chapter 3 Amino Acids

Question 1

Proteins: 
Main Agents of Biological Function

Answer 1

Catalysis
enolase (in the glycolytic pathway)
DNA polymerase (in DNA replication)

Transport
hemoglobin (transports O2 in the blood)
lactose permease (transports lactose across the cell membrane)

Structure
collagen (connective tissue)
keratin (hair, nails, feathers, horns)

Motion
myosin (muscle tissue)
actin (muscle tissue, cell motility)

Question 2

Proteins serve a

Answer 2

Proteins serve a wide range of
biological functions

Question 3

Amino Acids: 
Building Blocks of Protein

Proteins are linear

Amino acids have properties that are well-suited to carry out a variety of biological functions

Answer 3

Proteins are linear heteropolymers of α-amino acids

Amino acids have properties that are well-suited to carry out a variety of biological functions
-Capacity to polymerize
-Useful acid-base properties
-Varied physical properties
-Varied chemical functionality

Question 4

Amino acids share many features, differing only at
-General structure
of an amino acid- common to all except
-R group or side chain is

Answer 4

Amino acids share many features, differing only at the R substituent

General structure
of an amino acid- common to all except proline; all are α amino acids

R group or side chain is different in each amino acid; vary in structure, size and electric charge

Question 5

Most α-amino acids are chiral

The α-carbon always has four
-All (except proline) have:

-The fourth substituent (R) is

Answer 5

-The α-carbon always has four substituents and is tetrahedral

-All (except proline) have:
–an acidic carboxyl group
–a basic amino group
–an α-hydrogen connected to the α-carbon

-The fourth substituent (R) is unique
–In glycine, the fourth substituent is also hydrogen

Question 6

All amino acids are chiral (except

Answer 6

glycine)
Proteins only contain L amino acids

Question 7

L amino acid
D amino acid

Answer 7

left
right

Question 8

Amino Acids: Atom Naming

Answer 8

-Organic nomenclature: start from one end
-Biochemical designation:
start from α-carbon and go down the R-group

Question 9

Amino acids
-R- groups influence
-Common amino acids are assigned
-The α carbon is chiral in all common amino acids except for
-Amino acid residues in protein molecules are almost exclusively
-L stereoisomer is synthesized because the enzyme active site is

Answer 9

-R- groups influence solubility in water
In addition to the 20 common, there are less common ones
-Common amino acids are assigned a 3 letter abbreviation and a one letter symbol
-The α carbon is chiral in all common amino acids except for glycine
-Amino acid residues in protein molecules are almost exclusively L stereoisomers- exceptions in some small peptides of bacterial cell walls and some peptide antibiotics
-L stereoisomer is synthesized because the enzyme active site is asymmetric so the reactions catalyzed are stereospecific

Question 10

Amino Acids: Classification

Answer 10

Common amino acids can be placed in five basic groups depending on their R substituents:
-Nonpolar – aliphatic (7: G, A, P, V, L, I, M); aromatic (1: F)
-Aromatic (3: Y, W, F. NOTE: Y and W more polar than F)
-Polar, uncharged (5: S, T, C, N, Q)
Positively charged (3, K, R, H*) (pH 7.0)
Negatively charged (2: D, E) (pH 7.0)

Question 11

• pKR = 6.0. Why is Histidine positively charged at pH7, but then also have a charge at 0 at the same pH?

Answer 11

Best Answer:first of all, the reason its only 10% is because of H-HpH = pKa +log (b/a)7 = 6 + log (b/a)1 = log (b/a)10 = b/aso there are 10 b’s for every a.H-H tells you that when pH is MUCH greater than pKa, the population of molecules will be entirely deprotonated, but it also tells you that when the pKa and pH are somewhat close, the population is only partially deprotonated. (remember that pH/pKa is logarithmic; every 1 point difference between pH-pKa is a 10 fold increase of one acid-base species over the other.) in order to determine exactly how deprotonated, you use the H-H eq as shown above. for your problem, which histadine loses its side-chain proton, the molecule goes from +1 to 0. so if your population is 90% deprotonated on the side chain, the net charge of the population will be +0.1.that website simply says that histidine has a “net positive charge”, but the net is not very great, as you can see. arginine and lysine (their other examples) are fully positive at neutral pH because they are much weaker acids (stronger bases), with their pKa’s closer to 10.

