1. 1. Once you've isolated a protein, what do you do? What are the benefits of using this process?

    2. What do you do with the sequence? Why?

    3. Name the different parts of the sequence and describe what you can determine based on the similarities or differences.
    Image Upload 1
    - What can you say about the yellow regions? Blue? Pink?
    1. Sequence it using Edman degradation, automated process, 99% accuracy.

    2. Do sequence alignment - based on homology you can guess the putative function of the protein b/c proteins that perform the same function will have related sequences.

    3. Yellow regions are invariant/conserved regions - based on evolution, these are (1) closely related and (2) critical to function and structure.

    Pink - conservative substitution - one AA is replaced with AA from same R group  (same properties) - we have conserved the same basic chemical and physical properties

    Blue - non-conservative substitution - this is what makes organisms & proteins differ.
  2. 1. When would positions be considered critical to protein structure & function in seq alignment?

    2. How do you know if a position can tolerate conservative substitutions?

    3. Where is Mb found? What is its function? What structure does it have a lot of?

    4. What is in the center of the Mb and Hb? Why do we need this ion?

    5. Why do we need heme?
    1. only in conserved regions NOT conservative substitution

    2. If it has conservative substitutions

    3. Muscle, to bind/store oxygen until it is needed for ETC use. A-helices.

    4. Fe2+ b/c none of the other AA side chains were suitable for reversible O2 binding and because if O2 just travels on its own, it'll bubble out of solution.

    5. We need heme to hold Fe2+ to prevent it from forming free radicals.
  3. 1. What does Fe bind to? Why is it set up this way?

    2. Why do we need Hb and Mb?

    3. What is the purpose of dissociation curves?

    4. How is Image Upload 2 measured? What is Kd?
    1. 4 bonds to porphyrin ring system in heme, and two free bonds - one is usually occupied by His and the other is free to bind O2. To prevent Fe2+ from binding to two heme/O2 at once --> Fe3+.

    2. To keep heme deep within protein structure to keep O2 solubilized and to restrict access to the Fe2+ that can become a free radical.

    3. To show affinity of protein for ligand.

    4. Image Upload 3
  4. 1. What does the shape of Mb's curve tell us? (2)
    Image Upload 4
    2. It levels off. What does this tell us?
    3. Name the 4 subunits of Hb?
    4. What does Mb have such a high affinity?
    1. One binding site on the protein (no sigmoidicity). Based on the small p50, Mb has a tight binding affinity for O2.

    2. 100% saturation of Mb. No matter how much more O2 you add, we cannot increase binding to ligand b/c there is no more available protein.

    3. A1,2 and B1,2

    4. B/c Mb picks up O2 from Hb in muscles --> low pO2 --> requires high affinity.
  5. 1. What is the function of Hb? What is its saturation in arterial blood? In venous blood?

    2. What forces are responsible for holding Hb together? Why is this important?

    3. What do interactions among subunits of Hb do? What does this allow Hb to do?

    4. How does B2 interact w/ a1? How does B1 interact with a2?

    5. How does a1 and a2 interact?

    6. What other interaction plays a major role?
    1. To carry O2 from lungs to tissues and to carry CO2 back (but for now focus on O2). 96% and 64%

    2. Mostly electrostatic (noncovalent forces). This is important b/c it gives Hb flexibility, which is the key to the function of Hb.

    3. Causes conformational changes within Hb to adjust affinity to O2 based on surrounding conditions. Allows Hb to respond to change sin oxygen demand by tissues (lungs vs. tissue)

    4. B2 and a1 (and B1 and a2) interact COO- (B) salt bridging with Lys+ on alphas.

    5. Through Asp- Arg+ salt bridges.

    6. Hydrophobic
  6. 1. What would happen if Hb resembled Mb? What would happen if it had low affinity? How does Hb fix this conundrum?

    2. Draw dissociation curves for high-affinity state, low affinity state and transition from low to high affinity state. What letters do these correspond with?

    3. Draw a graph comparing Mb vs. Hb. How do the p50 compare? Shapes?

    4. Define sigmoidity and cooperativity (allostericity). What does the latter permit?
    1. If like Mb, it would bind to O2 tightly and would not release in tissues. With low binding affinity, would bea ble to release in tissues but would not be able to pick up in lungs.

