Biochem Exam 2 Practice

What prevents the water channel inAquaporin 5 from functioning as a "proton wire" without H2O molecules moving through the channel?

The two alpha helices at the constriction point have inverted dipoles that cause the H2O molecules to reorient and break H-bonds between H2O molecules.

Which of the experimental steps listed at the right is considered the most challenging in X-ray crystallography?

determining the phases of the diffracted X-rays.

The HbS mutation (Glu6 --> Val6) ______________.

is thought to provide heterozygous individuals protections from malaria.

In isoelectric focusing, a protein that is initially located in the pH gradient that is below the pI of the protein would_______________.

migrate toward the cathode.

Consider a system where a passive transport channel is available for a neutral molecule X. If RTln(C2/C1) is zero, then

no net transport will occur.

What type of transport is depicted in the figure below?

passive transport of molecules down a concentration gradient

Choose the five correct labels for the figure to the right. "Transition State" = TS.

(1)ES (2)TS (3)EP (4)E+P (5)substrate

In an uncatalyzed reaction, kF(uncatalyzed) = 2.5 X 10-8/s and Keq is 5 X 102. For the same reaction in the presence of an enzyme, kF(catalyzed) = 5 X 106/s. Remember that Keq = kF/kR = k1/k-1. (a) What is the rate enhancement of the catalyzed reaction in the forward direction? (b) What is the value for kR(catalyzed)?

(a) 2x1014; (b) 1x104/s

There are three main types of metabolite transformation reactions. (a) Which type produces the same molecular formula for the product and the substrate? (b) Which type involves the removal of H2O?

(a) Isomerization, (b) Dehydration.

Hydrophobic substrate channels provide a path for fatty acids to enter enzyme active sites buried deep within the protein. (a) Which amino acid is most likely found in a hydrophobic substrate channel? (b) What biomolecule is most likely excluded from entering a hydrophobic substrate channel? (a) Asp; (b) NH4+ (a) Val; (b) fatty acid (a) Val; (b) O2 (a) Val; (b) NH4+ (a) Lys; (b) H2O

(a) Val; (b) NH4+

In the sliding filament model of muscle contraction, the (a)______ filaments are made of (b) ______ protein and the (c) ______ filaments are made of actin and other proteins. These two filaments slide past each other during muscle (d) ______, shortening the distance between (e) ______ proteins. This contraction is initiated by the binding of Ca2+ to (f) ______ thin myosin thick contraction Z-disk actin

(a) thick, (b) myosin, (c) thin, (d) contraction, (e) Z-disk, (f) troponin.

Glucose transporters in the kidney aresecondary active symporters (see figure) in whichsodium ions (circles) are transported down a gradient from outside to the inside, and glucose (squares) is transported up a gradient outside to inside. Concentrations are [glucose]outside = 5 mM, [glucose]inside = 120 mM, [Na+]outside = 150 mM, and [Na+]inside=10 mM. The membrane potential is 70 mV (negative inside). What is the DG for glucose transport into kidney cells at 37 °C? Is this secondary active symport function favorable in kidney cells under the above conditions?

+8.2 kJ/mol; yes

What is the free energy change for transport of glucose from outside the cell to inside the cell at 37ºC when the glucose concentrations are 5 mM outside and 0.1 mM inside?

-10.1 kJ/mol

Glucose transporters in the kidney aresecondary active symporters (see figure) in whichsodium ions (circles) are transported down a gradient from outside to the inside, and glucose (squares) is transported up a gradient outside to inside. Concentrations are [glucose]outside = 5 mM, [glucose]inside = 120 mM, [Na+]outside = 150 mM, and [Na+]inside=10 mM. The membrane potential is 70 mV (negative inside). What is the DG for Na+ transport into kidney cells at 37 °C? Is this secondary active symport function favorable in kidney cells under the above conditions?

-13.6 kJ/mol; yes

A transporter protein transports Ca2+ ions across a membrane at 37 °C in a cell in which the membrane potential is 100 mV (inside of the cell is negative relative to the outside) and the Ca2+ ion concentrations are 150 mM inside and 15 mM outside. What is the delta G when transporting 2 Ca2+ from the outside of the cell to the inside of the cell?

-26.2

What are the THREE key assumptions of Michaelis-Menten kinetics. 1. The reaction is analyzed at a time in the reaction that negligible product is formed. 2. Product release is assumed to be a rapid step in the process. 3. The steady state condition is reached quickly under conditions where [S] >> [E]. 4. Enzymes have higher binding affinities for products than the transition state. 5. Catalysts alter the free energy of the reaction without altering the equilibrium constant 6. Substrates and products are chemically inert until enzymes are added, catalysts = life.

