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NAME ________________________________
EXAM II
October 15, 2002
Biochemistry I
I.
__________________/ 45
II.
__________________/ 26
III.
__________________/ 15
IV.
__________________/ 14
TOTAL
/100
I. MULTIPLE CHOICE. (45 points)
Choose the BEST answer to the question by circling the appropriate letter.
1.
Which of the following statements about proteins is false?
A.
B.
C.
D.
E.
2.
Which of the following statements about oligomeric proteins is false?
A.
B.
C.
D.
E.
3.
a Ramachandran plot.
molecular model building.
x-ray diffraction.
light microscopy.
electron microscopy.
A D-amino acid would interrupt an b-strand made of L-amino acids.
Another naturally occurring constraint on the formation of an bstrand is the presence of:
A.
B.
C.
D.
E.
5.
All subunits must be identical.
Some subunits may have nonprotein cofactors.
A subunit may be very similar to other proteins.
Some oligomeric proteins can further associate into large fibers.
Subunits can exist in dozens or even hundreds of genetic variations.
Determination of the precise spacing of atoms within a large protein is
possible only through the use of:
A.
B.
C.
D.
E.
4.
Primary structure determines tertiary structure.
Globular proteins are generally very compact.
Proteins are sometimes conjugated with carbohydrates.
Nonpolar amino acid side chains generally are arranged on the
surface where they interact with water.
Proteins consists of amino acids linked by peptide bonds.
a negatively charged Arg residue.
a positively charged Lys residue.
two Gly residues side by side.
a nonpolar residue near the carboxyl terminus.
a Pro residue.
Which of the following groups correctly shows intrachain hydrogen
bonding (denoted by |||) in an a-helix?
A.
B.
C.
D.
E.
-N-H|||H-N-C=O|||H-C-C=O|||H-N-N-H|||H-R-C=O|||O=C-
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interactions between two adjacent hydrophobic Val residues.
7.
All of the following are considered "weak" interactions in proteins
except:
A.
B.
C.
D.
E.
8.
Which of the following types of bonds or interactions are LEAST likely
to be involved in stabilizing the tertiary structure of most proteins?
A.
B.
C.
D.
E.
9.
Prolyl hydroxlyase
Tyrosine hydroxylase
Carboxypeptidase Y
Chymotrypsin
Tryptophan oxygenase
The role of an enzyme in an enzyme-catalyzed reaction is to:
A.
B.
C.
D.
E.
11.
hydrogen bonds
electrostatic bonds
hydrophobic interactions
disulfide bonds
ester bonds
Which of the following enzymes plays a direct role in the biosynthesis
of collagen?
A.
B.
C.
D.
E.
10.
van der Waals forces.
hydrogen bonds.
ionic bonds.
peptide bonds.
hydrophobic interactions.
ensure that the product is more stable than the substrate.
make the free-energy change for the reaction more favorable.
increase the rate at which substrate is converted into product.
ensure that all the substrate is converted to product.
do none of the above.
Which of the following statements is true of enzyme catalysts?
A.
B.
C.
D.
E.
They increase the stability of the reaction product by allowing
ionization, resonance, & isomerizations not available to substrates.
To be effective they must be present at the same concentration as
their substrates.
They increase the equilibrium constant for a reaction, thus favoring
product formation.
They lower the activation energy for the reaction of S to P.
They bind to substrates, but are never covalently attached to
substrate or product.
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NAME ________________________________
12.
The NEXT four MULTIPLE CHOICE questions refer to the following problem:
The protein a1-antitrypsin (a1AT) inhibits the action of the
proteolytic enzyme elastase in lung tissue by binding and mimicking the
normal substrate. A mutation has been described in which the only
change is the substitution of an Arg residue for a Met residue in a1AT.
The altered a1AT does not inhibit elastase but has a new property of
inhibiting the blood coagulation protein thrombin. The sequences
around the active site of a1AT, altered a1AT, and the natural thrombin
inhibitor (antithrombin) are given below:
a1AT ....Met-Ser-Ile-Pro-Pro-Glu-Leu
altered a1AT ....Arg-Ser-Ile-Pro-Pro-Glu-Leu
Antithrombin .....Arg-Ser-Leu-Asn-Pro-Asn-Gly
13.
Given the information above, which of the following would be the best
method to separate a1AT from altered a1AT? (2 pt)
A.
B.
C.
D.
E.
14.
From the information given, all of the following can correctly be
concluded EXCEPT: (3 pt)
A.