    By switching between a low-affinity state (T) with a high affinity state (R) based on pO2.

    2. Image Upload 5

    • 3. Image Upload 6
    • -Hb's p50 is much higher signaling lower affinity for O2. Hb is sigmoidal while Mb is hyperbolic.

    4. Sigmoidity - diagnostic presence of multiple binding site, but cna't tell how many and of cooperativity/allostericity

    Allostericity - binding of one subunit positively increases binding of ligand (O2) at another subunit.

    Sigmoidity/allostericity allows Hb to be much more sensitive to [ligand]
  7. 1. Describe the T and R state in terms of : tense vs. relaxed, O2 affinity, p50. Which predominates in high oxygen conditions? low?

    2. What causes tenseness or relaxness?

    3. Where does puckering occur?
    1. T (deoxy) - tense, lower O2 affinity; R (oxy) - relaxed, higher O2 affinity.

    2. Changes in ion pair interactions between the subunits that either stabilize or destabilize.

    3. During T state.  In R state, it's planar.
  8. 1. What happens when T state switches to R state and vice versa?

    2. What causes the Bohr Effect?

    3. What does increasing acidity do to p50? Curve? Affinity for O2? Bohr protons? Ion pairs? why? What state is stabliized?

    3.5  Do O2 and H+ bind in same places?
    4. Draw picture of increasing pH effects on dissociation curve.
    1. Large scale conformational changes, new interactions are made and old ones are broken to stabilize one state over the other based on O2 levels.

    2. Effect of pH and [CO2] on Hb binding to O2.

    3. Increasing acidity causes formation of salt bridges (b/c things get protonated

    p50 increases (b/c affinity decreases), curve shifts right, affinity for O2 decreases, Bohr protons are there (protonated) forming ion pairs promoting tense state (stabilizing deoxyHb) to increase release of O2 to tissues

    High acidity signals low O2: CO2 ratio, signaling the need for the release of O2.

    T state - relaxed state.

    3.5 No.

    Image Upload 7
  9. 1. What is the effect of 2,3BPG on T vs. R state classified as?

    2. What does the presence of 2,3 BPG do?

    3. What is the purpose of 2,3 BPG? Altitudes?

    3. Describe characteristic of 2,3 BPG. What does it bind to under low pO2? High pO2?
    1. Heterotrophic allosteric modulation.

    2. Decreases affinity of Hb for O2 by stabilizing the T state.

    3. TO regulate the O2 binding affinity of Hb in relation to the pO2 in the lungs (esp important for physiological adaptation to lower pO2 at higher altitudes).

    4. 2,3 BPG  is negatively charged (polar with net negative charge) that binds to BPG binding site (positively charged residues)

    Under low pO2 conditions, 2,3 BPG binds to non-O2 binding site of Hb, stabilizing T state over R state.

    Under high pO2, binding pocket disappears, allowing Hb to transition to R state.
  10. 1. At sea level, how much of total O2 is delivered to tissues? (%)

    2. How does this change when someone goes to higher altitude?

    3. What happens after a few hours at high altitude? What does this lead to? (2)
    4. What is [BPG] at sea level? At 4500 m?

    5. What happens when there's less BPG around?

    6. Describe relationship b/t elevation, pO2, 2,3 BPG conc, affinity for O2 by Hb and delivery of O2 to tissues?
    1. 40%

    2. Decreases to 30% due to lower pO2

    3. Increase in [BPG] leading to decreased affinity of Hb for O2 and increased delivery of O2 to tissues (restored to 40%)

    4. 5 mM vs. 8 mM

    5. Binding pocket for BPG disappears

    6. Increased elevation --> decreased pO2 --> increased BPG --> decreased affinity for O2 by Hb and increased delivery of O2 to tissues.
  11. 1. What happens on the amino acid level in sickle cell anemia (SCA)?

