VO Biophysikalische Chemie, Einführung, Prof. Schwarzenbacher, Ausgearbeiteter Fragenkatalog
Aus BioSalzburg
Biophysical Chemistry Exam Questions
1. Describe main features, terms and the equation of a buffer system on the example of the acetate buffer!
The acetate buffer is a buffer system consisting of acetic acid and acetate in same amounts (1:1). The pH of such a buffer is 4.75, which is the pKa of acetic acid. So in general we can say that this buffer consists of a weak acid and its conjugate base.
Mechanism:
Acetate buffer: HAc + H2O <-> Ac- + H3O+
Addition of H3O+: Ac- + H3O+ <-> HAc + H2O
Addition of OH-: HAc + OH- <-> Ac- + H2O
additon of acids is neutralized by Ac-, addition of bases is neutralized by HAc!
2. Define the pKa, equation and characteristics of a weak acid or base!
pKa = -log Ka; Ka = [H+][A-]/[HA]
The pKa is a measure for the strength of an acid (or base). The smaller the pKa, the stronger the acid is. Weak acids or bases in solution do not fully dissociate. Furthermore, acids are proton donors, bases are proton acceptors.
3. Draw the acid-base titration curves of 1L solutions of 1M acetic acid, H2PO4-, and NH4+ by a strong base. Indicate the buffer pH-range and buffer-concentration range for each acid.
(of interest are only the above curves) the buffer pH-ranges are indicated in colour starting point, midpoint (pH=pKa), end point
4. Describe the buffer system in blood, its physiological pH and the term acidosis!
The physiological pH of blood is 7,34 – 7,45 (at pH outside 6,8 and 7,8 you are dead). Acidosis means the decrease of pH in blood. This can happen, for instance, when the amounts of H+ and CO2 exceed the capacity of haemoglobin and thus lead to acidification.
In general, however, the blood buffer system is an interplay between four separate buffer systems:
- carbonate buffer system (carbonate/carbonic acid) ~75% of whole-buffer capacity
- hemoglobin buffer system (Hb picks up CO2 and H+) ~25% of whole-buffer capacity
- phosphate buffer system only about 1% of whole-buffer capacity
- protein buffer system (plasma proteins are buffers because they are amphoteric) only about 1% of whole-buffer capacity
5. What is the pH of pure water in contact with air? Name the main acid base species present and calculate their concentrations!
6. How does a pH indicator work?
pH indicators are frequently weak acids or bases that contain a chromophor which absorbs in the VIS area. When introduced into a solution, they bind to H+ or OH- ions. The different electron configuration of the bound indicator shifts the absorption maximum and thus causes the indicator’s colour to change.
7. Why is the solvent water so special? Describe biophysical properties and their reasons!
In biology, water has a special role, because almost everything takes place in aqueous solutions. The water molecule has an angle of 104,5° (because of repulsive effects between hydrogen and oxygen) and has partial charges: oxygen is partially negatively charged, the hydrogens are partially positively charged. So, water is a very polar molecule (dipole). Also, because of the hydrogen bonds between single water molecules, it has a very high boiling point in regards of the molecular mass. Hydrogen bonds are also the reason why some molecules are of great water-solubility, whereas others are not. Most important however is the fact the (pure) water has a pH of 7, which is the basic requirement in biological systems. It can act as an acid or a base!
8. What is the ratio of [A-]/[HA] for the carbonate system at the blood pH of 7,4? How can the carbonate system perform its task at physiological conditions?
At physiologic pH we find a ratio of carbonate to carbonic acid (=carbonate system) as follows:
pH = pKa(carbonic acid) + log [A-]/[HA]
7,4 = 6,1 + log [A-]/[HA]
1,3 = log [A-]/[HA]; therefore: [A-]/[HA] = 20
This ratio is very high and in general, this system would not be considered ideal for maintaining a pH of 7,4. However, physiologic conditions make this buffer ideal because:
- the infinite CO2 reservoir gives huge buffering capacity
- excess acid is produced by the body as a byproduct of exercise (lactic acid) making the higher concentration of the conjugate base (carbonate) an advantage
- the body has the ability to obtain more carbonic acid by reabsorbing carbon dioxide from the lungs
- in addition, the phosphate buffer as well as the buffering ability of proteins in plasma are also available to maintain blood pH
9. What are the physiological roles and differences of Myoglobin and Hemoglobin? Use the Hill-equation to describe binding characteristics!
