BCTB Quiz 1 Short Answer

Give an example of a biological reaction in which water participates as a reactant and a reaction in which it participates as a product.

See page 65 for examples such as condensation and hydrolysis reactions.

Name and briefly define five types of noncovalent interactions that occur between biological molecules.

(1) Hydrogen bonds: weak electrostatic attractions between one electronegative atom (such as oxygen or nitrogen) and a hydrogen atom covalently linked to a second electronegative atom; (2) electrostatic interactions: relatively weak charge-charge interactions (attractions of opposite charges, repulsions of like charges) between two ionized groups; (3) hydrophobic interactions: the forces that tend to bring two hydrophobic groups together, reducing the total area of the two groups that is exposed to surrounding molecules of the polar solvent (water); (4) van der Waals interactions: weak interactions between the electric dipoles that two close-spaced atoms induce in each other; (5) tightly bound water molecules can form as an essential part of the binding site in a protein for its ligand.

A biochemist wishes to determine the sequence of a protein that contains 123 amino acid residues. After breaking all of the disulfide bonds, the protein is treated with cyanogen bromide (CNBr), and it is determined that that this treatment breaks up the protein into seven conveniently sized peptides, which are separated from each other. It is your turn to take over. Outline the steps you would take to determine, unambiguously, the sequence of amino acid residues in the original protein.

(1) Use Edman degradation to determine the sequence of each peptide. (2) Create a second set of peptides by treatment of the protein with a specific protease (e.g., trypsin), and determine the sequence of each of these. (3) Place the peptides in order by their overlaps. (4) Finally, by a similar analysis of the original protein without first breaking disulfide bonds, determine the number and location of —S—S— bridges.

Define pKa for a weak acid in the following two ways: (1) in relation to its acid dissociation constant, Ka, and (2) by reference to a titration curve for the weak acid.

(1) pKa = -log Ka. (2) See Fig. 2-17, p. 59; pKa is the value of pH at the inflection point in a plot of pH vs. extent of titration of the weak acid. At the pKa, the concentration of ionized acid equals the concentration of un-ionized acid.

The amino acid histidine has three ionizable groups, with pKa values of 1.8, 6.0, and 9.2. (a) Which pKa corresponds to the histidine side chain? (b) In a solution at pH 5.4, what percentage of the histidine side chains will carry a positive charge?

(a) 6.0; (b) 80%. (See the previous problem for expanded solution to this problem.)

(a) Briefly define "isotonic," "hypotonic," and "hypertonic" solutions. (b) Describe what happens when a cell is placed in each of these types of solutions.

(a) An isotonic solution has the same osmolarity as the solution to which it is being compared. A hypotonic solution has a lower osmolarity than the solution to which it is being compared. A hypertonic solution has a higher osmolarity than the solution to which it is being compared. (b) Higher osmolarity results in osmotic pressure, which generally leads to movement of water across a membrane. In an isotonic solution, in which the osmolarity of the solution is the same as the cell cytoplasm, there will be no net water movement. In a hypotonic solution, water will move into the cell, causing the cell to swell and possibly burst. In a hypertonic solution, water will move out of the cell and it will shrink.

____(a) is most negatively charged at pH 7? ____(b) will yield DNP-tyrosine when reacted with l-fluoro-2,4-dinitrobenzene and hydrolyzed in acid? ____(c) contains the largest number of nonpolar R groups? ____(d) contains sulfur? ____(e) will have the greatest light absorbance at 280 n

(a) D; (b) A; (c) E; (d) A; (e) C

E. coli is known as a gram-negative bacterial species. (a) How is this determined? (b) How do gram-negative bacteria differ structurally from gram-positive bacteria?

(a) Gram-negative bacteria have little affinity for the dye gentian violet used in Gram's stain, but gram-positive bacteria retain Gram's stain. (b) Gram-negative bacteria have an outer membrane and a peptidoglycan layer; gram-positive bacteria lack an outer membrane and the peptidoglycan layer is much thicker.

For each of these methods of separating proteins, describe the principle of the method, and tell what property of proteins allows their separation by this technique. (a) ion-exchange chromatography (b) size-exclusion (gel filtration) chromatography (c) affinity chromatography

(a) Ion-exchange chromatography separates proteins on the basis of their charges. (b) Size-exclusion or gel filtration chromatography separates on the basis of size. (c) Affinity chromatography separates proteins with specific, high affinity for some ligand (attached to an inert support) from other proteins with no such affinity. (See Fig. 3-17, p. 87.)

(a) List the types of noncovalent interactions that are important in providing stability to the three-dimensional structures of macromolecules. (b) Why is it important that these interactions be noncovalent, rather than covalent, bonds?