    2. What is the difference between the heterozygous condition of this amino acid thing vs. homozygous?

    3. What does it cause to surface of Hb? What does it do to deoxy Hb?

    4. Symptoms of SCA?

    5. Treatment?
    1. Glu6Val - instead of glutamate (negatively charged AA), we have a nonpolar uncharged.

    2. Heterozygous = anti-malaria; homozygous = SCA

    3. Causes a nonpolar (hydrophobic) patch to form on surface of deoxy Hb (no H+ to form ion pairs) causing aggregation of fibers

    4. Blood clots, burning in peripheral tissues b/c of O2 blockages, shortness of breath

    5. 100% O2 to reduce deoxy Hb and administration of erythropoetin to increase RBC synthesis
  12. 1. What if there was a binding mutation in Hb so that 4 histidine residues --> 4 alanine residues in BPG binding pocket?

    Draw resulting dissociation curve in comparison to stripped Hb vs. normal Hb.

    2. Why does curve look the way it does?

    3. Can mutated Hb bind O2? can it release O2?

    4. How do we determine which AAs are important to protein function/structure?
    1. Image Upload 8

    2. It'll be in the middle between stripped and normal Hb. This is because the O2 binding site is fine (BPG site is separate from O2 binding site), so it'll have no problem binding, but will have trouble releasing.

    This is b/c Hb's affinity to O2 will increase since Hb can't bind anymore (replacing positively charged histidine w/ nonpolar alanines) to negative BPG.

    4. By mutating certain AAs
  13. 1. What do enzymes allow for?  (broadly)
    2. How do enzymes function? (1-0, 2-2) examples of 2?
    • 1. Increased rxn rates - makes favorable rxns go 10^3-10^17 faster.
    • 2. Increase frequency of collision between reactive molecules - serves as platform for reactive molecules to react in correct orientation and proximity.
    • 3. Allow rxns to occur under mild conditions
    • 4. Regulation of rxns

    2. Alone or with cofactors (transient - present only during rxn) or integrated into 3D structure (ex B vitamins, ions , etc).
  14. Name the six general types of enzymes and what they do.
    Oxidoreductases, transferases, hydrolases (break bond with H2O), lyases (add or remove groups to make or break C=C bonds), isomerases (rearrange C skeleton), and ligases (form C-C, C-N, C-O, C-S bonds at the expense of ATP hydrolysis)
  15. 1. Which k do enzymes change?
    1. NOT Keq, but they change k (rxn constant) and therefore rxn rate of the rxn.
  16. 1. How do you change Keq of rxn?

    2. How does Keq affect dG?

    3. Can an endothermic rxn be spontaneous?
    1. You don't b/c you cannot change dG. Image Upload 9. If you have Keq, you have to change dG, and tat's impossible!  Breaks law of thermodynamics.

    2. Lower Keq (less products vs. reactants) equals greater dG (more exergonic and spontaneous).
  17. 1. What factors contribute to activation energy? (4)

    2. What can be used to overcome all of these barriers?
    1. Entropy (reduces possibility that substrates will interact with each other)

    2. Solvation shell of H-bonded H2O that surrounds and stabilizes most biomolecules in an aq solution

    3. Distortion of substrates that must occur in many rxns

    4. The need for proper alignment of catalytic functional groups on enzyme.

    Binding energy (energy released during bond formation) can be used to overcome all these barriers
  18. 1. How do enzymes work? Source of energy?
    2. Where does binding energy come from? (2 main sources)
    3. How does substrate binding affect entropy?
    4. Why are some enzymes so large?
    1. By lowering activation barrier using binding energy.

    2. (1) Rearrangement of covalent bonds during enzyme-catalyzed rxn - covalent rxns on active site b/t enzyme & substrate lower activation energy by providing alternate, lower-energy path

    (2) weak non-covalent interactoins b/t substrate and enzyme - formation of each weak interaction in ES complex is accompanied by small release of energy that stabilizes interaction. This binding energy contributes to a lot of the specificity and catalysis. These weak interactions are optimized in the transition state

    3. Increases entropy due to displacement of solvent molecules from binding molecule.

    4. B/c increased weak interactions require increased surface area --> enzyme must provide platform for interactions while being precisely positioned to optimize dGb
  19. 1. When are weak interactions optimized?
    2. What factors contribute to enzymes catalytic activity? (first 2 out of 6)
    • 1. During transition state to stabilize it.
    • 2. Proximity & orientation - enzymes bind so that reactive groups are close and in correct orientation (constrain motion) increasing possibility of rxn

    Preferential transition state binding - enzymes will preferentially stabilize transition state (TS) not substrate by induced fit
  20. 1. What would energy diagram look like if enzyme preferentially stabilized substrate over TS? What would be the resulting energy barrier?