Hb consists of 4 polypeptide chains and 4 hemes and has a total of 4 O2 binding sites. It is found in red blood cells and its function is to deliver O2 from lungs to tissues, but also to carry CO2 from tissues to lungs. Its O2 affinity increases with each O2 molecule bound. In general, the O2 affinity is strong enough to bind O2 in the lungs and weak enough to give it away to Mg. So, Hb main function is the transport of O2 via blood. Mb instead, is a single polypeptide with one heme and thus one O2 binding site. It is found in tissues and its function is to store and transport O2 from blood to tissues. Its O2 affinity is very high; that to guarantee the O2 intake from Hb (at low p(O2)). So, Mb main function is to facilitate diffusion of O2 from blood to tissues and to store it.
- Y(O2) = O2 saturation
- p(O2) = partial pressure of O2
- p(50) = pressure at which saturation reaches half-maximum
- n = number of ligand binding places
10. Describe the effect of Bisphosphoglycerate on the oxygenbinding of Hemoglobin!
Bisphosphoglycerate binds in the subunit interface of Hb and causes a change in structure. As a consequence, the O2 affinity of Hemoglobin is decreased.
11. Describe the effect of CO2 on the oxygen binding capacity of Hemoglobin!
Hb, in addition to O2, also binds CO2 – namely to transport it from tissue to lung, where it can be exhaled. So, when CO2 is bound by amino groups of Hb, the Hb binding capacity for O2 decreases. This can be a problem when the amount of CO2 in the air is too high.
12. Describe oxygen binding characteristics of Myoglobin and Hemoglobin and sketch fractional saturation Y(O2)/1-Y(O2) vs p(O2)!
One can see that at low partial O2 pressure, the O2 affinity of Mb exceeds the O2 affinity of Hb. This is very important for the handover of O2 from Hb to Mb in tissues. At high partial O2 pressure, the O2 affinity of Hb increases as is the case in the lungs: O2 has to displace CO2 and H+. In tissues, it’s just the opposite: There, CO2 has to displace O2!
13. Describe cooperativity of oxygen binding to Hemoglobin and the characteristics of Y(O2) vs p(O2)!
cooperativity of Hb is strongly dependent on pH: a decrease of 0,2 pH results in a loss of affinity of about 20%!
14. How can Hemoglobin bind O2 in the lung and release it in tissues?
Well, the O2 affinity of Hemoglobin is as strong as necessary to bind O2 in the lung, but as weak as necessary to give O2 away to Myoglobin in tissues. In other words, the O2 affinity of Myoglobin tops the O2 affinity of Hemoglobin at low p(O2).
15. Describe the effect of high altitude exposure on the O2 binding capacity of haemoglobin in healthy individuals!
The O2 affinity of Hemoglobin depends on the partial pressure of O2 (pO2). In higher altitudes pO2 is small, leading to the effect that Hemoglobin doesn’t give bound O2 away to tissues well enough. To guarantee the tissue’s supply with O2, the level of BPG is increased. As we know, BPG shortens the O2 affinity so that bound O2 can be given away to tissues again. This is “altitude-adjustment”.
16. Describe the O2 binding site in Hemoglobin and the differences between the oxygen bound and unbound state!
When oxygen binds to the iron center, it causes contraction of the iron atom, and causes it to move back into the center of the porphyrin ring plane (plane = Ebene). At the same time, the porphyrin ring plane itself is pushed away from the oxygen and toward the imidizole side chain of the histidine residue interacting at the other pole of the iron. The interaction here forces the ring plane sideways toward the outside of the tetramer, and also induces a strain (strain = Belastung) on the protein helix containing the histidine as it moves nearer to the iron. This causes a tug (tug = Zug) on the peptide strand which tends to open up heme units in the rest of the molecule, so that there is more room for oxygen molecules to bind at other heme sites. In the tetrameric form of normal adult hemoglobin, the binding of oxygen is thus a cooperative process.
17. What is the molecular reason for Sickle Cell anaemia?
The molecular reason for Sickle Cell anaemia is a mutation in the beta-chain gene, leading to the substitution Glu6Val in HbS (at position 6, the amino acid glutamate is substituted by valine). Due to this, a hydrophobic patch at the surface is formed, that causes polymerization of HbS molecules into fibers.
18. Describe the equation for Internal Energy and its units!
- U = Q – W
- U…….internal energy
- Q…….heat added to the system
- W……work done by the system
- the change in internal energy thus depends on the addition of heat minus the work done
19. Describe the equation for Enthalpy and its units!
- H = U + pV
- H……enthalpy
- U……internal energy
- pV…..pressure-volume work
- the change in enthalpy depends on internal energy as well as on pressure-volume work
20. Describe the equation for Gibbs Free Energy and its units!
- G = U + pV – TS
- G……Gibbs free energy
- U……internal energy
- pV…..pressure-volume work
- TS…..absolute temperature*entropy
21.What are the main thermodynamic potentials and their equations and units?
