[John_L._Tymoczko,_Jeremy_M.

_Berg,_Lubert_Stryer]
ANSWERS TO PROBLEMS
Chapter 2
1. Identify. Examine the following four amino acids (A–D):

What are their names, three-letter abbreviations, and one letter symbols?
1. (A) Proline, Pro, P;
(B) tyrosine, Tyr, Y;
(C) leucine, Leu, L;
(D) lysine, Lys, K.

2. Properties. In reference to the amino acids shown in Problem 1,

which are associated with the following characteristics?
(a) Hydrophobic side chain ______________
(b) Basic side chain ______________
(c) Three ionizable groups ______________
(d) p Ka of approximately 10 in proteins ______________
(e) Modified form of phenylalanine ______________
2. (a) C, B, A; (b) D; (c) D, B; (d) B, D; (e) B.

3. Match ’em. Match each amino acid in the left-hand column with the
appropriate side-chain type in the right-hand column.
(a) Leu (1) hydroxyl-containing
(b) Glu (2) acidic
(c) Lys (3) basic
(d) Ser (4) sulfur-containing
(e) Cys (5) nonpolar aromatic
(f) Trp (6) nonpolar aliphatic
3. (a) 6; (b) 2; (c) 3; (d) 1; (e) 4; (f ) 5.
4. Solubility. In each of the following pairs of amino acids, identify which
amino acid would be more soluble in water:
(a) Ala, Leu;
(b) Tyr, Phe;
(c) Ser, Ala;
(d) Trp, His.
4. (a) Ala; (b) Tyr; (c) Ser; (d) His.

5. Bonding is good. Which of the following amino acids have R groups that
have hydrogen-bonding potential? Ala, Gly, Ser, Phe, Glu, Tyr, Ile, and Thr.
5. Ser, Glu, Tyr, Thr

6. Name those components. Examine the segment of a protein shown here.

(a) What three amino acids are present?

(b) Of the three, which is the N-terminal amino acid?
(c) Identify the peptide bonds.
(d) Identify the a -carbon atoms.
6. (a) Alanine-glycine-serine;
(b) Alanine;
(c and d):

7. Who’s charged? Draw the

structure of the dipeptide
GlyHis. What is the charge
on the peptide at pH 5.5?
pH 7.5?
8. Alphabet soup . How many different polypeptides of 50 amino acids in
length can be made from the 20 common amino acids?

9. Sweet tooth, but calorie conscious . Aspartame (NutraSweet), an artificial

sweetener, is a dipeptide composed of Asp-Phe in which the carboxyl
terminus is modified by the attachment of a methyl group. Draw the
structure of Aspartame at pH 7.
10. Vertebrate proteins? What is meant by the term polypeptide backbone?
10. The (nitrogen– a carbon–carbonyl carbon) repeating unit.

11. Not a sidecar. Define the term side chain in the context of amino acid or
protein structure.
11. Side chain is the functional group attached to the a -carbon
atom of an amino acid.
12. One from many. Differentiate between amino acid composition and
amino acid sequence .
12. Amino acid composition refers simply to the amino acids
that make up the protein. The order is not specified. Amino acid
sequence is the same as the primary structure—the sequence of
amino acids from the amino terminal to the carboxyl terminal
of the protein. Different proteins may have the same amino acid
composition, but amino acid sequence identifies a unique protein.

13. Shape and dimension .

(a) Tropomyosin, a 70-kDa muscle protein, is a two-stranded a -helical coiled
coil. Estimate the length of the molecule.
(b) Suppose that a 40-residue segment of a protein folds into a two-stranded
antiparallel b structure with a 4-residue hairpin turn. What is the longest
dimension of this motif?

13. (a) Each strand is 35 kDa and hence has about 318 residues
(the mean residue mass is 110 daltons). Because the rise per residue in an a
helix is 1.5 Å, the length is 477 Å. More precisely, for an a -helical coiled coil,
the rise per residue is 1.46 Å; so the length is 464 Å.
(b) Eighteen residues in each strand (40 minus 4 divided by 2) are in a b
-sheet conformation. Because the rise per residue is 3.5 Å, the length is 63 Å.
14. Contrasting isomers .
Poly- L -leucine in an organic solvent such as dioxane is a helical, whereas
poly- L -isoleucine is not. Why do these amino acids with the same number
and kinds of atoms have different helix-forming tendencies? 14. The methyl
group attached to the b -carbon atom of isoleucine sterically interferes with a
-helix formation. In leucine, this methyl group is attached to the g -carbon
atom, which is farther from the main chain and hence does not interfere.