Question 12

Nonpolar, aliphatic R groups

Answer 12

Glycine, alanine, proline, valine, leucine, isoleucine, methionine (thioether)

Nonpolar, hydrophobic, tend to cluster together by hydrophobic interactions

Proline- imino group is rigid and reduces structural flexibility

Question 13

Nonpolar, Aliphatic R Groups

These amino acid side chains are

Answer 13

These amino acid side chains are hydrophobic, tend to cluster together by hydrophobic interactions

Question 14

Aromatic R groups
These amino acid side chains absorb UV light at

Answer 14

Phenylalanine, Tyrosine, Tyrpotphan,

These amino acid side chains absorb UV light at 270–280 nm

Question 15

Polar, uncharged R groups

Answer 15

serine, threonine, cysteine(sulfhydryl), asparagine, glutamine

Question 16

Polar, Uncharged R Groups
-Hydrophilic and can form
-Polarity due to
-Cysteine can be oxidized to form a

Answer 16

Hydrophilic and can form hydrogen bonds with water;

Polarity due to OH or sulfhydryl group or amide group;

Cysteine can be oxidized to form a covalently linked dimeric amino acid called cystine. Disulfide bonds are very hydrophobic.

Question 17

Positively charged R groups

Answer 17

lysine, arginine, histidine

Question 18

Positively Charged (Basic) R Groups
-Hydrophilic due to
-Lysine has positive charge at
-Arginine has a positively charged
-Histidine has a imidazole group that is

Answer 18

-Hydrophilic due to charge

-Lysine has positive charge at pH7 due to the second primary amino group in ε position

-Arginine has a positively charged guanido group

-Histidine has a imidazole group that is ionizable at a pK near neutrality so can act as a proton donor/acceptor

Question 19

Negatively charged R groups

Answer 19

aspartate, glutamate

Hydrophilic; net negative charge at pH 7 due to carboxyl group

Question 20

Uncommon Amino Acids in Proteins
-Apart from the 20 common amino acids, there are some
-Not incorporated by
-Arise by
-Reversible modifications, especially phosphorylation, are

Answer 20

-Apart from the 20 common amino acids, there are some 300 other that have been found in cells
-Not incorporated by ribosomes
except for Selenocysteine
-Arise by post-translational modifications of proteins
-Reversible modifications, especially phosphorylation, are important in regulation and signaling

Question 21

Modified Amino Acids Found in Proteins

Answer 21

Some uncommon amino acids found in proteins. All are derived from common amino acids. Extra functional groups added by modification reactions are shown in red.

Plant cell walls and collagen

Fibrous protein elastin

FIGURE 3-8a Uncommon amino acids. (a) Some uncommon amino acids found in proteins. All are derived from common amino acids.
Extra functional groups added by modification reactions are shown in red. Desmosine is formed from four Lys residues (the carbon
backbones are shaded in light red). Note the use of either numbers or Greek letters in the names of these structures to identify the
altered carbon atoms.

Question 22

Ionization of Amino Acids
-At acidic pH, the carboxyl group is
-At neutral pH, the carboxyl group is
-At alkaline pH, the amino group is

Answer 22

-At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form.
-At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions.
-At alkaline pH, the amino group is neutral –NH2 and the amino acid is in the anionic form.