B.
C.
D.
E.
15.
The number of trypsin cleavage sites is greater in the altered than
in a1AT.
The number of cyanogen bromide cleavage sites is greater in a1AT
than in altered a1AT.
Individuals with this mutation might have a bleeding disorder.
Antithrombin differs from a1AT only at the active site.
Single-residue changes in proteins can alter substrate-binding
specificities.
Smoker's lungs are often damaged by elastase because the a1AT is
inactive. The best explanation for this is that tobacco smoke___. (2pt)
A.
B.
C.
D.
E.
16.
size-exclusion chromatography
ion-exchange chromatography
isopycnic density gradient centrifugation
SDS-polyacrylamide gel electrophoresis
affinity chromatography
causes the substitution of Met for Arg in a1AT.
causes the substitution of Asn for Pro in a1AT.
contains oxidants, which chemically modify the Met in a1AT.
contains carbon monoxide, which competes with a1AT for binding to
elastase.
contains nicotine, which serves as a cofactor for elastase.
The hexapeptide segment flanking the substitution in altered a1AT is
most likely involved in which type of secondary structure: (2 pt)
A.
B.
C.
D.
E.
a-helix
parallel b-sheet
anti-parallel b-sheet
a turn
a collagen helix
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NAME ________________________________
II. SHORT ANSWER (26 points)
Give a brief answer as directed to each problem or question below.
17.
Pauling and Corey showed that in small peptides, four atoms associated
with the peptide bond all lie in a plane. Draw a dipeptide of two
amino acids, indicate which four atoms are part of the planar structure
of the peptide bond, indicate the bond that rotates to change the
phi(f) and the psi(y) angles, and indicate the hydrogen bond donor and
acceptor atoms if this dipeptide were in an a-helix. (8 pts)
18.
Describe the four levels of protein structure and one technique that
can be used to determine or measure each type. (4 pts)
19.
You have a peptide with the following amino acid composition; 4Asp,
3Gly, 2His, Leu, Phe, 2Val. Draw the likely chromatograph from amino
acid analysis using ninhydrin as a colorometric reagent that would have
yielded these results. (6 pts)
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NAME ________________________________
20.
A biochemist purifies a new protein using the steps listed below.
After each step, he measures total protein and the units of enzyme
activity remaining as shown. What are the missing units for Specific
activity? For each method, give the basis for the separation. (8 pts)
Total
Specific Overall
protein
activity yield (%)
Activity
BASIS
(mg)
(
)
(units)
1. Crude extract
100
2,000
0.05
(100)
2. Ammonium sulfate
50
1,000
0.05
50
precipitation
3. Ion-exchange
45
180
0.25
45
chromatography
4. Affinity
12
40
0.30
12
chromatography
Are all of these step(s) useful?
would you eliminate, and why?
If so, which one(s)?
Which, if any,
III. TRUE/FALSE. (15 points; 1 point each) Choose the BEST answer to the
question by circling the appropriate letter.
T
F
21.
The turnover number of an enzyme refers to the number of cycles of
binding and catalysis the enzyme goes through per unit of time.
T
F
22.
Enzymes are thought to achieve their high rates of catalysis by
lowering the transition state energy barrier for the slow-step in the
reaction in going from substrate to product.
T
F
23.
Vitamin C is a necessary for the enzyme responsible for the Schiff base
cross-links in collagen.
T
F
24.
The protein a-keratin, found in hair, is an example of a globular
protein composed largely of a-helix.
T
F
25.
The Levinthal Problem refers to the incalculable number of different
possible conformations that a protein can have when including all
possible combinations.
T
F
26.
Carboxypeptidase Y is used for end-group analysis of proteins and
peptides.
T
F
27.
The basis by which proteins separate in gel filtration chromatography
using Sephadex is by size with the smallest proteins eluting from the
column before the larger proteins.
T
F
28.
The formation of a peptide bond is a type of hydrolysis reaction.
T
F
29.
The titration of water helps determine the pKa for the ionizations near
the extremes in pH.
T
F
30.
The buffer capacity of histidine is greatest at the equivalence point,
after the titration of the carboxylate and before the titration of the
imidazole.
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NAME ________________________________
T
F
31.
The titration of histidine had three ionizable groups, whereas if the
amino acid glycine were titrated there would be two ionizable groups.
T
F
32.
The pH meters used to measure [H+] have a special glass tip that is
permeable to only protons.
