Chapter 8 Homework

Isomerases

Catalyze intramolecular rearrangements

Oxidoreductases

Catalyze redox chemistry

Ligases

Catalyze the joining of two molecules together

k cat/K M

Enzyme efficiency

K M

Substrate concentration at 0.5 V max

A substrate binds to an enzyme at the ________, where the reaction occurs.

active site

At 37 ∘C, the serine protease subtilisin has kcat = 50 s−1 and KM=1.4×10−4M. It is proposed that the N155 side chain contributes a hydrogen bond to the oxyanion hole of subtilisin. J. A. Wells and colleagues reported (1986, Phil. Trans. R. Soc. Lond. A 317:415-423) the following kinetic parameters for the N155T mutant of subtilisin: kcat = 0.02 s−1 and KM=2×10−4M. Assuming that the T155 side chain cannot H-bond to the oxyanion intermediate, by how much (in kJ/mol) does N155 appear to stabilize the transition state at 37 ∘C?

20 kJ/mol

Considering both of the mechanisms proposed for catalysis by lysozyme, which of the following rate-enhancing features does lysozyme use to increase the rate of the hydrolysis reaction it catalyzes? 1) General acid-base catalysis 2) Electrostatic catalysis 3) Distortion of substrate 4) Covalent catalysis

All of them

Ammonia, NH3, is used in numerous industrial processes, including the production of pharmaceuticals such as sulfonamide and antimalarials and vitamins such as the B vitamins. The equilibrium equation for the synthesis of ammonia (sometimes known as the Haber process) is N2(g)+3H2(g)⇌2NH3(g) What will happen to the rates of the forward and reverse reaction when a catalyst is added?

Both forward and reverse rates increase. The Haber process can be cheaply catalyzed using porous iron. A much more effective catalyst for the Haber process is osmium; however, it is very expensive and toxic.

reaction rate vs substrate concentration

In region C of the graph, the reaction is independent of substrate concentration. The reaction rate will be constant in this region.

Ammonia, NH3, is used in numerous industrial processes, including the production of pharmaceuticals such as sulfonamide and antimalarials and vitamins such as the B vitamins. The equilibrium equation for the synthesis of ammonia (sometimes known as the Haber process) is N2(g)+3H2(g)⇌2NH3(g) Which of the following would increase the rate of the reverse reaction?

Increasing the concentration of ammonia. The concentration of NH3 affects how quickly N2 and H2 can be made.

In other words, would such a protein actually be a catalyst?

No

Would you expect an "enzyme" designed to bind to its target substrate as tightly as it binds the reaction transition state to show a rate enhancement over the uncatalyzed reaction?

No

k cat

Number of saturated molecules turned over by enzyme

An enzyme contains an active site aspartic acid with a pKa = 5.0, which acts as a general acid catalyst. Choose the curve of enzyme activity (reaction rate) versus pH for the enzyme (assume the protein is stably folded between pH 2-12 and that the active site Asp is the only ionizable residue involved in catalysis).

Similar to pepsin graph

At 37 ∘C, the serine protease subtilisin has kcat = 50 s−1 and KM=1.4×10−4M. It is proposed that the N155 side chain contributes a hydrogen bond to the oxyanion hole of subtilisin. J. A. Wells and colleagues reported (1986, Phil. Trans. R. Soc. Lond. A 317:415-423) the following kinetic parameters for the N155T mutant of subtilisin: kcat = 0.02 s−1 and KM=2×10−4M. Substilisin is used in some laundry detergents to help remove protein-type stains. What unusual kind of stability does this suggest for subtilisin?

The enzyme must be stable both to the presence of detergents and to moderately high temperatures.

ΔG‡

The higher point on the energy curve. Activation Energy

A ________, such as a vitamin, binds to an enzyme and plays a role in catalysis.

cofactor A cofactor, such as an inorganic ion or vitamin, may bind to the enzyme and assist in catalyzing the reaction.