Question 23

Zwitterion- dipolar ion that can act as a

Answer 23

Zwitterion- dipolar ion that can act as a H donor or acceptor

Dual nature- amphoteric; ampholyte

Question 24

Amino acid titration
pI is the arithmetic mean of the

Answer 24

Note two regions of buffering power

pI is the arithmetic mean of the pK values

Cation -> Zwitterion -> Anion

FIGURE 3-10 Titration of an amino acid. Shown here is the titration curve of 0.1 M glycine at 25°C. The ionic species predominating at key points in the titration are shown above the graph. The shaded boxes, centered at about pK1 = 2.34 and pK2 = 9.60, indicate the regions of greatest buffering power. Note that 1 equivalent of OH– = 0.1 M NaOH added

Question 25

Chemical Environment Affects pKa Values

Answer 25

pKa Values

α-carboxy group is much more acidic than in carboxylic acids
α-amino group is slightly less basic than in amines

FIGURE 3-11 Effect of the chemical environment on pKa. The pKa values for the ionizable groups in glycine are lower than those for simple, methyl-substituted amino and carboxyl groups. These downward perturbations of pKa are due to intramolecular interactions. Similar effects can be caused by chemical groups that happen to be positioned nearby—for example, in the active site of an enzyme.

Question 26

Amino acids can act as buffers
-Amino acids with uncharged side chains, such as glycine, have
-It can act as a buffer in

Answer 26

Amino acids with uncharged side chains, such as glycine, have two pKa values:

The pKa of the α-carboxyl group is 2.34
The pKa of the α-amino group is 9.6

It can act as a buffer in two pH regimes.

Question 27

Amino acids carry a
-Zwitterions predominate at pH values between
-For amino acids without ionizable side chains, the Isoelectric Point (equivalence point, pI) is

Answer 27

a net charge of zero 
at a specific pH (the pI)

-Zwitterions predominate at pH values between the pKa values of the amino and carboxyl groups
-For amino acids without ionizable side chains, the Isoelectric Point (equivalence point, pI) is

PI=Pk1+Pk2/2

-At this point, the net charge is zero
–AA is least soluble in water
–AA does not migrate in electric field

Question 28

Ionizable side chains can show up in
-Ionizable side chains can be
-Titration curves are now
-pKa values are

Answer 28

titration curves

-Ionizable side chains can be also titrated
-Titration curves are now more complex
-pKa values are discernable if two pKa values are more than two pH units apart

Why is the side chain pKa so much higher?

Question 29

How to Calculate the pI When the Side Chain is Ionizable
-Identify species that
-Take the average of

Answer 29

-Identify species that carries a net zero charge
-Identify pKa value that defines the acid strength of this zwitterion: (pK2)
-Identify pKa value that defines the base strength of this zwitterion: (pK1)
-Take the average of these two pKa values

What is the pI of histidine?

Question 30

Formation of Peptides

Answer 30

Peptides are small condensation products of amino acids
They are “small” compared to proteins (Mw < 10 kDa)

Hydrolysis- exergonic but slow due to high activation energy

α-amino group acts as a nucleophile to displace the OH group

Condensation- unfavorable, carboxyl group must be modified

Question 31

Peptide ends are not the same

Answer 31

Numbering (and naming) starts from the amino terminus

Question 32

Naming peptides: 
start at the N-terminus

Answer 32

Using full amino acid names
Serylglycyltyrosylalanylleucine

Using the three-letter code abbreviation
Ser-Gly-Tyr-Ala-Leu

For longer peptides (like
proteins) the one- letter code can be used
SGYAL

Question 33

Peptides: A Variety of Functions

Answer 33

Hormones and pheromones
insulin (think sugar)
oxytocin (think childbirth)
sex-peptide (think fruit fly mating)

Neuropeptides
substance P (pain mediator)

Antibiotics
polymyxin B (for Gram – bacteria)
bacitracin (for Gram + bacteria)

Protection, e.g., toxins
amanitin (mushrooms)
conotoxin (cone snails)
chlorotoxin (scorpions)

Question 34

Proteins are:

Answer 34

Polypeptides (covalently linked α-amino acids) + possibly:

-cofactors
A general term for functional non-amino acid component
metal ions or organic molecules

• coenzymes
  Used to designate an organic cofactors
  NAD+ in lactate dehydrogenase