    2. In energy diagram, are the peaks or valleys transition states? Which are the intermediates?

    3. Draw what energy diagram looks like if TS is stabilized instead.
    Image Upload 10

    Peaks = transition states; valleys = intermediates.

    Image Upload 11
  21. 1. What are the last four less important factors contributing to enzyme catalysis? Name examples of each.
    1. General acid-base chemistry (side chains function as proton donors/acceptors)

    2. Covalent catalysis - transient (eventually must be broken)

    3. Electrostatic catalysis (similar to acid-base, certain R groups stabilize charged high energy int)

    4. Metal ion catalysis (seen in redox rxns and involved in substrate binding).
  22. 1. For A<--> B what is the rate of the rxn?

    2. Is it unimolecular or bimolecular?

    3. What about 2A <--> B
    4. What is rare to see in bio systems? Why? What is never seen?
    • 1. rate = k[A]
    • 2. Unimolecular
    • 3. rate = k[A]^2 (biomolecular)
    • 4. Termolecular b/c the chances of three molecules hitting at the same time is low. 4th order and higher.
  23. 1. What happens to rxn rate when you slowly add  low levels of [S]?

    2. What happens when you increase [S] hugely and quickly?

    3. What are the two assumptions of the michaelis menten equation?

    4. What is the michaelis menten equation?
    • 1. The velocity will increase at a linear rate
    • 2. It will increase up until a certain point, and then it will flatten out at Vmax, b/c enzyme will be saturated.

    3. (1) Assumption of equilibirum - early in rxn when timepoints are taken, little product has accumulated. In this case, we will assume that equilibrium has NOT been reached to ignore K2

    (2) Steady state assumption - initial rxn rate reflects steady state in which [ES] remains constant over the course of the rxn (formation of ES must equal breakdown of ES).

    4. Image Upload 12
  24. 1. What is Km? What is unique about it?
    2. What does a high Km indicate? Low Km?
    3. How much [S] do you need to reach 1/2 Kmax with low Km? High Km?
    4. What is the best way to figure out Vmax?
    1. Measure of enzyme's affinity for substrate. It's unique for every enzyme-substrate pair.

    2. High Km = low affinity. Low Km = high affinity

    3. A lot. A little.

    4. To linearize the curve into a double reciprocal Linewaver Burk plot
  25. 1. In lineweakver burk plot, what does not change for each substrate & enzyme pair?
    2. Draw a picture of asymptotic Km and Vmax
    3. Draw a picture of double reciprocal plot. Label slope (what it equals), x axis, y axis, what y-int corresonds with and what x-int corresponds with.
    1. Km and Vmax

    2. Image Upload 13
  26. 1. What happens to Kmax and Vmax after adding a competitive inhibitor? Can it be overcome? If so, how?

    2. What about after adding a noncompetitive inhibitor?

    Draw pictures after adding a competitive inhibitor.

    Draw pictures after adding noncompetitive inhibitor
    1. VMax doesn't change (y-int), Km increases (decreased affinity for S) slope decreases.

    • Image Upload 14
    • 2. Vmax decreases (lower y-int), Km doesn't change. Increased slope.

    Image Upload 15
  27. 1. What is kcat? What is it most commonly? Another name?

    2. What is the equation? Units?

    3. What is the best indicator of enzyme efficiency? If it's larger or smalller?
    1. Number of rxns an enzyme can carry out, most commonly the rate determining step in an enzyme catalyzed rxn (formation of product: ES--> E+P). Turnover number

    2. Vmax/[Etotal] hertz.

    3. Kcat (1/s) and Km (M) alone are not sufficient indicators of enzyme efficiency.

    4. Kcat/Km is actually the best indicator. The greater it is the better.
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