Exam Biochemistry NWI-MOL008A
Friday 18 January 2019
• Write your name and student number on each piece of paper.
• Answer the multiple-choice questions on the appropriate form.
Rating: 50% of the final test grade is the open questions 1-7.
50% of the final test grade is the multiple-choice questions 1-40
Please write the answers to the open questions 1-2 (questions Cambi), 3-4 (questions Pruijn) and 5-7
(questions Heus) on separate sheets.
IT IS NOT ALLOWED TO USE A GRAPHIC CALCULATOR DURING THE TEST!
HAND IN THE QUESTIONS AFTERWARDS!
Open questions:
1. Proteins are large molecules usually composed of hundreds of amino acids sequentially linked together by
peptide bonds. Below, you find the sequence of a short peptide:
IAKTWPQECAAD
(a) Draw the chemical structure of this short peptide at pH 7, clearly indicating the N-terminus, the C-
terminus, the peptide bonds and the side chains of each amino acid residue. (3.5 points) The appendix table 3.1
might be useful for reference.
(b) What are the charges of the N-terminus, the C-terminus, and the side chains of each amino acid residue at
pH 12? Consult Table 3-1, draw the chemical structure at this basic pH and briefly motivate changes in the
amino acid residue charges, if they are present. (4 points)
(c) The primary structure of large polypeptides or proteins dictates their final three-dimensional architecture.
What is the tertiary structure of a protein and do filamentous proteins have a tertiary structure? (2.5 points)
2. Aspirin is a commonly used drug. In our body, absorption via the oral route normally occurs when the drug is
in the unionized state, as unionized molecules are more lipophilic and can better cross the cell membrane.
Knowing that Aspirin has a pKa of 3.5:
(a) Calculate the ratio of ionized/unionized of the drug in the stomach, where the pH is 1. (4 points)
(b) Calculate the ratio of ionized/unionized in the intestine, where the pH is 6. (4 points)
(c) Based on these calculations, is aspirin more likely absorbed in the stomach or in the intestine? (2 points)
Please hand in the answers to the following open questions 3 – 4 on a separate piece(s) of paper.
3. Mammalian antibodies of the immunoglobulin G type consist of 4 polypeptides, two heavy and two light
chains. Relatively high concentrations of antibodies can be found in the blood. When antibodies are elicited by
immunization of animals with a protein-of-interest, antibodies against this protein can be isolated from the
blood of the immunized animals. If, for example, rabbits are injected with human hemoglobin, antibodies to
human hemoglobin will be produced.
(a) Briefly describe a method that can be applied to separate immunoglobulins from the majority of other
proteins in the blood serum (the blood fraction from which the blood cells have been removed) of an
immunized rabbit. (4 points)
(b) Suppose the anti-hemoglobin antibodies that are produced by an immunized rabbit bind (very strongly) to
the beta-subunits of hemoglobin, but only when they are in the R-state. What will be the effect of antibody
binding to at least one of the beta-subunits of a hemoglobin tetramer on the structure of the alpha-subunits in
terms of R-state/T-state? And what will be the effect of oxygen binding? (4 points)
Page 1 of 10
, (c) Experiments with erythrocyte (red blood cell) ‘ghosts’ were carried out to learn more about the glucose
transporter in these cells. ‘Ghosts’ are prepared by lysing red blood cells in a hypotonic medium and washing
away the cytoplasmic contents. Suspension in an isotonic buffer allows the ghost membranes to reseal. If
ghosts are prepared so that the enzyme trypsin (a protease) is incorporated into the ghost interior, glucose
transport does not occur. But glucose transport is not affected if trypsin is located in the extracellular ghost
medium. What can you conclude about the glucose transporter, given these observations? (3 points)
4. During protein synthesis the genetic code present on an mRNA is translated into a polypeptide chain by the
ribosome, which catalyzes the formation of peptide bonds between the amino acids.