(a) Noncovalent interactions include hydrogen bonds, ionic interactions between charged groups, van der Waals interactions, and hydrophobic interactions. (b) Because noncovalent interactions are weak, they can form, break, and re-form more rapidly and with less energy input than can covalent bonds. This is important to maintain the flexibility needed in macromolecules.

(a) What is optical activity? (b) How did Louis Pasteur arrive at an explanation for the phenomenon of optical activity?

(a) Optical activity is the capacity of a substance to rotate the plane of plane-polarized light. (b) Using fine forceps, he was able to separate the two types of crystals found in tartaric acid (racemic acid) that are identical in shape, but mirror images of each other. One sample rotated polarized light to the left; the mirror image crystals rotated polarized light to the right.

A biochemist is attempting to separate a DNA-binding protein (protein X) from other proteins in a solution. Only three other proteins (A, B, and C) are present. The proteins have the following properties: What type of protein separation techniques might she use to separate: (a) protein X from protein A? (b) protein X from protein B? (c) protein X from protein C?

(a) Size-exclusion (gel filtration) chromatography to separate on the basis of size; (b) ion-exchange chromatography or isoelectric focusing to separate on the basis of charge; (c) specific affinity chromatography, using immobilized DNA.

You are trying to determine the sequence of a protein that you know is pure. Give the most likely explanation for each of the following experimental observations. You may use a simple diagram for your answer.

(a) The protein has some multiple of two subunits, with Ala and Leu as the amino-terminal residues. (b) The protein has two subunits (Mr 35,000 and 45,000), joined by one or more disulfide bonds. (c) The native protein (Mr 160,000) has two Mr 35,000 subunits and two Mr 40,000 subunits.

In one or two sentences, describe the usefulness of each of the following reagents or reactions in the analysis of protein structure: (a) Edman reagent (phenylisothiocyanate) (b) protease (c) reducing agent (dithiothreitol or ?-mercaptoethanol)

(a) Used in determination of the amino acid sequence of a peptide, starting at its amino terminus; (b) used to produce specific peptide fragments from a polypeptide; (c) used to break disulfide bonds or "bridges" or to keep them from forming and to keep Cys residues in their reduced form.

(a) On the reaction coordinate diagram shown below, label the transition state and the overall free-energy change (?G) for the uncatalyzed reaction A ? B. (b) Is this an exergonic or endergonic reaction? (c) Draw a second curve showing the energetics of the reaction if it were enzyme-catalyzed.

(a) and (c) (See Fig. 1-27, p. 27.) (b) exergonic reaction

Name two functions of (a) proteins, (b) nucleic acids, (c) polysaccharides, (d) lipids.

(a) proteins function as enzymes, structural elements, signal carriers, transporters; (b) nucleic acids store and transmit genetic information and act as both structural and catalytic elements; (c) polysaccharides serve as energy-yielding fuel stores and cellular and extracellular structural and recognition elements; (d) lipids function as membrane components, fuel stores, and cellular signals.

All cells are surrounded by a plasma membrane composed of lipid and protein molecules. What is the function of the plasma membrane?

-barrier to the free passage of inorganic ions and most other charged or polar compounds into or out of the cell. -contains proteins that can transport specific ions or molecules. -Other membrane proteins act as receptors that transmit signals from the outside to the inside of the cell.

Most cells of higher plants have a cell wall outside the plasma membrane. What is the function of the cell wall?

-provides a rigid, protective shell for the cell. -allowing water and small molecules to pass readily -resist the swelling of the cell caused by the accumulation of water.

Consider the reaction: A + B ? C + D. If the equilibrium constant for this reaction is a large number (say, 10,000), what do we know about the standard free-energy change (?G'°) for the reaction? Describe the relationship between Keq' and ?G'°.

?G'° = -RT ln Keq'. If Keq' is a large (positive) number, the term -RT ln Keq' (and therefore ?G'°) has a relatively large, negative value.

As more OH- equivalents (base) are added to an amino acid solution, what titration reaction will occur around pH = 9.5?

Around pH = 9.5, the —NH3+ group will be titrated according to the reaction: —NH3+ + OH- --> —NH2 + H2O.

Why are glycine and proline often found within a ? turn?

A ? turn results in a tight 180° reversal in the direction of the polypeptide chain. Glycine is the smallest and thus most flexible amino acid, and proline can readily assume the cis configuration, which facilitates a tight turn.

Provide a brief definition for a polymorphic protein.

A polymorphic protein is one whose amino acid sequence varies among the human population. The variants have little or no differences in the function or activity of the protein.

Explain the difference, if any, between a proteome and a proteasome.

A proteome is the list of all proteins that function in a given cell. A proteasome is a molecular machine or supramolecular structure responsible for protein degradation in a cell.