1, 2, 3

Chymotrypsin is a (1) serine / (2) cysteine protease used to prepareoligopeptides for Edman Degradation protein sequencing studies. This enzymecleaves on the carboxyl side of (3) F or W / (4) L or R amino acid residues. Theamino acid residue (5) histidine / (6) glycine has a central role in the reactionmechanism, which is described as an acid-base catalysis mechanism. The reactionsequence results in the release of the (7) amino / (8) carboxyl terminal fragment first as a result of the (9) Ser 197 / (10) His 57 residue donating a proton to the substrate. The chymotrypsin reaction relies on a hydroxyl ion to function as a (11) electrophile / (12) nucleophile that attacks the carbonyl carbon of the acyl- enzyme intermediate.

1, 3, 5, 8, 10, 12

Although most protein sequencing is now doneby mass spectrometry (MS), Edman degradation (ED) is useful if large amounts of a protein can be obtained from species for which genomic sequence data are not available. Answer the True/False questions and choose the set of all correct answers. 1. True / False - MS data provides probabilities of the most likely match, but ED provides chemical data. 2. True / False - Protein sequencing by ED does not require purified protein, but sequencing by MS does. 3. True / False - Genomic sequences are needed to predict protein sequences by the MS method. 4. True / False - The N-terminal residue removed by acid treatment in ED is always a methionine. 5. True / False - Trypsin and chymotrypsin treatment provide unique peptide fragments for ED analysis.

1. True 2. False 3. True 4. False 5. True

Calculate the purification of the target protein when there is a 30% decrease in activity and a 55% decrease in total protein after centrifugation.

1.6-fold

You start with 100 units of protein activity and 100 grams of total protein. After the first centrifugation step, you have 5% less activity and 95% less total protein. What is your fold-purification of the target protein?

19-fold

What is the rate enhancement as a result of the presence of an enzyme if the uncatalyzed rate of the reaction is 1.2 102 mmol/sec and the catalyzed rate is 2.4 104 mmol/sec?

200

Match each representative protein with the most correct specific major protein functional class. Record your answer in the order of a, b, c, d, and e, by choosing the numbers 1, 2, 3, 4, and 5. a. Cell signaling b. Metabolic enzyme c. Genomic caretaker d. Structural protein e. Transport protein 1. Malate dehydrogenase 2. Na+/K+ ATPase 3. Tubulin 4. G protein-coupled receptor 5. RNA polymerase

4,1,5,3,2

You start with 100 units of protein activity and 100 grams of total protein. After the first centrifugation step, you have 15% less activity and 80% less total protein. What is your fold-purification of the target protein?

4.2-fold

The change in free energy for Solute A concentration across a membrane is calculated to be -10 kJ/mol at 37ºC. If Solute B is transported across the same membrane against its own concentration gradient using a secondary active symporter transport mechanism linked to Solute A, what is the maximum concentration ratio that can be achieved for Solute B?

50:1

What is the theoretical yield using solid phase peptide synthesis of a pentapeptide with the sequence Asn-Phe-Ser-Tyr-Gly---[Resin] if you start with 20 mmol of each amino acid (100 mmol total) and the coupling efficiency is 90%? Assume the first coupling of the Gly to the resin is already done.

65 mmol

Consider the following interaction between a protein and its ligand: [P] + [L] mc021-1.jpg [PL]. If [L] = 5 mM, [P] = 3 mM, and [PL] = 2 mM, what is the dissociation constant?

7.5 mM

For this problem, assume the cell has a membrane potential of -60 mV with the interior negative with respect to the exterior, and is being kept at a constant temperature of 25 °C. The concentration of glucose outside of a given cell is 5 mM and inside the cell is 150 mM. What is the free energy change for the transport of glucose into the cell at 25 °C?

8.43 kJ/mol

Which of the following would shift the oxygen binding curve for hemoglobin to the left?

A higher pH

What distinguishes P-type transporters from ABC transporters?

ATP hydrolysis by P-type transporters results in a phosphorylated intermediate while ATP hydrolysis causes ABC transporters to convert from an outward facing transporter to an inward facing transporter.