T
F
33.
A multisubstrate, enzyme-catalyzed reaction in which a product is
released before all substrates have been bound to the enzyme is an
example of a sequential kinetic mechanism.
T
F
34.
A plot of binding data, Y (fraction of total enzyme as ES) vs. [S],
that produces a sigmoidal curve indicates that cooperative binding
occurs between active sites on the enzyme.
T
F
35.
Use of acid/base catalysis is one strategy employed by enzymes at their
active sites to increase the rate of a reaction.
IV. PROBLEM. (14 points)
Solve the sequence problem with the information given.
36.
A small peptide hormone was found to contain amounts of the following
amino acids with the given stoichiometries: 3Ala, Arg, 2Cys, Glu, Gly,
Lys, Met, Phe, Pro, Trp, Val. Individual samples of the peptide were
treated with the following agents with the results noted (where
possible Edman degradation was performed with the results of each round
given as E#=X):
(a) amino-terminal analysis gave two amino acids: Lys and Ala.
(b) carboxyl-terminal analysis gave a mixture of Val and Trp.
(c) reduction and alkylation followed by peptide separation gave a
decapeptide and a tetrapeptide, which was fluorescent (E1=Ala).
(d) trypsin: Lys, a tripeptide (E1=Gly, E2=Glu, E3=Val), and a
decapeptide
(e) cyanogen bromide: a tetrapeptide (E1=Lys, E2=Ala, E3=Phe,
E4=homoserine lactone) and a decapeptide (E1=Ala, E2=Cys).
(f) chymotrypsin: a tripeptide, and an 11-mer.
What is the primary structure of the peptide? (14 points)
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Answer Sheet for Exam II
Test
Question
Multiple Choice
1
2
3
4
5
6
7
8
9
10
11
13
14
15
16
Short Answer
17
10/15/02
Correct
Answer
D
A
C
E
C
C
D
E
A
C
D
B
D
C
D
Hydrogen bond
acceptor
O
Y
+
HN
3
C
Ca
R
H
N
H
Ca
R
F COO
H
Hydrogen bond donor
18
The structure of the "R" will vary and should correspond
to one of the 20 amino acids. In the box the
intermediate resonance structure imparts a partial
double bond characteristic to the C-N bond, thereby
prohibiting rotation. The N and H of the peptide and the
C and O of the carbonyl are all in the same plane, or
box. The N-Ca is the f-bond and the Ca-Carbonyl is the
y-bond. In an a-helix the carbonyl is an acceptor and
the NH is a donor.
-Primary-Edman degradation, protein sequence analysis,
etc.
Secondary-Circular dichroism (CD) spectroscopy
Tertiary- X-ray crystallography or NMR
Quaternary-gel filtration/SDS PAGE, or
ultracentrifugation/SDS PAGE
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Test
Question
Correct
Answer
--
19
absorbance or amount
Asp
Gly
Val
His
Leu
Phe
fractions
-1. Units of Specific activity are units/mg
2. Basis: 1) solubility, 2) charge, 3) binding
3. Yes, step 3, there has been a 5-fold purification
relative to prior step and only 5% loss in activity.
4. Both step 2 and 4 are not good. Each has little to
no gain in specific activity and anywhere from 50% loss
(#2) to nearly 75% loss (#4). Affinity column may not
be working because this method is usually quite
effective.
--
20
True/False
21
22
23
24
25
26
27
28
29
30
31
32
T
T
F
F
T
T
F
F
T
F
T
F
33
34
35
F
T
T
(hydroxlyl-proline synthesis)
(fibrous)
(larger proteins elute before the smaller)
(condensation)
(also permeable to other salts if high enough
concentration)
(ping-pong)
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Test
Correct
Question Answer
Problems
36
Lys-Ala-Phe-Met-Ala-Cys|-Arg-Gly-Glu-Val
Ala-Cys|-Pro-Trp
After experiments (a-c) you should realize that there
are two peptides joined by a disufide with the 2 Cys
residues. The fluorescence puts the Trp on the C-term
of the smaller 4-mer with the Val then on the C-term of
the 10-mer. The GEV sequence of the trypsin puts this
at the other C-term with the R adjacent and the K at the
N-term of the 10-mer with the Ala then at the N-term of
the 4-mer. CNBr gives more of the 10-mer; KAFM, but the
AC of the decapeptide means that BOTH N-term must be
Ala-Cys and that only leaves Pro as third residue in the
4-mer.
--
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