Severe diarrhea is accompanied by a loss of HCO3-. If untreated, will the condition result in acidosis or alkalosis? Use the bicarbonate buffer system given in the scheme below and Le Chatelier's Principle to explain your answer.

Acidosis. The removal of HCO3- will pull the equilibria in the direction of HCO3-, which will produce H+, thereby lowering the pH.

Suppose you have just added 100 mL of a solution containing 0.5 mol of acetic acid per liter to 400 mL of 0.5 M NaOH. What is the final pH? (The pKa of acetic acid is 4.7.)

Addition of 200 mmol of NaOH (400 mL × 0.5 M) to 50 mmol of acetic acid (100 mL × 0.5 mM) completely titrates the acid so that it can no longer act as a buffer and leaves 150 mmol of NaOH dissolved in 500 mL, an [OH-] of 0.3 M. Given [OH-], [H+] can be calculated from the water constant: [H+][OH-] = 10-14 [H+] = 10-14 M2 / 0.3 M pH is, by definition, log (1/[H+]). pH = log (0.3 M /10-14 M2) = 12.48.

Briefly describe the five major groupings of amino acids.

Amino acids may be categorized by the chemistry of their R groups: (1) nonpolar aliphatics; (2) polar, uncharged; (3) aromatic; (4) positively charged; (5) negatively charged. (See Fig. 3-5, p. 79.)

Why is an asymmetric carbon atom called a chiral center?

An asymmetric carbon has four different substituents attached, and cannot be superimposed on its mirror image—as a right hand cannot fit into a left glove. Thus, a molecule with one chiral carbon will have two stereoisomers, which may be distinguishable from one another in a biological system.

What is meant by endosymbiotic association? How can this concept explain the evolution of eukaryotic cells that are capable of carrying out photosynthesis and/or aerobic metabolism?

An endosymbiotic association is the envelopment of one organism by another to form a relationship that is beneficial to both organisms. It is believed that primitive eukaryotic cells, which were incapable of photosynthesis or aerobic metabolism, formed endosymbiotic associations with photosynthetic and/or aerobic bacteria. The aerobic bacteria then evolved into the mitochondria found in modern eukaryotic cells, and the photosynthetic bacteria evolved into the chloroplasts found in plant cells. (See Fig. 1-36, p. 35.)

Conjugated proteins contain chemical substituents in addition to amino acids. List three classes of conjugated proteins and identify the type of prosthetic group associated with each one.

Any of the following are acceptable answers: Lipoproteins, with lipid groups Glycoproteins, with carbohydrate groups Phosphoproteins, with phosphoryl groups Hemoproteins, with heme groups Flavoproteins, with flavin nucleotide groups Metalloproteins, with metal ions (zinc, iron, calcium, etc.)

Name four factors (bonds or other forces) that contribute to stabilizing the native structure of a protein, and describe one condition or reagent that interferes with each type of stabilizing force.

Any of the following forces stabilize native protein structures and are disrupted by the listed conditions or reagents: (a) disulfide bonds by reducing conditions or mercaptoethanol or dithiothreitol, (b) hydrogen bonds by pH extremes, (c) hydrophobic interactions by detergents or urea or guanidine hydrochloride, (d) ionic interactions by changes in pH or ionic strength, and (e) van der Waals interactions by any unfolding condition.

Draw the structures of the amino acids phenylalanine and aspartate in the ionization state you would expect at pH 7.0. Why is aspartate very soluble in water, whereas phenylalanine is much less soluble?

Aspartate has a polar (hydrophilic) side chain, which forms hydrogen bonds with water. In contrast, phenylalanine has a nonpolar (hydrophobic) side chain. (See Fig. 3-5, p. 79 for structures.)

Phosphoric acid (H3PO4) has three dissociable protons, with the pKa's shown below. Which form of phosphoric acid predominates in a solution at pH 4? Explain your answer.

At pH 4, the first dissociable proton (pKa = 2.14) has been titrated completely, and the second (pKa = 6.86) has just started to be titrated. The dominant form at pH 4 is therefore H2PO4-, the form with one dissociated proton (see Fig. 2-15).

A chemist working in a pharmaceutical lab synthesized a new drug as a racemic mixture. Why is it important that she separate the two enantiomers and test each for its biological activity?

Biomolecules such as receptors for drugs are stereospecific, so each of the two enantiomers of the drug may have very different effects on an organism. One may be beneficial, the other toxic; or one enantiomer may be ineffective and its presence could reduce the efficacy of the other enantiomer.

Explain how circular dichroism spectroscopy could be used to measure the denaturation of a protein.

Circular dichroism spectroscopy measures the amount of ?-helix in a given protein. As the protein denatures, the amount of ?-helix should decrease as the protein chain becomes disordered; this change would be detectable using CD spectrography.