When properly aligned, the enzyme and substrate form an enzyme-substrate (ES)_________.

complex

An enzyme is ________ when it loses its native conformation and its biological activity.

denatured The reaction environment must be appropriate for catalysis to proceed. An enzyme will denature, or change its shape and lose its biological activity, at too high a temperature or at a pH outside the enzyme's optimal range.

In some reactions, in which a protein molecule is binding to a specific site on DNA, a rate greater than that predicted by the diffusion limit is observed. Suggest an explanation. [Hint: The protein molecule can also bind weakly and nonspecifically to any DNA site.] If the protein can bind to a random location on the DNA and slide along until it finds the specific site, a more efficient __________ search replaces an ___________ less favorable ___________ search.

one-dimensional, entropically, three-dimensional

An enzyme is considered ______ because of its ability to recognize the shape of a particular molecule.

specific A substrate binds at an enzyme's active site; the enzyme typically recognizes the specific shape of its substrate.

Consider the following reaction: A-->B Which of the following statements about this reaction are correct? 1) If you double the concentration of reagent A, you will double the rate. 2) The reaction rate (or velocity) is dependent only on the concentration of A. 3) The units for the first-order rate constant are Ms^-1. 4) A plot of the log[A] versus time yields an inverse hyperbola.

1) If you double the concentration of reagent A, you will double the rate. 2) The reaction rate (or velocity) is dependent only on the concentration of A.

The enzyme urease catalyzes the hydrolysis of urea to ammonia plus carbon dioxide. At 21 ∘C the uncatalyzed reaction has an activation energy of about 125 kJ/mol, whereas in the presence of urease the activation energy is lowered to about 46 kJ/mol. By what factor does urease increase the velocity of the reaction?

1.09 x 10^14

Which of the following statements about the various methods of enzyme inhibition is NOT true? 1) Irreversible inhibitors, primarily because they covalently modify an enzyme, are often toxic. 2) Competitive inhibitors bind at a different site than substrate, altering the ability of the enzyme to bind its native target. 3) Noncompetitive inhibitors bind at a different site than substrate, altering the ability of the enzyme to bind its native target. 4) Uncompetitive inhibitors bind at a different site than substrate but only after enzyme has bound its native target.

2) Competitive inhibitors bind at a different site than substrate, altering the ability of the enzyme to bind its native target.

Which of the following statements about the oxyanion hole is NOT true? 1) It represents an enthalpic interaction that helps stabilize the transition state. 2) It represents an electrostatic interaction that leads to a lower energy state. 3) This preformed loop is properly positioned to stabilize the negatively charged tetrahedral intermediate involved in amid bond hydrolysis. 4) The backbone NH bonds of S195 and G193 create H-bonding interactions with the developing negative charge as S195 attacks the scissile carbon bond.

3) This preformed loop is properly positioned to stabilize the negatively charged tetrahedral intermediate involved in amid bond hydrolysis.

Arrange the events for the hydrolysis of amid bonds by chymotrypsin in their correct order. 1) attack by Ser 195 to give a tetrahedral intermediate 2) enzyme returns to initial state 3) protonation by His 57 and then cleavage of the C-N bond release the C-terminal fragment 4) protonation by His 57 and then cleavage of the C-O bond release the N-terminal fragment 5) attack by water, leading to formation of a tetrahedral intermediate 6) binding of substrate to properly position the scissile bond for cleavage

6, 1, 3, 5, 4, 2 6) Binding of substrate to properly position the scissile bond for cleavage. 1) Attack by Ser 195 to give a tetrahedral intermediate. 3) Protonation by His 57 and then cleavage of the C-N bond release the C-terminal fragment. 5) Attack by water, leading to formation of a tetrahedral intermediate. 4) Protonation by His 57 and then cleavage of the C-O bond release the N-terminal fragment. 2) Enzyme returns to initial state.

Which of the following describes a mechanism that enzymes use to achieve their rate enhancement of reactions? 1) Preferential binding to the transition state through complementary non-covalent interactions. 2) Altering the reaction pathway to include intermediate states. 3) Distortion of the substrate or active site, which, in effect, lowers the energy of activation. 4) All of the listed choices describe mechanisms used by enzymes to achieve their rate enhancement of reactions.