15. Exceptions to the rule.

Ramachandran plots for two amino acids differ significantly from that
shown in Figure 2.23. Which two, and why?
15. Proline and glycine. The cyclic side chain of proline linking
the nitrogen and a -carbon atoms limits to a very narrow range
(around 260 degrees). The lack of steric hindrance exhibited by
the side chain hydrogen atom of glycine enables this amino acid to access a
much greater area of the Ramachandran plot.
16. Active again.
A mutation that changes an alanine residue in the interior of a protein to
valine is found to lead to a loss of activity. However, activity is regained when
a second mutation at a different position changes an isoleucine residue to
glycine. How might this second mutation lead to an restoration of activity?
16. The first mutation destroys activity because valine occupies
more space than alanine does, and so the protein must take a
different shape, assuming that this residue lies in the closely
packed interior. The second mutation restores activity because
of a compensatory reduction of volume; glycine is smaller
than isoleucine.

17. Exposure issues . Many of the loops on proteins are composed of

hydrophilic amino acids. Why might this be the case?
17. Loops invariably are on the surface of proteins, exposed to the
environment. Because many proteins exist in aqueous environments, the
exposed loops will be hydrophilic to interact with water.

18. Shuffle test . An enzyme that catalyzes disulfide–sulfhydryl exchange

reactions, called protein disulfide isomerase (PDI), has been isolated. PDI
rapidly converts inactive scrambled ribonuclease into enzymatically active
ribonuclease. In contrast, insulin is rapidly inactivated by PDI. What does
this important observation imply about the relation between the amino acid
sequence of insulin and its three-dimensional structure?
18. The native conformation of insulin is not the thermodynamically most
stable form, because it contains two separate chains linked by disulfide
bonds. Insulin is formed from proinsulin, a single-chain precursor, that is
cleaved to form insulin, a 51-residue molecule, after the disulfide bonds have
formed.

19. Stretching a target . A protease is an enzyme that catalyzes the hydrolysis

of the peptide bonds of target proteins. How might a protease bind a target
protein so that its main chain becomes fully extended in the vicinity of the
vulnerable peptide bond?
19. A segment of the main chain of the protease could hydrogen
bond to the main chain of the substrate to form an extended parallel or
antiparallel pair of b strands.
20. Often irreplaceable. Glycine is a highly conserved amino acid residue in
the evolution of proteins. Why?
20. Glycine has the smallest side chain of any amino acid. Its size is often
critical in allowing polypeptide chains to make tight turns or to approach one
another closely.

21. Potential partners . Identify the groups in a protein that can form
hydrogen bonds or electrostatic bonds with an arginine side chain at pH 7.
21. Glutamate, aspartate, and the terminal carboxylate can form salt bridges
with the guanidinium group of arginine. In addition, this group can be a
hydrogen-bond donor to the side chains of glutamine, asparagine, serine,
threonine, aspartate, tyrosine, and glutamate and to the main-chain carbonyl
group. Histidine can form hydrogen bonds with arginine at pH 7.

22. Permanent waves . The shape of hair is determined in part by the pattern
of disulfide bonds in keratin, its major protein. How can curls be induced?
22. Disulfide bonds in hair are broken by adding a thiol-containing reagent
and applying gentle heat. The hair is curled, and an oxidizing agent is added
to re-form disulfide bonds to stabilize the desired shape.

23. 1. Most proteins have hydrophilic exteriors and hydrophobic interiors.

Would you expect this structure to apply to proteins embedded in the
hydrophobic interior of a membrane? Explain.
23. Some proteins that span biological membranes are “the exceptions that
prove the rule” because they have the reverse distribution of hydrophobic
and hydrophilic amino acids. For example, consider porins, proteins found in
the outer membranes of many bacteria. Membranes are built largely of
hydrophobic chains. Thus, porins are covered on the outside largely with
hydrophobic residues that interact with the neighboring hydrophobic chains.
In contrast, the center of the protein contains many charged and polar amino
acids that surround a water-filled channel running through the middle of the
protein. Thus, because porins function in hydrophobic
environments, they are “inside out” relative to proteins that
function in aqueous solution.

24. Location is everything 2 . Proteins that span biological membranes often

contain a helices. Given that the insides of membranes are highly
hydrophobic (Section 12.2), predict what type of amino acids would be in
such an a helix. Why is an a helix particularly suited to existence in the
hydrophobic environment of the interior of a membrane?
 24. The amino acids would be hydrophobic in nature. An a helix
is especially suited to crossing a membrane because all of the amide hydrogen
atoms and carbonyl oxygen atoms of the peptide backbone take part in
intrachain hydrogen bonds, thus stabilizing these polar atoms in a
hydrophobic environment.

25. Neighborhood peer pressure? Table 2.1 shows the typical p Ka values for
ionizable groups in proteins. However, more than 500 p Ka values have been
determined for individual groups in folded proteins. Account for this
discrepancy
25. This example demonstrates that the p Ka values are affected by the
environment. A given amino acid can have a variety of p Ka values,
depending on the chemical environment inside the protein.