A weak acid HA, has a pKa of 5.0. If 1.0 mol of this acid and 0.1 mol of NaOH were dissolved in one liter of water, what would the final pH be?

Combining 1 mol of weak acid with 0.1 mol of NaOH yields 0.9 mol of weak acid and 0.1 mol of salt. pH = pKa + log = 5.0 + log (0.1/0.9) = 4.05

Differentiate between configuration and conformation.

Configuration denotes the spatial arrangement of the atoms of a molecule that is conferred by the presence of either double bonds, around which there is no freedom of rotation, or chiral centers, which give rise to stereoisomers. Configurational isomers can only be interconverted by temporarily breaking covalent bonds. Conformation refers to the spatial arrangement of substituent groups that, without breaking any bonds, are free to assume different positions in space because of the freedom of bond rotation.

Explain the relationships among the change in the degree of order, the change in entropy, and the change in free energy that occur during a chemical reaction.

Entropy is a measure of disorder. Thus, if there is an increase in order there is a decrease in entropy. The greater the entropy of a system, the smaller its free energy. Thus, an increase in entropy during a reaction will result in a decrease in free energy.

Explain the fact that ethanol (CH3CH2OH) is more soluble in water than is ethane (CH3CH3).

Ethanol can form hydrogen bonds with water molecules, but ethane cannot. When ethanol dissolves, the decrease in the system's entropy that results from formation of ordered arrays of water around the CH3CH2- group is partly compensated by the favorable interactions (hydrogen bonds) of the hydroxyl group of ethanol with water molecules. Ethane cannot form such hydrogen bonds.

What is meant by feedback inhibition and why is it important in a living organism?

Feedback inhibition is the regulation of a biochemical pathway in which a reaction product inhibits an earlier (usually the first) step in the pathway. It is an important type of regulation because it ensures that energy is not wasted by an organism producing molecules it does not need.

You are given a solution containing an enzyme that converts B into A. Describe what you would do to determine the specific activity of this enzyme solution.

First, add a known volume of the enzyme solution (say, 0.01 mL) to a solution of its substrate B and measure the initial rate at which product A is formed, expressed as ?mol/mL of enzyme solution/min. Then measure the total protein concentration, expressed as mg/mL. Finally, divide the enzyme activity (?mol/min/mL) by the protein concentration (mg/mL); the quotient is the specific activity.

If the average molecular weight of the 20 standard amino acids is 138, why do biochemists divide a protein's molecular weight by 110 to estimate its number of amino acid residues?

For each peptide bond formed, a molecule of water is lost, bringing the average molecular weight down to 120. To reflect the preponderance of low-molecular-weight amino acids, the average molecular weight is lowered further to 110.

Lys residues make up 10.5% of the weight of ribonuclease. The ribonuclease molecule contains 10 Lys residues. Calculate the molecular weight of ribonuclease.

From the structure of lysine, we can calculate its molecular weight (146); when it condenses (loses H2O, Mr = 18) to form a peptide bond, the resulting residue contributes 146 - 18 = 128 to the protein's molecular weight. If 10 Lys residues contribute 10.5% of the protein's molecular weight, each Lys residue is 1.05%. To calculate the total molecular weight, divide 128 by 1.05% (0.0105); the result is 12,190. (The actual value is 13,700.)

Hereditary transmission of genetic information can be viewed as a balance between stability and change. Explain.

Hereditary transmission of genetic information occurs via replication of DNA, the information-containing molecule. This process is very accurate and thus results in relatively few changes in genetic information. This stability is important to maintain individual and species characteristics over long periods of time. On the other hand, regular changes in genetic information (mutations) do occur, primarily as a result of infrequent errors in replication. These mutations are essential for generating genetic diversity, which allows for adaptation of species.

Distinguish between homologs, paralogs, and orthologs as classes of related proteins.

Homologs are any members of a particular protein family, paralogs are two homologs present in the same species, and orthologs are are two homologs present in different species.

Draw the hydrogen bonding typically found between two residues in an ? helix.

Hydrogen bonds occur between every carbonyl oxygen in the polypeptide backbone and the peptide —NH of the fourth amino acid residue toward the amino terminus of the chain. (See Fig. 4-2, p. 116.)

If ice were denser than water, how would that affect life on earth?

Ice that formed at the surface of bodies of water would sink; hence, streams, ponds, lakes, and so on would freeze from the bottom up. With a reservoir of ice at the bottom, they would be perpetually cold, and in the limit they would freeze solid, precluding life as we know it.

How does the shape of a titration curve confirm the fact that the pH region of greatest buffering power for an amino acid solution is around its pK's?