-prosthetic groups
covalently attached cofactors
heme in myoglobin

-other modifications

Question 35

-Some proteins are
-If the subunits are identical then the protein is

Answer 35

-Some proteins are multisubunit
-If the subunits are identical then the protein is oligomeric and the units are protomers e.g. hemoglobin made of 2 α chains and 2 β chains held by noncovalent interactions. Each α paired with a β identically so it is a dimer of α β protomers

Question 36

Average molecular weights

Answer 36

-Average molecular weight of the 20 amino acids is about 138
-Smaller amino acids predominate in most proteins
-Lose 1 molecule of H2O in bond formation (MW 18)
-Taking the proportions into account, the average molecular weight of protein amino acids is 128
-Average MW of a residue is about 110
-Note that this is just a rough guide!!!

Question 37

Levels of protein structure
-Most important aspect of primary structure is the

Answer 37

-Most important aspect of primary structure is the residue sequence. Primary Structure also includes a description of the peptide and disulfide bonds
-Secondary structure-stable arrangements giving rise to recurring structural patterns

Question 38

Working with Proteins

Answer 38

-Need to purify proteins to determine properties and activities
-Separation methods take advantage of differing properties such as size, charge and binding properties
-First, break cells open to make crude extract
-If necessary, differential centrifuge to prepare subcellular fractions or isolate organelles
-Then fractionate using differing properties

Question 39

A mixture of proteins can be separated
Separation relies on differences in physical and chemical properties

Answer 39

Separation relies on differences in physical and chemical properties
-Charge
-Size
-Affinity for a ligand
-Solubility
-Hydrophobicity
-Thermal stability

Chromatography is commonly used for preparative separation

Question 40

Salting out
column chromatography

Answer 40

(Precipitatioin by Salts)
FIGURE 3-16 Column chromatography. The standard elements of a chromatographic column include a solid, porous material (matrix) supported inside a column, generally made of plastic or glass. A solution, the mobile phase, flows through the matrix, the stationary phase. The solution that passes out of the column at the bottom (the effluent) is constantly replaced by solution supplied from a reservoir at the top. The protein solution to be separated is layered on top of the column and allowed to percolate into the solid matrix. Additional solution is added on top. The protein solution forms a band within the mobile phase that is initially the depth of the protein solution applied to the column. As proteins migrate through the column, they are retarded to different degrees by their different interactions with the matrix material. The overall protein band thus widens as it moves through the column. Individual types of proteins (such as A, B, and C, shown in blue, red, and green) gradually separate from each other, forming bands within the broader protein band. Separation improves (i.e., resolution increases) as the length of the column increases. However, each individual protein band also broadens with time due to diffusional spreading, a process that decreases resolution. In this example, protein A is well separated from B and C, but diffusional spreading prevents complete separation of B and C under these conditions.

Question 41

Separation by Charge

Answer 41

Cation-Exchanger
FIGURE 3-17a Three chromatographic methods used in protein purification. (a) Ion-exchange chromatography exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH.
proteins with a more negative charge move faster down.

Question 42

Sepration by size

Answer 42

FIGURE 3-17b Three chromatographic methods used in protein purification. (b) Size-exclusion chromatography, also called gel filtration, separates proteins according to size. Larger proteins pass more freely and move faster down.

Question 43

Separation by affinity

Answer 43

FIGURE 3-17c Three chromatographic methods used in protein purification. (c) Affinity chromatography separates proteins by their binding specificities. Further details of these methods are given in the text.

Question 44

Protein purification steps

Answer 44

Note that at each step, protein is lost and the total starting amount decreases, the total activity decreases but the specific activity increases- Why?