26. Greasy patches. The a and b subunits of hemoglobin bear a remarkable

structural similarity to myoglobin. However, in the subunits of hemoglobin,
certain residues that are hydrophilic in myoglobin are hydrophobic. Why
might this be the case?

26. Recall that hemoglobin exists as a tetramer while myoglobin

is a monomer. Consequently, the hydrophobic residues on
the surface of hemoglobin subunits are probably involved in
van der Waals interactions with similar regions on the other
subunits, and will be shielded from the aqueous environment
by this interaction.

27 . Maybe size does matter . Osteogenesis imperfecta displays a wide range

of symptoms, from mild to severe. On the basis of your knowledge of amino
acid and collagen structure, propose a biochemical basis for the variety of
symptoms.

27. A possible explanation is that the severity of the symptoms corresponds to

the degree of structural disruption. Hence, substitution of alanine for glycine
might result in mild symptoms, but substitution of the much larger
tryptophan may result in little or no collagen triple-helix formation.

28. Issues of stability . Proteins are quite stable. The lifetime of a peptide
bond in aqueous solution is nearly 1000 years. However, the free energy of
hydrolysis of proteins is negative and quite large. How can you account for
the stability of the peptide bond in light of the fact that hydrolysis releases
much energy?

28. The energy barrier that must be crossed to go from the polymerized state
to the hydrolyzed state is large even though the reaction is
thermodynamically favorable.

29. Minor species . For an amino acid such as alanine, the major species in
solution at pH 7 is the zwitterionic form. Assume a p Ka value of 8 for the
amino group and a p Ka value of 3 for the carboxylic acid. Estimate the ratio
of the concentration of the neutral amino acid species (with the carboxylic
acid protonated and the amino group neutral) to that of the zwitterionic
species at pH 7 (Section 1.3).

29. Using the Henderson–Hasselbach equation, we find the ratio

of alanine-COOH to alanine-COO2 at pH 7 to be 10 24 . The ratio of alanine-
NH 2 to alanine-NH 3 1 , determined in the same fashion, is 10 21 . Thus, the
ratio of neutral alanine to the zwitterionic species is 1024 3 1021 5 1025

30. A matter of convention . All L amino acids have an S absolute

configuration except L -cysteine, which has the R configuration. Explain why
L -cysteine is designated as having the R absolute configuration.

30. The assignment of absolute configuration requires the assignment of

priorities to the four groups connected to a tetrahedral carbon atom. For all
amino acids except cysteine, the priorities are:
(1) amino group; (2) carbonyl group; (3) side chain; (4) hydrogen. For
cysteine, because of the sulfur atom in its side chain, the sidechain has a
greater priority than does thecarbonyl group, leading to the assignment of an
R rather than S configuration.
31. Hidden message . Translate the following amino acid sequence into one-
letter code: Glu-Leu-Val-Ile-Ser-IleSer-Leu-Ile-Val-Ile-Asn-Gly-Ile-Asn-Leu-
Ala-Ser-ValGlu-Gly-Ala-Ser.
31. ELVISISLIVINGINLASVEGAS

32. Who goes first? Would you expect Pro}X peptide bonds to tend to have
cis conformations like those of X}Pro bonds? Why or why not?
32. No, Pro–X would have the characteristics of any other peptide bond. The
steric hindrance in X–Pro arises because the R group of Pro is bonded to the
amino group. Hence, in X–Pro, the proline R group is near the R group of X,
which would not be the case in Pro–X.

33. Matching . For each of the amino acid derivatives shown here (A–E), find
the matching set of and values (a–e).

33. A, c; B, e; C, d; D, a; E, b.

34. Scrambled ribonuclease. When performing his experiments on protein

refolding, Christian Anfinsen obtained a quite different result when reduced
ribonuclease was reoxidized while it was still in 8 M urea and the preparation
was then dialyzed to remove the urea. Ribonuclease reoxidized in this way
had only 1% of the enzymatic activity of the native protein. Why were the
outcomes so different when reduced ribonuclease was reoxidized in the
presence and absence of urea?

34. The reason is that the wrong disulfides formed pairs in urea.
There are 105 different ways of pairing eight cysteine molecules
to form four disulfides; only one of these combinations is enzymatically
active. The 104 wrong pairings have been picturesquely termed “scrambled”
ribonuclease.

Chapter 3
PROBLEMS
1. Valuable reagents. The following reagents are often used
in protein chemistry:
CNBr Trypsin
Urea Performic acid
Mercaptoethanol 6 N HCl
Chymotrypsin Phenyl isothiocyanate
Which one is the best suited for accomplishing each of the following tasks?
(a) Determination of the amino acid sequence of a small peptide.
(b) Reversible denaturation of a protein devoid of disulfide bonds. Which
additional reagent would you need if disulfide bonds were present?
(c) Hydrolysis of peptide bonds on the carboxyl side of aromatic residues.
(d) Cleavage of peptide bonds on the carboxyl side of methionines .
(e) Hydrolysis of peptide bonds on the carboxyl side of lysine and arginine
residues.