In a certain range around the pKa's of an amino acid, the titration curve levels off. This indicates that for a solution with pH ? pK, any given addition of base or acid equivalents will result in the smallest change in pH—which is the definition of a buffer.

Describe three of the important features of a ? sheet polypeptide structure. Provide one or two sentences for each feature.

In the ? sheet structure, several extended polypeptides, or two regions of the same polypeptide, lie side by side and are stabilized by hydrogen bonding between adjacent chains. Adjacent chains may be either parallel (with a repeat distance of about 6.5 Å) or antiparallel (7 Å repeat). The R groups are often small and alternately protrude from opposite faces of the ? sheet.

How can isoelectric focusing be used in conjunction with SDS gel electrophoresis?

Isoelectric focusing can separate proteins of the same molecular weight on the basis of differing isoelectric points. SDS gel electrophoresis can then separate proteins with the same isoelectric points on the basis of differing molecular weights. When combined in two-dimensional electrophoresis, a great resolution of large numbers of proteins can be achieved.

When a polypeptide is in its native conformation, there are weak interactions between its R groups. However, when it is denatured there are similar interactions between the protein groups and water. What then accounts for the greater stability of the native conformation?

In the unfolded polypeptide, there are ordered solvation shells of water around the protein groups. The number of water molecules involved in such ordered shells is reduced when the protein folds, resulting in higher entropy. Hence, the lower free energy of the native conformation.

Describe the "RNA world" hypothesis.

Initially, RNA molecules were both genes and catalysts. Self-replication of these molecules over long periods of time produced variants that were able to catalyze polymerization of amino acids to form peptides that assumed the function of catalysts. Eventually, genomic RNA was copied into DNA, which assumed the function of genetic information storage.

Explain why living organisms are able to produce particular chiral forms of different biomolecules while laboratory chemical synthesis usually produces a racemic mixture.

Laboratory syntheses usually use achiral reagents and thus produce racemic mixtures of products. In contrast, because all enzymes are made of chiral precursors, all enzymes are inherently chiral catalysts. Thus, they will show strong stereoselectivity in reactants and mechanisms, leading to the production of chiral products.

What six characteristics distinguish living organisms from inanimate objects?

Living organisms (1) are chemically complex and highly organized; (2) extract, transform, and use energy from their environment; (3) have the capacity to precisely self-replicate and self-assemble; (4) exploit a chemical interplay with their environment; (5) possess programmatically defined functions; (6) evolve to new forms over many generations.

Describe the relationship between a living organism and its surroundings in terms of both matter and energy.

Living organisms are open systems and exchange both matter and energy with their surroundings. They are not at equilibrium with their surroundings; that is, the concentrations of molecules inside the cells of the organism are not the same as their concentrations in the surroundings. To maintain this situation, the organism must acquire energy from its surroundings, either in the form of chemical energy or directly from sunlight.

What is the underlying, organizing biochemical principle that results in the chemical similarity of virtually all living things? Given this biochemical similarity, how is the structural and functional diversity of living things possible?

Living things are composed primarily of macromolecules, polymers of simple compounds of just a few different types. The properties of these polymers are determined by their sequence of monomers and these can be combined in many different ways. Diversity is thus achieved through the nearly limitless variety of sequences that can exist when amino acids are linked to form proteins, nucleotides are linked to form nucleic acids, and monosaccharides are linked to form polysaccharides. Branching in the latter can contribute additional heterogeneity. Each type of organism constructs a unique set of macromolecules from these monomeric units, resulting in the structural and functional diversity among species.

Explain with an appropriate diagram why amphipathic molecules tend to form micelles in water. What force drives micelle formation?

Micelle formation minimizes the area of the hydrophobic part of amphipathic molecules that contacts the polar solvent, water. Hydrophobic interactions between hydrophobic moieties are the driving force for micelle formation. When amphipathic molecules form micelles in water, the entropy decrease due to the formation of ordered arrays of water molecules around the hydrophobic moieties is minimized. (See Fig. 2-7, p. 48.)

Describe Stanley Miller's experiment (1953) and its relevance.

Miller subjected a gaseous mixture of ammonia, methane, water vapor, and hydrogen to electrical sparks for periods of a week or more. When he analyzed the contents of the closed reaction vessel, the gas phase contained CO and CO2, as well as unreacted starting materials. The water phase contained a variety of organic compounds, including some amino acids, hydroxy acids, aldehydes, and hydrogen cyanide. This experiment established the possibility of abiotic production of biomolecules in relatively short times under relatively mild conditions.

Why do amino acids, when dissolved in water, become zwitterions?

Near pH = 7, the carboxylic acid group (—COOH) will dissociate to become a negatively charged —COO- group, and the —NH2 amino group will attract a proton to become a positively charged —NH3+ group.