(a) Describe the possible outcomes that could occur because of a single base change in an mRNA. (4 points)
(b) Indicate for each factor or enzyme below at which stage(s) of protein synthesis, amino acid activation,
initiation, elongation and termination, it acts. If a factor or enzyme participates in more than one stage of
protein synthesis, indicate all of them. (2 points)
b1. tRNAAla
b2. RF
b3. aminoacyl-tRNA synthetase
b4. 40S ribosomal subunit
(c) Regarding translation in eukaryotes versus that in prokaryotes (bacteria), indicate whether each of the
following statements is true (T) or false (F). (4 points)
c1. Assembly of a complete ribosome onto an mRNA requires ATP hydrolysis.
c2. Aminoacylation or “charging” of tRNA requires the formation of an aminoacyl-AMP intermediate.
c3. Aminoacyl-tRNA binding to the A site of the ribosome requires the accessory factor EF-G and GTP
hydrolysis.
c4. Translocation of a growing polypeptide from the A to the P site on the ribosome requires EF-G and
GTP hydrolysis.
Please hand in the answers to the following open questions 5 – 7 on a separate piece(s) of paper.
5. (a) Describe briefly the biochemical role of the following enzymes and/or enzymatic activities in DNA
replication in E. coli: (1) DNA helicase; (2) primase; (3) 3' ® 5' exonuclease activity of DNA polymerase; (4)
DNA 1igase; (5) topoisomerase. (3 points)
(b) Name the four general types of processes that can occur in RNA after RNA synthesis. For each type of
process give an example of an RNA in which this process occurs, and if the process is specific for eukaryotes,
prokaryotes or both. (4 points)
(c) Describe the main differences in the mechanism of group I and of group II intron splicing. What is the main
similarity? (3 points)
6. The velocity of a catalyzed reaction is usually described using the Michaelis-Menten equation, in which the
functioning of the enzyme is characterized by the parameters Km and Vmax.
(a) Give the Michaelis-Menten equation and the definition of the parameters Km and Vmax. Include in your
answer the importance of these parameters for characterizing the functioning of an enzyme. (6 points)
(b) How does the total enzyme concentration affect the turnover number and Vmax? (2 points)
(c) Why is the Lineweaver-Burk (double reciprocal) plot more useful than the standard V vs. [S] plot in
determining kinetic constants for an enzyme? (2 points)
Page 2 of 10
Friday 18 January 2019
• Write your name and student number on each piece of paper.
• Answer the multiple-choice questions on the appropriate form.
Rating: 50% of the final test grade is the open questions 1-7.
50% of the final test grade is the multiple-choice questions 1-40
Please write the answers to the open questions 1-2 (questions Cambi), 3-4 (questions Pruijn) and 5-7
(questions Heus) on separate sheets.
IT IS NOT ALLOWED TO USE A GRAPHIC CALCULATOR DURING THE TEST!
HAND IN THE QUESTIONS AFTERWARDS!
Open questions:
1. Proteins are large molecules usually composed of hundreds of amino acids sequentially linked together by
peptide bonds. Below, you find the sequence of a short peptide:
IAKTWPQECAAD
(a) Draw the chemical structure of this short peptide at pH 7, clearly indicating the N-terminus, the C-
terminus, the peptide bonds and the side chains of each amino acid residue. (3.5 points) The appendix table 3.1
might be useful for reference.
(b) What are the charges of the N-terminus, the C-terminus, and the side chains of each amino acid residue at
pH 12? Consult Table 3-1, draw the chemical structure at this basic pH and briefly motivate changes in the
amino acid residue charges, if they are present. (4 points)
(c) The primary structure of large polypeptides or proteins dictates their final three-dimensional architecture.
What is the tertiary structure of a protein and do filamentous proteins have a tertiary structure? (2.5 points)
2. Aspirin is a commonly used drug. In our body, absorption via the oral route normally occurs when the drug is
in the unionized state, as unionized molecules are more lipophilic and can better cross the cell membrane.
Knowing that Aspirin has a pKa of 3.5:
(a) Calculate the ratio of ionized/unionized of the drug in the stomach, where the pH is 1. (4 points)
(b) Calculate the ratio of ionized/unionized in the intestine, where the pH is 6. (4 points)
(c) Based on these calculations, is aspirin more likely absorbed in the stomach or in the intestine? (2 points)
Please hand in the answers to the following open questions 3 – 4 on a separate piece(s) of paper.