1. (a) Phenyl isothiocyanate; (b) urea; b -mercaptoethanol to

reduce disulfides; (c) chymotrypsin; (d) CNBr; (e) trypsin.

2. The only constant is change. Explain how two different cell types from the
same organism will have identical genomes but may have vastly divergent
proteomes.
2. For each cell within an organism, the genome is a fixed property. However,
the proteome is dynamic, reflecting different environmental conditions and
external stimuli. Two different cell types will likely express different subsets
of proteins encoded within the genome.

3. Crafting a new breakpoint. Ethyleneimine reacts with cysteine side chains in

proteins to form S - aminoethyl derivatives. The peptide bonds on the carboxyl
side of these modified cysteine residues are susceptible to hydrolysis by trypsin .
Why?
3. The S -aminoethylcysteine side chain resembles that of lysine.
The only difference is a sulfur atom in place of a methylene group.

4. Spectrometry. The absorbance A of a solution is defined as

in which I 0 is the incident-light intensity and I is the transmitted- light intensity.

The absorbance is related to the molar absorption coefficient (extinction
coefficient) _ (in M _ 1 cm _ 1 ), concentration c (in M), and path length l (in cm)
by A = elc
The absorption coefficient of myoglobin at 580 nm is 15,000 M _ 1 cm _ 1 . What
is the absorbance of a 1 mg ml _ 1 solution across a 1-cm path? What percentage
of the incident light is transmitted by this solution?
4. A 1 mg ml 21 solution of myoglobin (17.8 kDa; Table 3.2) corresponds to
5.62 3 1025 M. The absorbance of a 1-cm path length is 0.84, which
corresponds to an I 0 /I ratio of 6.96. Hence 14.4% of the incident light is
transmitted.

5. It’s in the bag. Suppose that you precipitate a protein with 1 M (NH4 )2 SO4
and that you wish to reduce the concentration of the (NH 4 )2 SO 4 . You take 1
ml of your sample and dialyze it in 1000 ml of buffer. At the end of dialysis, what
is the concentration of (NH 4 )2 SO4 in your sample? How could you further
lower the (NH 4 ) 2 SO 4 concentration?
5. The sample was diluted 1000-fold. The concentration after
dialysis is thus 0.001 M, or 1 mM. You could reduce the salt concentration by
dialyzing your sample, now 1 mM, in more buffer free of (NH 4 ) 2 SO 4 .

6. Too much or not enough. Why do proteins precipitate at high salt

concentrations? Although many proteins precipitate at high salt concentrations,
some proteins require salt to dissolve in water. Explain why some proteins require
salt to dissolve.

6. If the salt concentration becomes too high, the salt ions interact with the
water molecules. Eventually, there will not be enough water molecules to
interact with the protein, and the protein will precipitate. If there is lack of
salt in a protein solution, the proteins may interact with one another—the
positive charges on one protein with the negative charges on another or
several others. Such an aggregate becomes too large to be solubilized by
water alone. If salt is added, the salt neutralizes the charges on the proteins,
preventing protein–protein interactions.

7. A slow mover . Tropomyosin , a 70-kDa muscle protein, sediments more slowly

than does hemoglobin (65 kDa ). Their sedimentation coefficients are 2.6S and
4.31S, respectively. Which structural feature of tropomyosin accounts for its slow
sedimentation?
7. Tropomyosin is rod shaped, whereas hemoglobin is approximately
spherical
8. Sedimenting spheres. What is the dependence of the sedimentation?
coefficient s of a spherical protein on its mass? How much more rapidly does an
80 -kDa protein sediment than does a 40-kDa protein?
8. The frictional coefficient, f , and the mass, m , determine s .
Specifically, f is proportional to r (see equation 2 on p. 71). Hence, f is
proportional to m1/3 , and so s is proportional to m2/3 (see the equation on p.
76). An 80-kDa spherical protein undergoes sedimentation 1.59 times as
rapidly as a 40-kDa spherical protein.

9. Frequently used in shampoos. The detergent sodium dodecyl sulfate (SDS)

denatures proteins. Suggest how SDS destroys protein structure.
9. The long hydrophobic tail on the SDS molecule (see p. 72) disrupts the
hydrophobic interactions in the interior of the protein. The protein unfolds,
with the hydrophobic R groups now interacting with SDS rather than with
one another.