What is the uniquely important acid-base characteristic of the histidine R group?

Only the imidazole ring of the histidine R group has a pKa near physiological pH (pKa = 6.0), which suggests that histidine may provide buffering power in intercellular and intracellular fluids.

Hydrolysis of peptide bonds is an exergonic reaction. Why, then, are peptide bonds quite stable?

Peptide bonds are stable because hydrolysis of peptide (or amide) bonds has a high activation energy and as a result occurs very slowly.

The artificial sweetener NutraSweet®, also called aspartame, is a simple dipeptide, aspartylphenylalanine methyl ester, on which the free carboxyl of the dipeptide is esterified to methyl alcohol. Draw the structure of aspartame, showing the ionizable groups in the form they have at pH 7. (The ionizable group in the side chain of aspartate has a pKa of 3.96.)

See the structure on p. 83.

Leucine has two dissociable protons: one with a pKa of 2.3, the other with a pKa of 9.7. Sketch a properly labeled titration curve for leucine titrated with NaOH; indicate where the pH = pK and the region(s) in which buffering occurs.

See the titration curve for glycine in Fig. 3-10, p. 79.

Instant cold packs get cold when the contents, usually solid urea and liquid water, are mixed, producing an aqueous solution of urea. Although this process is clearly spontaneous, the products are colder than the reactants. Explain how this is possible in terms of the difference between ?G and ?H.

Since the dissolution reaction is spontaneous, the ?G must be negative. Since the reaction absorbs heat, the ?H must be positive. Given ?G = ?H - T?S, this is possible if the ?S is very large and positive, as one would expect for a solid dissolving.

A polypeptide is hydrolyzed, and it is determined that there are 3 Lys residues and 2 Arg residues (as well as other residues). How many peptide fragments can be expected when the native polypeptide is incubated with the proteolytic enzyme trypsin?

Six fragments would be expected, unless the carboxyl-terminal residue is Lys or Arg; in which case there would be five.

Provide a brief explanation for the statement "Soluble globular proteins can be distinguished from soluble intrinsically disordered proteins on the basis of their amino acid content."

Soluble globular proteins contain significant percentages of hydrophobic amino acids that are usually clustered to facilitate packing into the core of the structure. Intrinsically disordered proteins are enriched in charged amino acids and small amino acids such as Gly and Ala.

Name two uncommon amino acids that occur in proteins. By what route do they get into proteins?

Some examples are 4-hydroxyproline, 5-hydroxylysine, ?-carboxyglutamate, N-methyllysine, desmosine, and selenocysteine. Uncommon amino acids in proteins (other than selenocysteine) usually result from chemical modifications of standard amino acid R groups after a protein has been synthesized.

Discuss how a mutation in DNA could be harmful or beneficial to an organism.

Some mutations lead to the synthesis of an inactive or defective enzyme or other protein that can no longer carry out its proper function, which is thus harmful to the organism. However, other mutations may lead to a more stable enzyme or to a protein that is better able to carry out its function in a particular environment, making it beneficial to the organism.

As a protein is purified, both the amount of total protein and the activity of the purified protein decrease. Why, then, does the specific activity of the purified protein increase?

Specific activity is the units of enzyme activity (?mol of product/min) divided by the amount of protein (mg). As the protein is purified, some of it is lost in each step, resulting in a drop in activity. However, other contaminating proteins are lost to a much greater extent. Therefore, with each purification step, the purified protein constitutes a greater proportion of the total, resulting in an increase in specific activity. (See also Table 3-5, p. 88.)

How are "signature sequences" useful in analyzing groups of functionally related proteins?

Such sequences are often present in one taxonomic group or shared by closely related taxonomic groups but are absent in evolutionarily more distant groups. They thus aid in constructing more elaborate evolutionary trees based on protein sequences.

Describe three of the important features of the ?-helical polypeptide structure predicted by Pauling and Corey. Provide one or two sentences for each feature.

The ?-helical structure of a polypeptide is tightly wound around a long central axis; each turn of the right-handed helix contains 3.6 residues and stretches 5.4 � along the axis. The peptide NH is hydrogen-bonded to the carbonyl oxygen of the fourth amino acid along the sequence toward the amino terminus. The R groups of the amino acid residues protrude outward from the helical backbone.

Pauling and Corey showed that in small peptides, six atoms associated with the peptide bond all lie in a plane. Draw a dipeptide of two amino acids in trans linkage (side-chains can be shown as —R), and indicate which six atoms are part of the planar structure of the peptide bond.

The N and H of the amino and the C and O of the carbonyl are all in the same plane with the two C? atoms, which are diagonally opposite relative to the C—N bond. (See Fig. 4-2, p. 119.)