3. Mammalian antibodies of the immunoglobulin G type consist of 4 polypeptides, two heavy and two light
chains. Relatively high concentrations of antibodies can be found in the blood. When antibodies are elicited by
immunization of animals with a protein-of-interest, antibodies against this protein can be isolated from the
blood of the immunized animals. If, for example, rabbits are injected with human hemoglobin, antibodies to
human hemoglobin will be produced.
(a) Briefly describe a method that can be applied to separate immunoglobulins from the majority of other
proteins in the blood serum (the blood fraction from which the blood cells have been removed) of an
immunized rabbit. (4 points)
(b) Suppose the anti-hemoglobin antibodies that are produced by an immunized rabbit bind (very strongly) to
the beta-subunits of hemoglobin, but only when they are in the R-state. What will be the effect of antibody
binding to at least one of the beta-subunits of a hemoglobin tetramer on the structure of the alpha-subunits in
terms of R-state/T-state? And what will be the effect of oxygen binding? (4 points)
Page 1 of 10
, (c) Experiments with erythrocyte (red blood cell) ‘ghosts’ were carried out to learn more about the glucose
transporter in these cells. ‘Ghosts’ are prepared by lysing red blood cells in a hypotonic medium and washing
away the cytoplasmic contents. Suspension in an isotonic buffer allows the ghost membranes to reseal. If
ghosts are prepared so that the enzyme trypsin (a protease) is incorporated into the ghost interior, glucose
transport does not occur. But glucose transport is not affected if trypsin is located in the extracellular ghost
medium. What can you conclude about the glucose transporter, given these observations? (3 points)
4. During protein synthesis the genetic code present on an mRNA is translated into a polypeptide chain by the
ribosome, which catalyzes the formation of peptide bonds between the amino acids.
(a) Describe the possible outcomes that could occur because of a single base change in an mRNA. (4 points)
(b) Indicate for each factor or enzyme below at which stage(s) of protein synthesis, amino acid activation,
initiation, elongation and termination, it acts. If a factor or enzyme participates in more than one stage of
protein synthesis, indicate all of them. (2 points)
b1. tRNAAla
b2. RF
b3. aminoacyl-tRNA synthetase
b4. 40S ribosomal subunit
(c) Regarding translation in eukaryotes versus that in prokaryotes (bacteria), indicate whether each of the
following statements is true (T) or false (F). (4 points)
c1. Assembly of a complete ribosome onto an mRNA requires ATP hydrolysis.
c2. Aminoacylation or “charging” of tRNA requires the formation of an aminoacyl-AMP intermediate.
c3. Aminoacyl-tRNA binding to the A site of the ribosome requires the accessory factor EF-G and GTP
hydrolysis.
c4. Translocation of a growing polypeptide from the A to the P site on the ribosome requires EF-G and
GTP hydrolysis.
Please hand in the answers to the following open questions 5 – 7 on a separate piece(s) of paper.
5. (a) Describe briefly the biochemical role of the following enzymes and/or enzymatic activities in DNA
replication in E. coli: (1) DNA helicase; (2) primase; (3) 3' ® 5' exonuclease activity of DNA polymerase; (4)
DNA 1igase; (5) topoisomerase. (3 points)
(b) Name the four general types of processes that can occur in RNA after RNA synthesis. For each type of
process give an example of an RNA in which this process occurs, and if the process is specific for eukaryotes,
prokaryotes or both. (4 points)
(c) Describe the main differences in the mechanism of group I and of group II intron splicing. What is the main
similarity? (3 points)
6. The velocity of a catalyzed reaction is usually described using the Michaelis-Menten equation, in which the
functioning of the enzyme is characterized by the parameters Km and Vmax.
(a) Give the Michaelis-Menten equation and the definition of the parameters Km and Vmax. Include in your
answer the importance of these parameters for characterizing the functioning of an enzyme. (6 points)
(b) How does the total enzyme concentration affect the turnover number and Vmax? (2 points)
(c) Why is the Lineweaver-Burk (double reciprocal) plot more useful than the standard V vs. [S] plot in
determining kinetic constants for an enzyme? (2 points)
Page 2 of 10