10. Size estimate. The relative electrophoretic mobilities of a 30-kDa protein and a
92-kDa protein used as standards on an SDS– polyacrylamide gel are 0.80 and
0.41, respectively. What is the apparent mass of a protein having a
mobility of 0.62 on this gel?
10. 50 kDa

11. Unexpected migration. Some proteins migrate anomalously

in SDS-PAGE gels. For instance, the molecular weight determined from an SDS-
PAGE gel is sometimes very different from the molecular weight determined
from the amino acid sequence. Suggest an explanation for this discrepancy.
11. The protein may be modified. For instance, asparagine residues in the
protein may be modified with carbohydrate units
(Section 2.6).

12. Sorting cells. Fluorescence-activated cell sorting (FACS) is a powerful

technique for separating cells according to their content of particular molecules.
For example, a fluorescence- labeled antibody specific for a cell-surface protein
can be used to detect cells containing such a molecule.
Suppose that you want to isolate cells that possess a receptor enabling them to
detect bacterial degradation products. However, you do not yet have an antibody
directed against this receptor. Which fluorescence-labeled molecule would you
prepare to identify such cells?
12. A fluorescence-labeled derivative of a bacterial degradation
product (e.g., a formylmethionyl peptide) would bind to cells containing the
receptor of interest.

13. Column choice.

(a) The octapeptide AVGWRVKS was digested with the enzyme trypsin. Which
method would be most appropriate for separating the products: ion-exchange or
gel-filtration chromatography? Explain.
13. (a) Trypsin cleaves after arginine (R) and lysine (K), generating AVGWR,
VK, and S. Because they differ in size, these products could be separated by
molecular exclusion chromatography.
(b) Suppose that the peptide was digested with chymotrypsin. What would
be the optimal separation technique? Explain.
(b) Chymotrypsin, which cleaves after large aliphatic or aromatic R groups,
generates two peptides of equal size (AVGW) and (RVKS). Separation based
on size would not be effective. The peptide RVKS has two positive charges (R
and K), whereas the other peptide is neutral. Therefore, the two products
could be separated by ion-exchange chromatography.

14. Power( ful ) tools. Monoclonal antibodies can be conjugated to an insoluble

support by chemical methods. Explain how these antibody-bound beads can be
exploited for protein purification.
14. Antibody molecules bound to a solid support can be used for
affinity purification of proteins for which a ligand molecule is not known or
unavailable

15. Assay development. You wish to isolate an enzyme from its native source and
need a method for measuring its activity throughout the purification. However,
neither the substrate nor the product of the enzyme-catalyzed reaction can be
detected by spectroscopy. You discover that the product
of the reaction is highly antigenic when injected into mice. Propose a strategy to
develop a suitable assay for this enzyme.
15. If the product of the enzyme-catalyzed reaction is highly antigenic, it may
be possible to obtain antibodies to this particular molecule. These antibodies
can be used to detect the presence of product by ELISA, providing an assay
format suitable for the purification of this enzyme.
16. Making more enzyme? In the course of purifying an enzyme, a researcher
performs a purification step that results in an increase in the total activity to a
value greater than that present in the original crude extract. Explain how
the amount of total activity might increase.
16. An inhibitor of the enzyme being purified might have been
present and subsequently removed by a purification step. This
removal would lead to an apparent increase in the total amount of enzyme
present.

17. Divide and conquer. The determination of the mass of a protein by mass
spectrometry often does not allow its unique identification among possible
proteins within a complete proteome, but determination of the masses of all
fragments produced by digestion with trypsin almost always allows unique
identification. Explain.
17. Many proteins have similar masses but different sequences and different
patterns when digested with trypsin. The set of masses of tryptic peptides
forms a detailed “fingerprint” of a protein that is very unlikely to appear at
random in other proteins regardless of size. (A conceivable analogy is: “Just
as similarly sized fingers will give different individual fingerprints, so also
similarly sized proteins will give different digestion patterns with trypsin.”)
18. Know your limits. Which two amino acids are indistinguishable
in peptide sequencing by the tandem mass spectrometry
method described in this chapter and why?
18. Isoleucine and leucine are isomers and, hence, have identical
masses. Peptide sequencing by mass spectrometry as described in this
chapter is incapable of distinguishing these residues. Further analytical
techniques are required to differentiate these residues.