22. Describe the second law of thermodynamics and its definition and main use!
“You can’t break even!”==== The total entropy of any isolated thermodynamic system tends to increase over time, approaching a maximum value.
23. Describe the first law of thermodynamics and its definition and main use!
“You can’t win!” The increase in the energy of a closed system is equal to the amount of energy added to the system by heating, minus the amount lost in the form of work done by the system on its surroundings.
24. Consider the variation of reaction spontaneity (Sign of dG) with the signs of dH and dS.
Will the reaction be spontaneous at all temperatures and will Keq>1 if
- a)dH = negative dS = negative dG = dH - TdS
The reaction is enthalpically favored but entropically opposed. It is spontaneous only at temperatures below T = dH/dS Keq>1 only under certain circumstances
- b)dH = positive dS = negative
The reaction is both enthalpically and entropically opposed. It is unspontaneous (endergonic) at all temperatures. Keq<1
25. Consider the variation of reaction spontaneity (Sign of dG) with the signs of dH and dS.
Will the reaction be spontaneous at all temperatures and will Keq>1 if
- a)dH = positive dS = positive
The reaction is enthalpically opposed but entropically favored. It is spontaneous only at temperatures above T = dH/dS Keq>1 only under certain circumstances
- b)dH = negative dS = positive
The reaction is both enthalpically and entropically favored. It is spontaneous (exergonic) at all temperatures. Keq>1
26. What are the energy yields for the hydrolysis reactions of ATP, ADP and PPi?
- ATP + H2O -> ADP + Pi + H+ -30,5 kJ/mol
- ADP + H2O -> AMP + Pi + H+ -31,1 kJ/mol (according to http://www.sp.uconn.edu/~bi107vc/sp03/terry/metabolism.html -> -7.2kcal/mol = -30,14kJ/mol)
- PPi + H2O -> 2 Pi + H+ -33 kJ/mol
27. What kind of reactions are catalyzed by ATPases and ATP synthases? Where in the cell are these enzymes located?
ATP synthase (=FoF1 ATPase) catalyzes the reaction for building ATP out of ADP + Pi. The energy comes from a proton gradient (proton motive force). However, depending on supplies, it can also function the other way ‘round, using the hydrolysis of ATP to “pump” protons. It is located in the inner mitochondrion membrane (eukaryotes) and in the thylakoid membrane of chloroplasts (plants). Also it is referred to as the smallest motor existing. ATPase is an integral membrane protein (enzyme) that uses the energy coming from the decomposition of ATP into ADP and Pi to drive chemical reactions that would not otherwise occur. In most cases, such chemical reactions are the transport of ions against their gradients, which is very essential for cell metabolism – so in the case of the sodium-potassium exchanger (also referred to Na+/K+ ATPase). It is located in the cell membrane to guarantee contact with the “outer world”.
28. How is ATP generated in mitochondria?
Via the enzyme ATP synthase and using an existing proton gradient, ATP is generated out of ADP + Pi in the inner mitochondrion membrane of eukaryotes.
29. Name a high energy and a low energy phosphate compound in metabolism! Which one can be used to drive the synthesis of ATP from ADP and Pi?
High energy phosphate compounds: -Phosphoenolpyruvate -1,3-Bisphosphoglycerate -Phosphocreatine all of these can be used to drive ATP synthesis Low energy phosphate compounds: -Glucose-6-phosphate -Glycerol-3-phosphate
30. Which experimental method can be used to obtain dH and dS? Briefly describe its principle!
31. What is an enzyme and how does it accelerate chemical reactions?
An enzyme is a protein with a well-defined and specific biological function. It acts as a catalyst that accelerates most of the chemical reactions taking place in living cells or under biological conditions. That, an enzyme does by stabilizing the transition state and thus reducing the activation energy of a specific reaction. enzymes accelerate the adjustment of the equilibrium of reactions, but don’t have influence on the equilibrium itself enzymes interact with their reaction partners temporarily and come off unaltered
32. Sketch dG vs reaction progress for an uncatalyzed and a catalyzed exothermic chemical reaction!
33. Describe the Michaelis-Menten model, its assumptions and limitations!
- describes the influence of enzymes on the reaction velocity
- reaction is split into two “separate” reactions (building of ES, dissociation into E + P)
- building of ES (reaction 1) is reversible (in contrast to reaction 2)
- enzyme finally comes off unaltered
Limitations: The MM theory is a good model to describe simple enzyme reactions (single substrate-reactions). However, if we have allosteric enzymes (their activity can be regulated through cooperativity), the MM theory does not conform to it anymore.