All

Activation energy with enzymes

Enzymes lower the activation energy of a chemical reaction. This means that a catalyzed reaction is more likely to proceed than an uncatalyzed reaction, and it forms products more rapidly than an uncatalyzed reaction.

At 37 ∘C, the serine protease subtilisin has kcat = 50 s−1 and KM=1.4×10−4M. It is proposed that the N155 side chain contributes a hydrogen bond to the oxyanion hole of subtilisin. J. A. Wells and colleagues reported (1986, Phil. Trans. R. Soc. Lond. A 317:415-423) the following kinetic parameters for the N155T mutant of subtilisin: kcat = 0.02 s−1 and KM=2×10−4M. Is the effect of the N155T mutation what you would expect for a residue that makes up part of the oxyanion hole? How do the reported values of Kcat and KM support your answer?

For a mutation of a residue that only interacts with the oxyanion intermediate, one would expect Km to change significantly Kcat should be reduced due to the loss of enthalpic stabilization of the transition state The oxyanion is formed after S binds

ΔG°

Free energy of the reaction

At 37 ∘C, the serine protease subtilisin has kcat = 50 s−1 and KM=1.4×10−4M. It is proposed that the N155 side chain contributes a hydrogen bond to the oxyanion hole of subtilisin. J. A. Wells and colleagues reported (1986, Phil. Trans. R. Soc. Lond. A 317:415-423) the following kinetic parameters for the N155T mutant of subtilisin: kcat = 0.02 s−1 and KM=2×10−4M. Subtilisin does have a problem in that it becomes inactivated by oxidation of a methionine close to the active site. Suggest a way to make a better subtilisin. Replace the methionin, by site-directed mutagenesis, with another residue. Because mehtionine is quite __________, a ___________ replacement would seem appropriate. A single base change in the Met codon could yield Phe, Leu, Ile, or _____.

Hydrophobic, hydrophobic, Val

Consider a situation in which the enzyme is operating at optimum temperature and pH, and has been saturated with substrate. What is your best option for increasing the rate of reaction? 1) increase pH 2) increase temperature 3) increase enzyme concentration 4) increase substrate concentration

Increase enzyme concentration If an enzyme is saturated with substrate, and it is operating at optimum pH and optimum temperature, there is very little that can be done except to increase the enzyme concentration. Some enzymes can be activated further by allosteric activators, in which case one might add some activator to the reaction. But otherwise, increasing the enzyme concentration is the only option.

Classify each of the characteristics as lock-and-key model, induced-fit model, or both. Active site is rigid Active site-substrate interaction induces an optimal fit for catalysis Active site is flexible Substrate shape is modified Enzyme returns to its initial state after catalysis Enzyme is substrate specific

Lock-and-key model Active site is rigid Induced-fit model Active site-substrate interaction induces an optimal fit for catalysis Active site is flexible Substrate shape is modified Both Enzyme returns to its initial state after catalysis Enzyme is substrate specific All enzymes are substrate specific, and like all catalysts, they return to their original shape when the reaction is complete. However, the method by which an enzyme works is what varies between the lock-and-key and induced-fit models. The lock-and-key model explains the mechanism as one involving two rigid structures, and the induced-fit model explains the mechanism as one involving two flexible species that form around each other as they interact.

Review - Enzymes are a type of protein, and they act as biocatalysts to carry out thousands of reactions in our bodies. Enzymes increase the rate of chemical reactions taking place in a living system. Such reactions are a billion times faster than uncatalyzed reactions because enzymes lower the energy barrier, also called the activation energy, of a reaction by inducing strain in the substrate molecule. Although the energy barrier decreases, the energy difference between the reactants and products remains the same.

Review - While all enzymes increase the rate of a chemical reaction for a specific type of substrate, the models regarding how this occurs differ. Two of the models that explain how the enzyme works are: The lock-and-key model: The substrate and the enzyme are analogous to a lock and a key, that is, only a specific key can open the lock. In this model, both the enzyme and the substrate are rigid molecules that fit together much like tow puzzle pieces. The induced-fit model: Both the enzyme and the substrate are flexible, and the enzyme can change shape when interacting with the substrate to ensure greater compatibility. In this model, the substrate's interaction with the enzyme induces a change that allows for interactions between the pair.