19. Protein purification problem. Complete the following table.

Purification Total Total Specific Purification Yield
Procedure protein activity activity level (%)
 (mg) (units) (units mg_1)

Crude extract 20,000 4,000,000 1 100

(NH4)2SO4 1,500 3,000,000

Precipitation

DEAE-cellulose 500 1,000,000

Chromatography

Gel-filtration 5,000 750,000

Chromatography

Affinity 45 675,000
chromatography

20. Part of the mix. Your frustrated colleague hands you a mixture of four proteins
with the following properties:

Isoelectric point (pI) Molecular weight (in kDa)

Protein A 4.1 80
Protein B 9.0 81
Protein C 8.8 37
Protein D 3.9 172

(a) Propose a method for the isolation of Protein B from the other proteins. (b) If
Protein B also carried a His tag at its N-terminus, how could you revise your
method?
(b) 20. (a) Ion exchange chromatography will remove Proteins A and
(c) D, which have substantially lower isoelectric point; then gel filtration
chromatography will remove Protein C, which has a lower molecular
weight.
(d) (b) If Protein B carries a His tag, a single affinity chromatography step
with an immobilized nickel(II) column may be sufficient to isolate the
desired protein from the others.

21. The challenge of flexibility. Structures of proteins comprising domains

separated by flexible linker regions can be quite difficult to solve by x-ray
crystallographic methods. Why might this be the case? What are possible
experimental approaches to circumvent this barrier?

21. Protein crystal formation requires the ordered arrangement of identically

positioned molecules. Proteins with flexible linkers can introduce disorder
into this arrangement and prevent the formation of suitable crystals. A
ligand or binding partner may induce an ordered conformation to this linker
and could be included in the solution to facilitate crystal growth.
Alternatively, the individual domains separated by the linker may be
expressed by recombinant methods and their crystal structures solved
separately.

Chapter Integration Problems

22. Quaternary structure. A protein was purified to homogeneity.

Determination of the mass by gel-filtration chromatography
yields 60 kDa . Chromatography in the presence of 6 M urea yields a 30-kDa
species. When the chromatography is repeated in the presence of 6 M urea and 10
mM b - mercaptoethanol , a single molecular species of 15 kDa results. Describe
the structure of the molecule.
22. Treatment with urea will disrupt noncovalent bonds. Thus the original
60-kDa protein must be made of two 30-kDa subunits. When these subunits
are treated with urea and b -mercaptoethanol, a single 15-kDa species
results, suggesting that disulfide bonds link the 30-kDa subunits.

23. Helix–coil transitions. (a) NMR measurements have shown that poly- L
-lysine is a random coil at pH 7 but becomes a helix as the pH is raised above 10.
Account for this dependent conformational transition. (b) Predict the pH
dependence of the helix–coil transition of poly- L -glutamate.
23. (a) Electrostatic repulsion between positively charged ´ -amino groups
hinders a -helix formation at pH 7. At pH 10, the side chains become
deprotonated, allowing a -helix formation.
(b) Poly- L -glutamate is a random coil at pH 7 and becomes a helical below
pH 4.5 because the g -carboxylate groups become protonated.

24. Peptide mass determination. You have isolated a protein from the bacterium
E. coli and seek to confirm its identity by trypsin digestion and mass
spectrometry. Determination of the masses of several peptide fragments has
enabled you to deduce the identity of the protein. However, there is a
discrepancy with one of the peptide fragments, which you believe should have the
sequence MLNSFK and an (M 1 H ) _ value of 739.38. In your experiments, you
repeatedly obtain an (M 1 H ) _ value of 767.38. What is the cause of this
discrepancy and what does it tell you about the region of the protein from which
this peptide is derived?
24. The difference between the predicted and the observed masses for this
fragment equals 28.0, exactly the mass shift that would be expected in a
formylated peptide. This peptide is likely formulated at its amino terminus,
and corresponds to the most N-terminal fragment of the protein

Exchange rate. The amide hydrogen atoms of peptid .26

bonds within proteins can exchange with protons in the solve
In general, amide hydrogen atoms in buried regions of
proteins and protein complexes exchange more slowly than
those on the solvent-accessible surface do. Determination of
,these rates can be used to explore the protein-folding reaction
probe the tertiary structure of proteins, and identify the
regions of protein–protein interfaces. These exchange reactions
can be followed by studying the behavior of the protein
in solvent that has been labeled with deuterium ( 2 H), a stable
isotope of hydrogen. What two methods described in this
chapter could be readily applied to the study of hydrogen
deuterium exchange rates in proteins?
26. Mass spectrometry is highly sensitive and capable of detecting
the mass difference between a protein and its deuterated counterpart.
Fragmentation techniques can be used to identify the amino
acids that retained the isotope label. Alternatively, NMR spectroscopy
can be used to detect the isotopically labeled atoms because the
deuteron and the proton have very different nuclear-spin properties.

