ● If all enzymes are bound to substrate than the enzyme is working at its maximal rate ● It measures how fast the reaction catalyzed by the enzyme can proceed once [ES] is formed ● Why is the michaelismenten formula useful? ● Why is it beneficial to use an equation that only applies to a small part of the reaction before a significant portion of the substrate is depleted? ○ A.) because when we define these conditions we can view them as a standard and compare different enzymes, or compare the activity of an enzyme in different experimental conditions (pH, temperature, inhibitor presence) ○ B.) because in vivo the product usually does not accumulate in many reactions (because it is used as the substrate for another reaction) and the substrate of the reaction is being constantly replenished so the conditions are actually similar to the initial conditions ● use this formula to determine Km instead of the hyperbolic curve . This is a linear curve with the slope being Km/Vmax and the y intercept being 1/Vmax ● If the values for an enzyme are being held constant, than the rate of formation = the rate of breakdown ● Catalytic constant or turnover number Kcat: number of molecules of substrate converted into product per molecule of enzyme at saturating substrate concentration (Vmax conditions) ● “measure of processitivity” ● Kcat is often thousands of magnitudes larger than Km for certain enzymes (km gives you the best estimate for real world performance) ● making the affinity for E and S larger doesn’t exactly speed up a reaction because when ES becomes more stable, the activation energy rises and the rate of the reaction slows ● When [S] is much less than Km, Vo depends on both the turnover number and the fraction of active sites occupied. (Vo is proportional to both Kcat and 1/Km) ● Catalytic efficiency: Kcat/Km is the rate constant for the conversion of E+S to E+P when [S] is much less than Km ● kcat/Km = V0/([S][Et]) so the higher the catalytic efficiency the greater the initial velocity ● Kcat/Km is limited by the rate at which E and S can diffuse together, catalytic perfection is more easily reached in solution ● an enzyme inactivator reacts irreversibly with an enzyme while an inhibitor reversibly binds ● competitive inhibitors binds only to E and competes with S for the active site, this has an effect of lowering the affinity for E to S (thus raising apparent Km remember Km parallels the dissociation constant) ○ a smaller fraction of active sites are occupied by substrates ○ the Km increases ○ Vmax is not affected ● uncompetitive inhibition binds only to ES and distorts the active site to prevent conversion of ES to E+P. SInce the inhibitor only binds ES it stimulates the formation of ES which decreases Km. ○ decreases Km ○ ESI complex is ineffective so Vmax is lowered ● mixed inhibition inhibitor binds to both E and ES ○ Vmax is lowered because concentration of [ES] is lowered at every substrate concentration ○ raising or lowering Km depends on the relative concentrations of the competitive and uncompetitive inhibitors ● a noncompetitive inhibitor is when the inhibitor binds equally well to E and ES so only Vmax is effected and not Km
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