Only one of the common amino acids has no free ?-amino group. Name this amino acid and draw its structure.

The amino acid L-proline has no free ?-amino group, but rather has an imino group formed by cyclization of the R-group aliphatic chain with the amino group (see Fig. 3-5, p. 79).

Why do smaller molecules elute after large molecules when a mixture of proteins is passed through a size-exclusion (gel filtration) column?

The column matrix is composed of cross-linked polymers with pores of selected sizes. Smaller molecules can enter pores in the polymer beads from which larger molecules would be excluded. Smaller molecules therefore have a larger three-dimensional space in which to diffuse, making their path through the column longer. Larger molecules migrate faster because they pass directly through the column, unhindered by the bead pores.

What is the difference, if any, between cytosol and cytoplasm?

The cytoplasm is the internal volume enclosed by the plasma membrane; the cytosol is the aqueous portion of the cytoplasm.

Provide a brief explanation for the observation that macromolecules diffuse at a slower rate in the cytosol than they do in dilute solution.

The cytosol is very crowded and gel-like. The diffusion of macromolecules is slowed by collisions with other large molecules and structures.

The free-energy change for the formation of a protein from the individual amino acids is positive and is thus an endergonic reaction. How, then, do cells accomplish this process?

The endergonic (thermodynamically unfavorable) reaction is coupled to an exergonic (thermodynamically favorable) reaction through a shared intermediate, so that the overall free-energy change of the coupled reactions is negative (the overall reaction is exergonic).

How is the genetic information encoded in DNA and how is a new copy of DNA synthesized?

The genetic information is encoded in the linear sequence (order) of the four different deoxyribonucleotides in the DNA. When a new copy of DNA is needed, the two strands of the DNA unwind and each strand serves as a template on which a new strand is synthesized.

Give the general Henderson-Hasselbalch equation and sketch the plot it describes (pH against amount of NaOH added to a weak acid). On your curve, label the pKa for the weak acid and indicate the region in which the buffering capacity of the system is greatest.

The inflection point, which occurs when the weak acid has been exactly one half titrated with NaOH, occurs at a pH equal to the pKa of the weak acid. The region of greatest buffering capacity (where the titration curve is flattest) occurs at pH values of pKa ±1. (See Fig. 2-17, p. 59.)

Draw the resonance structure of a peptide bond, and explain why there is no rotation around the C—N bond.

The intermediate resonance structure imparts a partial double bond characteristic to the C—N bond, thereby prohibiting rotation. (See Fig. 4-2, p. 116.)

What is the pI, and how is it determined for amino acids that have nonionizable R groups?

The pI is the isoelectric point. It occurs at a characteristic pH when a molecule has an equal number of positive and negative charges, or no net charge. For amino acids with nonionizable R groups, pI is the arithmetic mean of a molecule's two pKa values: pI = 1/2 (pK1 + pK2)

Draw the structure of Gly-Ala-Glu in the ionic form that predominates at pH 7.

The peptide must have an amino-terminal Gly residue, a carboxyl-terminal Glu residue, and ionized amino and carboxyl groups.

Draw the titration curve for a weak acid, HA, whose pKa is 3.2. Label the axes properly. Indicate with an arrow where on the curve the ratio of salt (A-) to acid (HA) is 3:1. What is the pH at this point?

The plot of pH versus added base should have the general shape of those shown in Fig. 2-17, p. 59, with the midpoint of the titration (inflection point) at pH 3.2. The ratio of A- to HA is 3 when 0.75 equivalents of base have been added. From the Henderson-Hasselbalch equation, the pH at this point can be calculated: pH = pKa + log = 3.2 + log 3 = 3.2 + 0.48 = 3.68

Explain the fact that triethylammonium chloride ((CH3CH2)3N-HCl) is more soluble in water than is triethylamine ((CH3CH2)3N).

The positive charge on the N atom in triethylammonium chloride is more polar than the uncharged N atom in triethylamine. This increased polarity leads to stronger interactions with water, leading to increased solubility.

In the amino acid glycine, what effect does the positively charged —NH3+ group have on the pKa of an amino acid's —COOH group?

The positively charged amino group stabilizes the negatively charged ionized form of the carboxyl group, —COO-, and repels the departing H+ thereby promoting deprotonation. The effect is to lower the pKa of the carboxyl group. (See Fig. 3-11, p. 80.)

Define the primary structure of a protein.

The primary structure of a protein is its unique sequence of amino acids and any disulfide bridges present in the native structure, that is, its covalent bond structure.

Proteins are constantly being synthesized in a living cell. Why doesn't the number of protein molecules become too great for the cell to contain, leading to cell destruction?