34. Sketch the reaction formula and the v vs [S] graph for a typical enzyme reaction and discuss the Michaelis-Menten constants Km and vmax!
vmax = maximum velocity of enzyme reaction
Km = that special substrate concentration, at which semi-saturation is reached (semi-saturation = vmax/2). Km is a measure for substrate-enzyme affinity
35. What is the difference between competitive and uncompetitive inhibition? How can you distinguish in a lineweaver-burk diagram?
Competitive inhibition: prevents the substrate from binding to the active site of the enzyme. (such inhibitors are chemically and structurally similar to the substrate). Increases Km, but leaves vmax unaltered.
Uncompetitive inhibition: the inhibitor binds exclusively to ES, thus decreases Km as well as vmax
36. What is a transition-state analogue in enzyme catalysis?
Transition-state analogues have played a major part in the verification of the transition-state hypothesis
Transition-state analogs are stable compounds that mimic key features of this highest-energy species. They are potent and specific inhibitors of enzymes. Proof that transition- state stabilization is a key aspect of enzyme activity comes from the generation of catalytic antibodies. Transition-state analogs are used as antigens, or immunogens, in generating catalytic antibodies.
37. Sketch the catalytic mechanism of trypsin and describe the active site triad!
Trypsin is a serine protease and thus cleaves peptide bonds in proteins. It is very important for the digestive system and comes into existence when trypsinogen is split. The active site triad consists of Asp-His-Ser and cleaves selectively after the amino acids lysine/arginine but also after modified cystein (the negatively charged Asp of the active site triad is responsible for attracting positively charged lysine and arginine).
Mechanism: -nucleophilic attack of Ser residue on the carboxyl carbon atom of the polypeptide -intermediate, at which the Ser breaks up hydrogen bond with His and peptide bond of polypeptide is cleaved -nucleophilic attack of water intermediate -reforming of hydrogen bond between Ser and His and dissociation of cleaved peptide
38. pKa values of active site residues in proteins often have values very different from the expected ones. Describe two reasons for this phenomenon!
39. Name three possible effects of pH on enzyme catalysis! How can you determine whether an aspartate residue (aspartate residue is negatively charged) is involved in catalysis?
-extreme changes in pH will denature folded structure due to repulsive effects/forces -milder changes in pH can dissociate the oligomeric state of enzyme into inactive monomers -even milder changes in pH will affect only the velocity with which the enzyme is working (so an enzyme needs certain pH conditions to reach optimum):
-therefore it is of great interest to be aware of protein stability issues due to pH conditions when characterizing enzyme activity
enzyme catalytic groups contain acid and basic groups; therefore, ionization state is critical for proper catalysis how the enzyme activity is altered by pH dependency may allow identification of the type of groups present at the active site that are important for catalysis (for instance aspartate)
one group of the enzyme has to be protonated for function other group of the enzyme has to be unprotonated for function
ALSO SEE QUESTIONS # 59-61!
40. How can you determine whether an aspartate (aspartate residue is negatively charged) and a histidine residue (histidine residue is positively charged) are involved in catalysis of your enzyme reaction?
check the pH dependence of the reaction rate!
41. What is the value of the activation energy Ea for a reaction where Keq doubles with a temperature increase of 10 degrees?
When K doubles with a temperature increase of 10 degrees the Ea is around 12,5 kcal/mol. When Ea is smaller the reaction rate is less temperature dependent. Arrhenius equation:
42. What is the value of the activation energy dH for a reaction where Keq doubles with a temperature increase of 10 degrees?
When K doubles with a temperature increase of 10 degrees, dH is around 12,5 kcal/mol.
43. How to make an unfavourable reaction like the phosphorylation of glucose go forward?
COUPLED REACTIONS: Not all biological reactions have negative dG values. To make it go forward nonetheless, the reaction is coupled with another reaction (which has a negative dG value)
44. Sketch the progress curves for the components of a simple enzyme reaction according to the Michaelis-Menten model
Progress curves for the components of a simple enzyme reaction according to the Michaelis-Menten model
45. How are turnover number and catalytic constant defined?
46. Sketch the plot of the initial velocity v0 of a simple Michaelis-Menten reaction versus the substrate concentration [S]!