Match each major class of protein with the best representative protein from each class. Protein Class: Actin Pepsin Adenylate cyclase Histone Aquaporin Representative Protein: Structural proteins Metabolic enzymes Cell signaling proteins Genomic caretaker proteins Transport proteins

Actin - Structural proteins Pepsin - Metabolic enzymes Adenylate cyclase - Cell signaling proteins Histone - Genomic caretaker proteins Aquaporin - Transport proteins

Saguaro cacti transport nutrients from the soil to the roots. If the mineral concentration in the root cells is 1000x higher than the surrounding soil, this is a form of _________ because ____________.

Active Transport; the minerals are being transported up their concentration gradient.

Enzyme active sites__________

Are usually a structural pocket or cleft

The mutation that would lead to the most dramatic effect on Ca2+ transport is a mutation of________________.

Asp351 in SERCA

Why don't normal Hbb subunits form oligomers considering they contain F85 and L88?

Because Hbb contains E6, which does not interact with the hydrophobic patch formed by F85/L88.

Refer to the reaction coordinate diagram below. At what point is there a maximum number of interactions between the enzyme and the compound that it is binding?

C

Carbon monoxide (CO) competes for binding of O2 to hemoglobin. Which of the following statements best explains the mechanism by which carbon monoxide poisoning occurs?

CO binds so tightly to the Fe2+ in the heme group that it gradually replaces all of the O2 in RBCs, starving tissues of oxygen.

After __________ induced changes in the structure of thin filament, myosin heads containing __________ in the nucleotide binding site will bind with high affinity to actin subunits.

Ca2+; ADP + Pi

Select the one TRUE statement regarding how catalysts increase reaction rates. Catalysts lower the amount of energy required to reach the transition state Catalysts lower the ground state energy of both the product and reactant. Catalysts raise the ground state energy of both the product and reactant, thereby lowering the activation energy Catalysts lower the ground state energy of the product, thereby lowering deltaG of the reaction.

Catalysts lower the amount of energy required to reach the transition state

Select the most correct statement regarding how catalysts increase reaction rates. Catalysts lower the amount of energy required to reach the transition state. Catalysts raise the ground state energy of the product and the reactant to lower the activation energy. Catalysts lower the ground state energy of the product, thereby lowering the DG of the reaction. Catalysts lower the ground state energy of the product and the reactant to increase the activation energy. Catalysts lower the DG of the reaction and the Keq of the reaction to increase reaction rates.

Catalysts lower the amount of energy required to reach the transition state.

Nitrite reductase contains two histidine amino acids that coordinate a Cu2+ ion. When the ion is present in the enzyme, the ion is a __________ and the enzyme is a __________.

Cofactor; holoenzyme

Which condition is required to elute a protein from a column using affinity chromatography?

Competing ligand at high concentration.

A kinase adds a phosphate group to a target enzyme, altering the catalytic efficiency of the enzyme. This is an example of_________________.

Covalent modification

Bird hemoglobins are tetrameric and very similar in structure and function to mammalian hemoglobins. However, in some bird species, O2 binding affinity to hemoglobin is not regulated by 2,3-bisphosphoglycerate (BPG), but rather by a different compound that functions as a BPG analog. Considering the chemical and physical properties of the following compounds, which is the most likely candidate for the BPG analog in bird red blood cells?

D

Predict the fragments of the following peptide after cleavage by trypsin DASRTYPECHI

DASR TYPECHI

The sarcomere length of isolated heart myoblasts can be measured over time in the laboratory. The myoblasts are maintained in a buffer during the experiment. In a control experiment, Ca2+ and ATP are added to the buffer and the change in sarcomere length is analyzed. If EDTA, a chelating agent that sequesters Ca2+, was included in the buffer, the length change would be __________ compared with a control experiment without EDTA.

Decreased

For chymotrypsin, which amino acid is involved in forming the oxyanion hole, but is not part of the catalytic triad?

Gly 193

Although most protein sequencing is now done by mass spectrometry (MS), Edman degradation (ED) is useful if large amounts of a protein can be obtained even if from an uncharacterized species. Answer the True/False questions regarding methods used to sequence a polypeptide fragment and choose the set of all correct answers. True / False - Protein sequencing by ED does not require purified protein but sequencing by MS does. True / False - The N-terminal residue removed by acid treatment in ED is always a methionine. True / False - Trypsin and chymotrypsin treatment often provides identical peptide fragments for ED. True / False - Genomic sequences are needed to predict protein sequences by the MS method. True / False - Trypsin treatment provides useful sequence information when analyzing data from MS.