The proteins in a cell are continuously being synthesized and degraded. The cell maintains a dynamic steady state in which the amount of each protein remains fairly constant at the level required under given conditions.

Describe how the rise of O2-producing bacteria might have led to the eventual predominance of aerobic organisms on earth.

The rise of O2-producing bacteria would result in an increase in the levels of O2 in the earth's atmosphere. This would give a selective advantage to aerobic organisms (which utilized O2 as electron acceptor) over anaerobic organisms for which O2 was toxic.

In superhelical proteins, such as collagen, several polypeptide helices are intertwined. What is the function of this superhelical twisting?

The superhelical twisting of multiple polypeptide helices makes the overall structure more compact and increases its overall strength.

Any given protein is characterized by a unique amino acid sequence (primary structure) and three-dimensional (tertiary) structure. How are these related?

The three-dimensional structure is determined by the amino-acid sequence. This means that the amino-acid sequence contains all of the information that is required for the polypeptide chain to fold up into a discrete three-dimensional shape.

Describe two major differences between chemical synthesis of polypeptides and synthesis of polypeptides in the living cell.

There are many such differences; here are a few. (1) Chemical synthesis proceeds from carboxyl terminus to amino terminus; in the living cell, the process starts at the amino terminus and ends at the carboxyl terminus. In the living cell, synthesis occurs under physiological conditions; chemical synthesis does not. Chemical synthesis is only capable of synthesizing short polypeptides; cells can produce proteins of several thousand amino acids.

How can a polypeptide have only one free amino group and one free carboxyl group?

This is possible only if the peptide has no side chains with carboxyl or amino groups. Then, with the exception of the single amino-terminal amino acid and the single carboxyl-terminal amino acid, all the other ?-amino and carboxyl groups are covalently condensed into peptide bonds.

In proteins, the amino acid histidine (His) plays an important role in many biological reactions. The pKa for the protonation of His to form HisH+ = 6.0. When pH = 7.0, what is the fraction of total histidine that will be in the HisH+ form?

Use the Henderson-Hasselbalch equation to determine the ratio of [His] to [HisH+]. pH = pKa + log ([His]/ [HisH+]) 7.0 = 6.0 + log ([His]/ [HisH+]) 1.0 = log ([His]/ [HisH+]) [His]/ [HisH+] = antilog (1) = 10 To determine the fraction of the total in the HisH+ form, [His]total = [His] + [HisH+], fraction = [HisH+]/[His]total= [HisH+]/([His] + [HisH+]) substitute from ratio calculated above = [HisH+]/(10[HisH+] + [HisH+])= 1/11, or 0.09

What factors would make it difficult to interpret the results of a gel electrophoresis of proteins in the absence of sodium dodecyl sulfate (SDS)?

Without SDS, protein migration through a gel would be influenced by the protein's intrinsic net charge—which could be positive or negative—and its unique three-dimensional shape, in addition to its molecular weight. Thus, it would be difficult to ascertain the difference between proteins based upon a comparison of their mobilities in gel electrophoresis.

What are the structural characteristics common to all amino acids found in naturally occurring proteins?

alpha carbon to which are attached a carboxylic acid, an amine, a hydrogen, and a variable side chain. All the amino acids are also in the L configuration.

The following reagents are often used in protein chemistry. Match the reagent with the purpose for which it is best suited. Some answers may be used more than once or not at all; more than one reagent may be suitable for a given purpose. ___ hydrolysis of peptide bonds on the carboxyl side of Lys and Arg ___ cleavage of peptide bonds on the carboxyl side of Met ___ breakage of disulfide (—S—S—) bonds ___ modification of sulfhydryl groups of Cys ___ determination of the amino acid sequence of a peptide ___ determining the amino-terminal amino acid in a polypeptide

g; a; d and e; h; b; c

The amino acid histidine has a side chain for which the pKa is 6.0. Calculate what fraction of the histidine side chains will carry a positive charge at pH 5.4. Be sure to show your work.

pH = pKa + log pKa - pH = log antilog (pKa - pH) = antilog (6.0 - 5.4) = 4 = [acid]/[conjugate base], or 4[conjugate base] = [acid] Therefore, at pH 5.4, 4/5 (80%) of the histidine will be in the protonated form.

What is the pH of a solution containing 0.2 M acetic acid (pKa = 4.7) and 0.1 M sodium acetate?

pH = pKa + log = 4.7 + log (0.1/0.2) = 4.7 - 0.3 = 4.4

You have just made a solution by combining 50 mL of a 0.1 M sodium acetate solution with 150 mL of 1 M acetic acid (pKa = 4.7). What is the pH of the resulting solution?

pH = pKa + log = 4.7 + log (5/150) = 4.7 - 1.48 = 3.22