47. Which equation describes the temperature dependence of Keq and what can you use it for?
this equation allows the calculation of dH° if K is know for two temperatures. Or let’s say if you know dH° and a K, you can calculate the K for the other temperature.
48. Which equation describes the temperature dependence of the activation energy Ea and what can you use it for?
this equation allows the calculation of Ea, if k (=velocity constant of reaction) is known at two temperatures Arrhenius plot lnk vs 1/T results in a straight line with slope = -Ea/R
49. Which equation describes the link between redox potential and Keq and what can you use it for?
n = number of electrons transferred F = Faraday constant E = redox potential E° = standard redox potential this equation allows the calculation of E or Keq (if one is know)
50. Where exactly in the cell is the photosynthetic reaction centre located? Describe its components and the sites where photons are absorbed, NAD+ is reduced and where water is split!
Components: -light reaction -dark reaction -NADP+ reductase -proton translocating ATP synthase
In the light reactions one molecule of the pigment chlorophyll absorbs one photon and loses one electron. This electron is passed to a modified form of chlorophyll called pheophytin, which passes the electron to a quinone molecule, allowing the start of a flow of electrons down an electron transport chain that leads to the ultimate reduction of NADP into NADPH. In addition, it serves to create a proton gradient across the chloroplast membrane; its dissipation is used by ATP Synthase for the concomitant synthesis of ATP. The chlorophyll molecule regains the lost electron by taking one from a water molecule through a process called photolysis, that releases oxygen gas as a waste product. In the Light-independent or dark reactions the enzyme RuBisCO captures CO2 from the atmosphere and in a process that requires the newly formed NADPH, called the Calvin-Benson cycle releases three-carbon sugars which are later combined to form sucrose and starch. Photosynthesis may simply be defined as the conversion of light energy into chemical energy by living organisms. It is affected by its surroundings and the rate of photosynthesis is affected by the concentration of carbon dioxide, the intensity of light, and the temperature.
51. Where exactly in the cell is the electron transport chain located? Describe its components and the sites where NADH is oxidized and where oxygen is reduced!
The electron transport chain is located in the inner mitochondrion membrane.
Components:
-Complex I oxidation of NADH through the enzyme NADH dehydrogenase
-Complex II succinate dehydrogenase electron transfer via FAD and Quinone
-Complex III cytochrome bc electron transfer onto cytochrome c (=electron carrier)
-Complex IV reduction of O2 into H2O by they enzyme cytochrome c oxidase; at the same time, protons are moved across the membrane producing a proton gradient
-Complex V ATP synthase (F0F1-ATPase)
52. Write the balanced chemical equation for the calvin cycle!
53. In oxidative phosphorylation, a certain potential is converted into the phosphoryl transfer potential of ATP. What is this potential and which compounds carry it?
The electron transport potential of the electron carriers (such as FADH2 and NADH) is converted into the phosphoryl transfer potential of ATP.
54. What are the measures of phosphoryl transfer potential and the electron transfer potential? Describe the equation for these potentials!
-electron transfer potential (given by the reduction potential):
-phosphoryl transfer potential (given by the hydrolysis of the activated phosphate compound):
55. Describe the electron transport chain, its main components, redox cofactors with values for dG°’ and dE°’!
56. The standard free-energy change is related to the change in reduction potential. Describe this equation and its parameters and units!
n = number of electrons transferred F = Faraday constant dE’° = standard redox potential
57. The standard free-energy change is related to the change in entropy. Describe this equation and its parameters and units!
58. The standard free-energy change is related to the equilibrium constant. Describe this equation and its parameters and units!
ΔG0 = − RT * lnKeq
[ΔG0] = kcal * mol − 1
R = gas constant (1.987cal/(mol*K))
T = Temperature in Kelvin Keq = Equilibrium Constant
59. What constant will be affected when the pH dependent process affects the transformation of ES -> E+P? Will it be dependent on the pKa of the E, S, and ES complex?
Vmax and kcat will be affected, dependent on the pKa of the ES complex
60. What will be affected when the pH dependent process affects the transformation of E+S <-> ES? Will it be dependent on the pKa of the ES complex?
Km of the reaction will be affected, dependent on the pKa of the E, S, (forward) and ES complex (reverse)
61. What will be affected when the pH dependent process affects the transformation of E+S <-> E+P? Will it be dependent on the pKa of E and S?
kcat/Km of the reaction will be affected, dependent on the pKa of E and S