Data Interpretation Problems

27. Protein sequencing 1. Determine the sequence of hexapeptide
on the basis of the following data. Note: When the sequence is not known, a
comma separates the amino acids (Table 3.3).
Amino acid composition: (2R,A,S,V,Y)
N-terminal analysis of the hexapeptide : A
Trypsin digestion: (R,A,V) and (R,S,Y)
Carboxypeptidase digestion: No digestion.
Chymotrypsin digestion: ( A,R,V,Y) and (R,S)

27. First amino acid: A

Last amino acid: R (not cleaved by carboxypeptidase).
Sequence of N-terminal tryptic peptide: AVR (tryptic peptide
ends in K)
Sequence of N-terminal chymotryptic peptide: AVRY
(chymotryptic peptide ends in Y)
Sequence: AVRYSR

28. Protein sequencing 2. Determine the sequence of a peptide

consisting of 14 amino acids on the basis of the following
data. Amino acid composition: (4S,2L,F,G,I,K,M,T,W,Y)
N-terminal analysis: S Carboxypeptidase digestion: L
Trypsin digestion: (3S,2L,F,I,M,T,W) (G,K,S,Y)
Chymotrypsin digestion: (F,I,S) (G,K,L) (L,S) (M,T) (S,W) (S,Y)
N-terminal analysis of (F,I,S) peptide: S
Cyanogen bromide treatment: (2S,F,G,I,K,L,M*,T,Y) (2S,L,W)
M*, methionine detected as homoserine
28. First amino acid: S
Last amino acid: L
Cyanogen bromide cleava ge: M is 10th position
C-terminal residues are: (2S,L,W)
Amino-terminal residues: (G,K,S,Y), tryptic peptide, ends in K
Amino-terminal sequence: SYGK
Chymotryptic peptide order: (S,Y), (G,K,L), (F,I,S), (M,T), (S,W), (S,L)
Sequence: SYGKLSIFTMSW

29. Applications of two-dimensional electrophoresis.

Performic acid cleaves the disulfide linkage of cystine and converts the
sulfhydryl groups into cysteic acid residues, which are then no longer capable of
disulfide-bond formation. Consider the following experiment: You suspect that a
protein containing three cysteine residues has a single disulfide bond. You digest
the protein with trypsin and subject the mixture to electrophoresis along one end
of a sheet of paper. After treating the paper with performic acid, you subject the
sheet to electrophoresis in the perpendicular direction and stain it with a reagent
that detects proteins. How would the paper appear if the protein did not contain
any disulfide bonds? If the protein contained a single disulfide bond? Propose an
experiment to identify which cysteine residues form the disulfide bond. Performic
acid

29. If the protein did not contain any disulfide bonds, then the
electrophoretic mobility of the trypsin fragments would be the same before and
after performic acid treatment: all the fragments would lie along the diagonal of
the paper. If one disulfide bond were present, the disulfide-linked trypsin
fragments would run as a single peak in the first direction, then would run as two
separate peaks after performic acid treatment. The result would be two peaks
appearing off the diagonal

These fragments could then be isolated from the chromatography paper and analyzed by mass
spectrometry to determine their amino acid composition and thus identify the cysteines
participating in the disulfide bond.

:Chapter 8 enzymes
PROBLEMS
1. Raisons d’être . What are the two properties of enzymes
that make them especially useful catalysts?

1. Rate enhancement and substrate specificity.

2. Partners. What does an apoenzyme require to become a holoenzyme?

2. A cofactor

3. Different partners . What are the two main types of cofactors?

3. Coenzymes and metals
4. One a day. Why are vitamins necessary for good health?
4. Vitamins are converted into coenzymes.

substrates5 . A function of state . What is the fundamental mechanism by which

enzymes enhance the rate of chemical reactions?

5. Enzymes facilitate the formation of the transition state.

6. Nooks and crannies. What is the structural basis for enzyme specificity?
6. The intricate three-dimensional structure of proteins allows
the construction of active sites that will recognize only specific
7. Give with one hand, take with the other. Why does the activation energy of a
reaction not appear in the final D G of the reaction?

8. The more things change, the more they stay the same . Suppose that, in the
absence of enzyme, the forward rate constant ( k F ) for the conversion of S into P
is 10 2 4 s 2 1 and the reverse rate constant ( k R ) for the conversion of P into Sis
10 2 6 s 2 1 .SΔ 1024 s21 026 s21 P
(a) What is the equilibrium for the reaction? What is the D G 8 9 ?
(b) Suppose an enzyme enhances the rate of the reaction 100 fold. What are the
rate constants for the enzyme catalyzed reaction? The equilibrium constant? The
D G 8 9?
8. (a) K 5 [P][S] 5 kFkR5 10241026 5 100. Using equation 5 in the text,DG89
5 2 11.42 kJ mol 21 ( 2 2.73 kcal mol 21 )
(b) kF 5 10 22 s 21 and kR 5 10 24 s 21 . The equilibrium constant and DG89
values are the same for both the uncatalyzed and catalyzed reactions.

9. Mountain climbing. Proteins are thermodynamically unstable. The D G of the

hydrolysis of proteins is quite negative, yet proteins can be quite stable. Explain
this apparent paradox. What does it tell you about protein synthesis?
9. Protein hydrolysis has a large activation energy. Protein synthesis must
require energy to proceed.

