CH 306 TEST 2

Question 1

Why is an assay required for protein purification?

Answer 1

An assay, which should be based on some unique biochemical property of the protein that is being purified, allows the detection of the protein of interest.

Question 2

What physical differences among proteins allow for

their purification?

Answer 2

Differences in size, solubility, charge, and the specific binding of certain molecules

Question 3

What are the two properties of enzymes that make them especially useful catalysts?

Answer 3

Rate enhancement and substrate specificity

Question 4

What are the general characteristics of enzyme active sites?

Answer 4

The active site is a three-dimensional crevice or cleft; it makes up only a small part of the total volume of the enzyme. Active sites have unique microenvironments. A substrate binds to the active site with multiple weak interactions. The specificity of the active site depends on the active site’s precise three-dimensional structure.

Question 5

What does an apoenzyme require to become a holoenzyme?

Answer 5

A cofactor

Question 6

What are the two main types of cofactors?

Answer 6

Coenzymes and metals

Question 7

Why are vitamins necessary for good health?

Answer 7

Vitamins are converted into coenzymes that are required for most biochemical reactions.

Question 8

What is the fundamental mechanism by

which enzymes enhance the rate of chemical reactions?

Answer 8

Enzymes facilitate the formation of the transition state.

Question 9

What is the structural basis for

enzyme specificity?

Answer 9

The intricate three-dimensional structure of proteins allows the construction of active sites that will recognize only specific substrates.

Question 10

What is meant by the term binding

energy?

Answer 10

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

Question 11

What is the role of binding energy in enzyme catalysis?

Answer 11

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.

Question 12

What would be the result of an enzyme having a greater binding energy for the substrate than for the transition state?

Answer 12

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.

Question 13

Protein catalyst

Answer 13

Enzyme

Question 14

Reactant in an enzyme-catalyzed reaction

Answer 14

Substrate

Question 15

A coenzyme or metal

Answer 15

Cofactor

Question 16

Enzyme minus its cofactor

Answer 16

Apoenzyme

Question 17

Enzyme plus cofactor

Answer 17

Holoenzyme

Question 18

Small vitamin-derived organic cofactors

Answer 18

Coenzymes

Question 19

Function of K′eq

Answer 19

ΔG°′

Question 20

The least-stable reaction intermediate

Answer 20

Transition state

Question 21

Site on the enzyme where catalysis takes place

Answer 21

Active site

Question 22

Change in enzyme structure

Answer 22

Induced fit

Question 23

Change in enzyme structure

Answer 23

Induced fit

Question 24

Why does the activation energy of a reaction not appear in the final ΔG of the reaction?

Answer 24

The energy required to reach the transition state (the activation energy) is returned when the transition state proceeds to product.

Question 25

Proteins are thermodynamically unstable. The ΔG 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?

Answer 25

Protein hydrolysis has a large activation energy. Protein synthesis requires energy to proceed.

Question 26

Suggest why the enzyme lysozyme, which degrades cell walls of some bacteria, is present in tears.

Answer 26

Lysozyme helps protect the fluid that surrounds eyes from bacterial infection.

Question 27

Transition-state analogs, which can be used as enzyme inhibitors and to generate catalytic antibodies, are often difficult to synthesize. Suggest a reason.

Answer 27

Transition states are very unstable. Consequently, molecules that resemble transition states are themselves likely to be unstable and, hence, difficult to synthesize.

Question 28

Suppose that a mutant enzyme binds a substrate 100 times as tightly as does the native enzyme. What is the effect of this mutation on catalytic rate if the binding of the transition state is unaffected?

Answer 28

The mutation slows the reaction by a factor of 100. The activation-free energy is increased by 11.4 kJ mol−1 (2.73 kcal mol−1). Strong binding of the substrate relative to the transition state slows catalysis.

Question 29

Separating proteins on the basis of size differences

Answer 29

Molecular exclusion chromatography

Question 30

Allows high resolution and rapid separation

Answer 30

High-pressure liquid chromatography (HPLC)

Question 31

Produced by hybridoma cells

Answer 31

Monoclonal antibodies

Question 32

An immunoassay technique preceded by gel electrophoresis

Answer 32

Western blotting

Question 33

A measure of the rate of movement due to centrifugal force

Answer 33

Sedimentation coefficient

Question 34

Separating proteins on the basis of net charge

Answer 34

Ion-exchange chromatography

Question 35

Specific site recognized by an antibody

Answer 35

Antigenic determinant (epitope)

Question 36

Based on the fact that proteins have a pH at which the net charge is zero

Answer 36

Isoelectric focusing

Question 37

Based on attraction to a specific chemical group or molecule

Answer 37

Affinity chromatography

Question 38

A means of identifying a protein based on a unique property of the protein

Answer 38

Assay

Question 39

Why do proteins precipitate at high salt concentrations?

Answer 39

If the salt concentration becomes too high, the salt ions interact with the water molecules. Eventually, there are not enough water molecules to interact with the protein, and the protein precipitates.

Question 40

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

Answer 40

If there is a 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, it neutralizes the charges on the proteins, preventing protein–protein interactions.

Question 41

What types of R groups would compete with salt ions for water of solvation?

Answer 41

Charged and polar R groups on the surface of an enzyme

Question 42

(a) The octapeptide AVGWRVKS was digested with the enzyme trypsin. Would ion-exchange or molecular exclusion chromatography be most appropriate for separating the products? Explain.
(b) Suppose that the peptide had, instead, been digested with chymotrypsin. What would be the optimal separation technique? Explain.

Answer 42

(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) Chymotrypsin, which cleaves a er 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.

Question 43

The detergent sodium dodecyl sulfate (SDS) denatures proteins. Suggest how SDS destroys protein structure.

Answer 43

The long hydrophobic tail on the SDS molecule (p. 73) 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.

Question 44

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.

Answer 44

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.