FPLC- fast protein liquid chromatography- use high pressure to speed movement down column, reduces transit time and reduces diffusional spreading of bands improving resolution

Dialysis- separates proteins from solvents, uses a semipermeable membrane with pore size that retains proteins but lets salts and buffers through- can only go to equilibrium, so buffer changes necessary

Question 45

Electrophoresis for Protein Analysis

Answer 45

Separation in analytical scale is commonly done by electrophoresis, PAGE
-Electric field pulls proteins according to their charge
-Gel matrix hinders mobility of proteins according to their size and shape
-Gel matrix: polyacrylamide

Question 46

Principle of Protein Separation by PAGE

Answer 46

FIGURE 3-18a Electrophoresis. (a) Different samples are loaded in wells or depressions at the top of the polyacrylamide gel. The proteins move into the gel when an electric field is applied. The gel minimizes convection currents caused by small temperature gradients, as well as protein movements other than those induced by the electric field.
small go faster down

Question 47

SDS (sodium dodecyl sulfate ) PAGE

Answer 47

SDS – sodium dodecyl sulfate – a detergent

SDS micelles bind to and unfold all the proteins
-SDS gives all proteins an uniformly negative charge
-The native shape of proteins does not matter
-Rate of movement will only depend on size: small proteins will move faster

In this case the effect of charge is eliminated by binding a negatively charged detergent, SDS, to all the proteins that are denatured.
The SDS binds uniformly per unit length of protein and therefore the force on the molecules from the field will be a uniform amount per unit length, and the only affect on the speed of travel will be the retarding force due to their size.

This is therefore a method to separate molecules based on their molecular weights; clearly not useful for oligomers because these will be forced apart by the SDS.

-can remove covlalent bond but can’t remove disulfide bonds, DTT is what breaks down disulfide bonds

Question 48

FIGURE 3-18b Electrophoresis. (b) Proteins can be visualized after electrophoresis by treating the gel with

Answer 48

FIGURE 3-18b Electrophoresis. (b) Proteins can be visualized after electrophoresis by treating the gel with a stain such as Coomassie blue, which binds to the proteins but not to the gel itself. Each band on the gel represents a different protein (or protein subunit); smaller proteins move through the gel more rapidly than larger proteins and therefore are found nearer the bottom of the gel. This gel illustrates purification of the RecA protein of Escherichia coli (described in Chapter 25). The gene for the RecA protein was cloned (Chapter 9) so that its expression (synthesis of the protein) could be controlled. The first lane shows a set of standard proteins (of known Mr), serving as molecular weight markers. The next two lanes show proteins from E. coli cells before and after synthesis of RecA protein was induced. The fourth lane shows the proteins in a crude cellular extract. Subsequent lanes (left to right) show the proteins present after successive purification steps. The purified protein is a single polypeptide chain (Mr ~38,000), as seen in the rightmost lane.

Question 49

SDS-PAGE can be used to calculate the

Answer 49

SDS-PAGE can be used to calculate the molecular weight of a protein

FIGURE 3–19 Estimating the molecular weight of a protein. The electrophoretic mobility of a protein on an SDS polyacrylamide gel is related to its molecular weight, Mr. (a) Standard proteins of known molecular weight are subjected to electrophoresis (lane 1). These marker proteins can be used to estimate the molecular weight of an unknown protein (lane 2). (b) A plot of log Mr of the marker proteins versus relative migration during electrophoresis is linear, which allows the molecular weight of the unknown protein to be read from the graph. (In similar fashion, a set of standard proteins with reproducible retention times on a size-exclusion column can be used to create a standard curve of retention time versus log Mr. The retention time of an unknown substance on the column can be compared with this standard curve to obtain an approximate Mr.)

Question 50

Specific activity (activity/total protein) can be used to

Answer 50

Specific activity (activity/total protein) can be used to assess protein purity

FIGURE 3–22 Activity versus specific activity. The difference between these terms can be illustrated by considering two flasks containing marbles. The flasks contain the same number of red marbles, but different numbers of marbles of other colors. If the marbles represent proteins,
both flasks contain the same activity of the protein represented by the red marbles. The second flask, however, has the higher specific activity
because red marbles represent a higher fraction of the total.