Briefly explain the shape of the curve.

The Asp must be protonated to act as a general acid catalyst; thus, activity will be higher when pH < pKa and lower when pH > pKa. At pH = pKa expect 50% of maximal activity because the Asp will be 50% protonated.

Explain why or why not.

The activation energy would be identical for both the catalyzed and uncatalyzed reactions.

Reaction Coordinate

The plot of the reaction progress as a function of time

Ammonia, NH3, is used in numerous industrial processes, including the production of pharmaceuticals such as sulfonamide and antimalarials and vitamins such as the B vitamins. The equilibrium equation for the synthesis of ammonia (sometimes known as the Haber process) is N2(g)+3H2(g)⇌2NH3(g) The Haber process is typically carried out at a temperature of approximately 500∘C. What would happen to the rate of the forward reaction if the temperature were lowered to 100∘C?

The reaction rate would decrease. Most reactions double in rate for each 10∘C increase in temperature.

Ammonia, NH3, is used in numerous industrial processes, including the production of pharmaceuticals such as sulfonamide and antimalarials and vitamins such as the B vitamins. The equilibrium equation for the synthesis of ammonia (sometimes known as the Haber process) is N2(g)+3H2(g)⇌2NH3(g) What would happen to the rate of the forward reaction if the concentration of nitrogen were decreased?

The reaction rate would increase. As the concentration of nitrogen decreases, collisions between nitrogen and hydrogen are less likely to occur.

Transferases

Transfer functional groups between molecules

An enzyme is considered a ________ because it speeds up chemical reactions without being used up.

catalyst

Consider the enzyme-catalyzed reaction with Vmax=164 (μmol/L)min−1 and KM=32 μmol/L. If the total enzyme concentration was 1 nmol/L, how many molecules of substrate can a molecule of enzyme process in each minute?

kcat = 1.64×105 min^−1 kcat = turnover number = Vmax/[Etotal] = ( 164*10-6 (mol/L)min^-1 )/(1*10-9 mol/L) = 164000 min^-1

Consider the enzyme-catalyzed reaction with Vmax=164 (μmol/L)min−1 and KM=32 μmol/L. Calculate kcat/KM for the enzyme reaction.

kcat/KM =8.50×10 (M⋅s)^−1 kcat/kM = ( 164000 min^-1 / 32*10-6 M ) = 5125*106 (M.min)^-1 = 85.416*106 (M.s)^-1

The catalytic efficiency of many enzymes depends on pH. Chymotrypsin shows a maximum value of kcat/KM at pH 8. Detailed analysis shows that kcat increases rapidly between pH 6 and 7 and remains constant at higher pH. KM increases rapidly between pH 8 and 10. The change in Kcat between pH 6 and pH 7 must involve _________ of a proton in the active site. The best candidate is _________. The increase in KM at higher pH must involve a change in the binding site. The group involved is probably the ___________ at Ile 16, created by the cleavage that activates chymotrypsin.

loss, His 57, N-terminus

At 37 ∘C, the serine protease subtilisin has kcat = 50 s−1 and KM=1.4×10−4M. It is proposed that the N155 side chain contributes a hydrogen bond to the oxyanion hole of subtilisin. J. A. Wells and colleagues reported (1986, Phil. Trans. R. Soc. Lond. A 317:415-423) the following kinetic parameters for the N155T mutant of subtilisin: kcat = 0.02 s−1 and KM=2×10−4M. The value you calculated in previous question represents the strength of the H-bond between N155 and the oxyanion in the transition state. This value is higher than typical H-bonds in water. How might this observation be rationalized? The dielectric constant, ε, is ________ in the enzyme active site than it is in water; thus, Coulomb's law predicts a ______ interaction between the H-bond donor and acceptor.

lower, stronger

V max

rate of a reaction when enzyme is saturated with substrate.

In a catalyzed reaction, a reactant is often called a ________.

substrate