False False False True True

A histidine residue at an enzyme's active site transfers a H+ to an amine group of the substrate as the first step of the reaction. The result is an increase in the rate of release of the product. This is an example of _____________.

General acid catalysis mechanism

In one type of hemoglobin mutant the amino acid change eliminates a hydrogen bond that normally stabilizes the T state. Would you expect this mutant hemoglobin to have a higher or lower O2 affinity compared to the normal protein and why?

Higher O2 affinity because the R-T equilibrium would be mostly shifted to the R state.

Which amino acid acts as a general acid and a general base in the mechanism of chymotrypsin?

His57

If an enzyme carries out acid-base catalysis, which of the following amino acids could act as general acid?

Histidine

If the Ca2+ transporter SERCA is inhibited and unable to remove Ca2+ from the cytoplasm and return it to the lumen of the sarcoplasmic reticulum, which of the processes described at the right is most likely to occur under these conditions?

Muscle contraction occurs, but not muscle relaxation.

Which of the following is true of sickle cell anemia?

It is caused by a mutation in the beta-globin gene

The K+ channel protein does not allow Na+ ions to pass through because______________.

It is unfavorable for the Na+ ions to lose their associated water molecules, which makes the solvated Na+ ions too large to fit through the channel.

Though oxygen only binds to a particular place in the hemoglobin molecule, the oxy- and deoxy- forms of hemoglobin differ in overall protein conformation. An example of a structural change that does NOT play a role in translating local binding of oxygen to global protein conformational change would be the difference ______ in the presence and absence of oxygen binding.

Location of the distal histidine

In the enolase reaction, the Lys 345 andGlu 211 residues participate in the reaction toconvert 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP). Based on the reaction scheme shown in the figure to the right, which statement below best describes the roles of Lys 345 and Glu 211 in the enolase reaction?

Lys 345 functions as general base and Glu 211 functions as a general acid.

A mutation in the beta subunit of hemoglobin is discovered that reduces the affinity of 2,3-BPG binding. Which of the following mutations is most likely to have this consequence?

Lys82 → Asp82

What are two examples of enzyme-mediated reversible chemical modifications?

Methylation of cytosine on nucleic acids and phosphorylation of phosphatidylinositols in the membrane.

In one type of hemoglobin mutant the amino acid change generates a strong ionic interaction stabilizing the T state conformation, but only under conditions of lower pH, e.g., at pH 7.2 compared to pH 7.6. What effect would this mutation have on the amount of O2 delivered to the tissue and why?

More O2 delivered to the tissue because the R-T equilibrium would be shifted to the T state in tissues.

What is the functional role of NAD+ in the glycolytic pathway reaction catalyzed by the enzyme glyceraldehyde 3-P dehydrogenase (GAPDH)?

NAD+ is a co-substrate in the reaction that functions to oxidize glyceraldehyde 3-P to generate the acyl thioester intermediate.

Which of the following is a positive effector for oxygen binding to hemoglobin?

O2

The figure below shows the coordination of heme, O2, and two critical histidine residues in globin proteins. Which of the following steps happens first in the oxygen binding process?

O2 binds to the iron of heme

Which of the following statements best explains how hemoglobin is able to exhibit cooperative binding once the first oxygen molecule is bound?

Once the first oxygen molecule binds to the heme group, a conformational change occurs in the hemoglobin complex that favors additional oxygen binding to the other heme groups.

If the histidine on the E helix (His E7) that coordinates the O2 that binds to the heme iron in hemoglobin is mutated to an alanine, what effect would be most likely?

Oxygen binding would not cause the movement of the F helix, since the alanine at E7 cannot coordinate oxygen.

In studying a transporter, it is found that no external energy input is needed to facilitate the movement of a neutral compound through the transporter. The translocation rate of the compound reaches a plateau at high levels of compound. It is a(n) ____________.

Passive transport carrier

Both passive and active transport proteins are needed to transport __________ across membranes.

Polar molecules and ions

Which of the protein purification methods listed below is the first step one needs to take after collecting a tumor cell tissue biopsy in which the objective is to purify a drug-resistant enzyme?

Preparation of a crude cell extract

Which of the following may result from a His143 Ala143 mutation in adult hemoglobin?

Reduced affinity for 2,3-BPG

A conformational change in troponin that opens the myosin-binding sites on actin is triggered by the _______.

Release of Ca2+ by the sarcoplasmic reticulum

Using SDS gel electrophoresis to separate proteins, _________ proteins migrate the fastest in the gel and move farther away from the ___________ because of their _________ charge.