Question 51

Protein Sequence
-Amino acid sequence determines how a
-Amino acid sequence of a protein is not always
-Some polymorphisms do not affect

Answer 51

-Amino acid sequence determines how a protein folds to give it a unique 3-D shape and this determines its function
-Amino acid sequence of a protein is not always fixes, and polymorphisms exist (20-30% proteins in humans)
-Some polymorphisms do not affect biological function while others can be debilitating or lethal

FIGURE 3–22 Activity versus specific activity. The difference between these terms can be illustrated by considering two flasks containing marbles. The flasks contain the same number of red marbles, but different numbers of marbles of other colors. If the marbles represent proteins,
both flasks contain the same activity of the protein represented by the red marbles. The second flask, however, has the higher specific activity
because red marbles represent a higher fraction of the total.

Question 52

-It is essential to further
-Actual sequence generally determined from
-Edman Degradation (_____method)
-Mass Spectrometry (_____ method)

Answer 52

-It is essential to further biochemical analysis that we know the sequence of the protein we are studying
-Actual sequence generally determined from DNA sequence

-Edman Degradation (Classical method)
-Successive rounds of N-terminal modification, cleavage, and identification
-Can be used to identify protein with known sequence

-Mass Spectrometry (Modern method)
-MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence
-Can be used to determine post-translational modifications

Question 53

Edman’s Degradation

Answer 53

FIGURE 3–27 The protein sequencing chemistry devised by Pehr Edman. The peptide bond nearest to the amino terminus of the protein or polypeptide is cleaved in two steps. The two steps are carried out under very different reaction conditions (basic conditions in step 1, acidic in
step 2), allowing one step to proceed to completion before the second is initiated.

Question 54

FIGURE 3–27 The protein sequencing chemistry devised by Pehr Edman.

Answer 54

FIGURE 3–27 The protein sequencing chemistry devised by Pehr Edman. The peptide bond nearest to the amino terminus of the protein or polypeptide is cleaved in two steps. The two steps are carried out under very different reaction conditions (basic conditions in step 1, acidic in
step 2), allowing one step to proceed to completion before the second is initiated.

Question 55

MS Procedures for Sequence IDs

Answer 55

FIGURE 3–30 Electrospray ionization mass spectrometry of a protein. (a) A protein solution is dispersed into highly charged droplets by passage through a needle under the influence of a high-voltage electric field. The droplets evaporate, and the ions (with added protons in this case) enter the mass spectrometer for m/z measurement. The spectrum generated (b) is a family of peaks, with each successive peak (from right to left) corresponding to a charged species increased by 1 in both mass and charge. The inset shows a computer-generated transformation of this spectrum.
FIGURE 3–31 Obtaining protein sequence information with tandem MS. (a) After proteolytic hydrolysis, a protein solution is injected into a
mass spectrometer (MS-1). The different peptides are sorted so that only one type is selected for further analysis. The selected peptide is further fragmented in a chamber between the two mass spectrometers, and m/z for each fragment is measured in the second mass spectrometer (MS-
2). Many of the ions generated during this second fragmentation result from breakage of the peptide bond, as shown. These are called b-type or
y-type ions, depending on whether the charge is retained on the amino- or carboxyl-terminal side, respectively. (b) A typical spectrum with peaks
representing the peptide fragments generated from a sample of one small peptide (21 residues). The labeled peaks are y-type ions derived from
amino acid residues. The number in parentheses over each peak is the molecular weight of the amino acid ion. The successive peaks differ by
the mass of a particular amino acid in the original peptide. The deduced sequence is shown at the top.

Question 56

What do identical sequence residues from different proteins tell us about the proteins?
homologs, paralogs, orthologs

Answer 56

Homologs-members of protein families
Paralogs-two proteins of the same family present in the same species
Orthologs- two proteins of the same family in different species

Question 57

Protein Sequences as Clues to Evolutionary Relationships

Answer 57

Sequences of homologous proteins from a wide range of species can be aligned and analyzed for differences

Differences indicate evolutionary divergences

Analysis of multiple protein families can indicate evolutionary relationships between organisms, ultimately the history of life on Earth