10. Protection. Suggest why the enzyme lysozyme, which degrades cell walls of
some bacteria, is present in tears.
10. The enzymes help protect the fluid that surrounds eyes from
bacterial infection.

11. Mutual attraction. What is meant by the term binding energy?

11. Binding energy is the free energy released when two molecules bind
together, such as when an enzyme and a substrate interact.
.

12. Catalytically binding . What is the role of binding energy in enzyme catalysis?
12. Binding energy is maximized when an enzyme interacts with
the transition state, thereby facilitating the formation of the transition state
and enhancing the rate of the reaction
1 3. Sticky situation . What would be the result of an enzyme having a greater
binding energy for the substrate than for the transition state?
13. There would be no catalytic activity. If the enzyme–substrate complex is
more stable than the enzyme–transition-state complex, the transition state
would not form and catalysis would not take place.

14. Stability matters . Transition-state analogs, which can be used as enzyme

inhibitors and to generate catalytic antibodies, are often difficult to synthesize.
Suggest a reason

. 14. Transition states are very unstable. Consequently, molecules

that resemble transition states are themselves likely to be unstable and,
hence, difficult to synthesize

15. Match’em . Match the K9eq values with the appropriate

D G 8 9 values.
K 9e q DG 89 (kJ mol2 1 )
(a) 1 28.53
(b) 10 2 5 2 11.42
(c) 10 4 5.69
(d) 10 2 0
(e) 10 2 1 2 22.84

16. Free energy! Assume that you have a solution of 0.1 M glucose 6-phosphate.
To this solution, you add the enzyme phosphoglucomutase, which catalyzes the
following reaction:
Phosphoglucomutase
Glucose 6-phosphate 3::::::::4 glucose 1- phosphate
The D G89 for the reaction is 1 7.5 kJ mol 2 1 ( 1 1.8 kcal mol 2 1 ).
(a) Does the reaction proceed as written? If so, what are the
final concentrations of glucose 6-phosphate and glucose 1-phosphate?
(b) Under what cellular conditions could you produce glucose
1-phosphate at a high rate?

17. Free energy, too! Consider the following reaction:

Phosphoglucomutase Glucose 6-phosphate 3::::::::4 glucose 1- phosphate
After reactant and product were mixed and allowed to
reach equilibrium at 25 8 C , the concentration of each compound
was measured:
[Glucose 1-phosphate]eq 5 0.01 M
[Glucose 6-phosphate]eq 5 0.19 M
Calculate Keq and DG89.

18. Keeping busy . Many isolated enzymes, if incubated at

37 8 C, will be denatured. However, if the enzymes are incubated
at 37 8 C in the presence of substrate, the enzymes are catalytically active.
Explain this apparent paradox.

19. Active yet responsive . What is the biochemical advantage of having a K M

approximately equal to the substrate concentration normally available to an
enzyme?

20. Affinity or not affinity? That is the question . The affinity between a protein
and a molecule that binds to the protein is frequently expressed in terms of a
dissociation constant K d .
Protein2small molecule complex Δ protein 1 small molecule Kd 5
[protein][small molecule]
[Protein 2 small molecule complex]
Does K M measure the affinity of the enzyme complex?
Under what circumstances might KM approximately equal K d ?

14. Transition states are very unstable. Consequently, molecules

that resemble transition states are themselves likely to be unstable and,
hence, difficult to synthesize
18.The three-dimensional structure of an enzyme is stabilized by interactions
with the substrate, reaction intermediates, and products. This stabilization
minimizes thermal denaturation.

Chapter 10
20. When phosphorylation takes place at the expense of ATP, sufficient
energy is expended to dramatically alter the structure and hence activity of a
protein. Moreover, because ATP is the cellular energy currency, protein
modification is linked to the energy status of the cell.

21. Covalent modification is reversible, whereas proteolytic cleavage is

irreversible.
22. Activation is independent of zymogen concentration because the reaction
is intramolecular.
23. Although quite rare, cases of enter peptidase deficiency have been
reported. The affected person has diarrhea and fails to thrive because
digestion is inadequate. In particular, protein digestion is impaired.
24. Add blood from the second patient to a sample from the first. If the
mixture clots, the second patient has a defect different from that of the first.
This type of assay is called a complementation test.
25. Activated factor X remains bound to blood-platelet membranes, which
accelerates its activation of prothrombin.
26. Antithrombin III is a very slowly hydrolyzed substrate of thrombin.
Hence, its interaction with thrombin requires a fully formed active site on the
enzyme.

27. Replacing methionine with leucine would be a good choice.

Leucine is resistant to oxidation and has nearly the same volume
and degree of hydrophobicity as methionine has.