Small; cathode; negative

Leghemoglobin is an oxygen-binding protein in root nodules that contain bacteria that fix atmospheric nitrogen. Which ONE of the following is TRUE if leghemoglobin is more like myoglobin than hemoglobin?

The O2 binding curve is hyperbolic

When oxygen binds to hemoglobin, which of the following occurs?

The heme group becomes planar

The figure below shows the oxygen binding curve for myoglobin in muscle. Based on this curve, which of the following is correct?

The lower pO2 in active muscle leads to release of O2 from myoglobin.

The initial velocity of an enzyme-catalyzed reaction is followed at various substrate concentrations. At very high substrate concentrations it is observed that the initial velocity no longer increases as more substrate is added. The velocity under these conditions is known as_____________.

The maximum velocity

When Pi is released from myosin_________________.

The power stroke occurs

The F helix moves toward heme Oxygen binding is monitored for a solution of hemoglobin. During the experiment, the curve changes from sigmodial to hyperbolic. Which of the following may be the reason for that change?

The protein dissociated into individual subunits

Which of the TWO following statements about x-ray crystallography are true?

The x-ray beam is scattered by the protein sample. Only crystallized proteins can be analyzed

Which one of the findings below would provide support for the transition state theory of enzyme catalysis?

Transition state analogs bind tightly to enzyme active sites

Biochemical assays are important in the process of isolating proteins because they___________.

Uniquely identify one protein

The fractional saturation of hemoglobin (Hb) binding to oxygen is illustrated by______________.

[HbO2] / ([HbO2] + [Hb])

For an uncatalyzed reaction in which Substrate is converted to Product, S<-->P, there is a forward rate constant, kF, of 10-5/s, and a reverse rate constant, kR, of 10-2/s. In addition, there is a forward rate constant in the presence of a catalyst, kF(cat), of 107/s. Answer the two questions below based on this information. Remember that Keq = kF/kR = k1/k-1. a) What is the Keq for the uncatalyzed reaction? b) What is the rate enhancement for the catalyzed forward reaction kF(cat) over the uncatalyzed reaction kF?

a) 10-3, b) 1012

Binding of the negatively charged allosteric effector 2,3-bisphosphoglycerate (2,3-BPG) stabilizes the T state of hemoglobin. A lysine residue in the central cavity of hemoglobin interacts with 2,3-BPG. In a hemoglobin variant, there is a substitution of this lysine by an asparagine. Relative to the wild-type hemoglobin, this variant would be expected to have _____________________.

an increased affinity for oxygen in the presence of 2,3-BPG, and an equivalent affinity in the absence of 2,3-BPG

Number the five steps in the actin-myosin reaction cycle; which STEP is the power stroke? a. ATP hydrolysis induces the recovery conformation. b. ADP release empties the nucleotide binding site in myosin. c. Ca2+ binding to troponin uncovers myosin binding sites on actin. d. ATP binding causes myosin to dissociate from actin. e. Pi release pulls the active filament toward the center

c,e,b,d,a; STEP e

An aspartate residue in a hemoglobin variant replaces a histidine residue that normally stabilizes 2,3-BPG binding. Relative to the wild-type hemoglobin, the variant has a(n) ________.

decreased affinity for 2,3-BPG and an increased ratio of hemoglobin in the R state relative to T state.

Below is a fractional saturation curve for O2 binding to adult hemoglobin. Assume that curve Y represents a system at pH 7.4 and with a normal physiological level of 2,3-BPG. Curve X represents a system that__________________.

has a higher pH with a normal physiological level of 2,3-BPG.

One explanation for the prevalence of the sickle cell anemia trait in Africa is _______________.

heterozygous individuals carrying the Val-6 mutation are less susceptible to malaria because their red blood cells are resistant to infection by the malarial parasite.

The lock-and-key model of enzyme catalysis _____________.

involves a rigid fit between the substrate and the enzyme

The figure below shows three proteins that are separated using gel filtration chromatography. Which protein is the largest?

squares

A reaction coordinate diagram comparing an uncatalyzed reaction with an enzyme-catalyzed reaction can directly illustrate that the enzyme __________, but will not directly illustrate that the enzyme __________.

stabilizes the transition state; orients the substrates appropriately for the reaction to occur

For reversible binding between a protein and a ligand,______________.

the larger the Ka, the higher the affinity between the protein and ligand.

The lactose permease from E. coli and the molybdate transporter from Archaeoglobus fulgidus both____________.

undergo a conformational change that occurs on substrate binding