Biochem 384 Exam 2 Review

The lactose permease transported of E.coli is a secondary active transporter protein that can accumulate lactose inside the cell against a concentration gradient. Thus symporter mechanism is driven by proton motive force that serves as the energy source for transport. If the ∆G for the proton gradient across the membrane is -10kJ/mol, what is the maximum concentration ratio of lactose that can be achieved at 37degressC? (ch6)

47.9:1 max concentration ratio

Which of the following is not a property or characteristic of both hemoglobin and myoglobin? (sw9)

A protein composed of four polypeptide chains

Considering that the Na+-K+ ATPase membrane protein is essential for maintaining an electrochemical gradient across cell membranes, which of the following is likely not a component of an electrolyte solution for treating dehydration? (sw10)

ATP

What distinguishes P-type transporters from ABC transporters? (sw10)

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.

Which one of these is not an amino acid that allows for the selectivity of it listed transporter protein (sw10)

Asp75 (aspartic acid at position 75) within the OMP32 porin protein

The constriction point within the water channel of aquaporin proteins is formed by two short α helices, each of which contains a conserved ______ residue that interacts with translocating water molecules. (sw10)

Asparagine

Briefly describe the chain of events in a skeletal muscle cell that occurs between a nerve impulse stimulating influx of Ca2+ from the sarcoplasmic reticulum into the cytosol and contraction of the muscle. (ch6)

Ca2+ binds to troponin C (TnC), changing the conformation of troponin complex subunits such that the TnC, TnI, and TnT all rotate tropomyosin away from the myosin binding site on actin, so that myosin can bind actin and initiate the concentration cycle.

Which of the following is best described as a prosthetic group? (sw11)

Heme

Which of the following is accurate in terms of the relationship between the velocity of a reaction and the rate constant (k) of a reaction? (sw12)

In a first-order reaction, the rate constant of a reaction is equal to the velocity of the reaction divided by the concentration of substrate.

First, view just the heme cofactor of myoglobin and locate the four amine nitrogen atoms that are in close proximity to the iron ion (bright blue color in the center). Next, click the dropdown menu in the upper right corner and click the "Measure Distance" icon. Now use the angstrom measurement tool to determine the approximate distance between any one of the four amine nitrogen atoms and the iron ion. Part 1- This distance is approximately ______ angstrom(s) Part 2- Identify the location within myoglobin where O2 interacts. Which best describes where and how O2 interacts in myoglobin? Part 3- Within this molecular structure, the polyatomic ion sulfate (SO42-) is observed. Which of the following best describes the interaction between myoglobin and this polyatomic ion? (sw9)

Part 1- 2 Part 2- on one side of the heme cofactor and associated with the iron ion Part 3- Sulfate was used during precipitation of myoglobin and is seen in this molecular structure interacting with amino acids.

Which of the following is not an assumption that is made when applying Michaelis-Menten kinetics to an enzyme? (sw11)

The rate constant of E + P re-associating to form the ES complex must be considered.

Which of the following is correct about turnover number (kcat) and the specificity constant for an enzyme? (sw12)

The specificity constant is defined as kcat/Km.

The contractile unit of skeletal muscle is the sarcomere, which is flanked by two Z disks. Sort the proteins listed based on their locations. (sw10)

Vimentin, desmin, and α -actinin are all proteins composing the Z disks. Troponin, tropomyosin, actin, and myosin are all proteins within the sarcomere structure.

Which of the following is not an assumption made in Michaelis-Menten kinetics? (sw12)

Working conditions have [E] >> [S].

In isoelectric focusing, a protein that is physically located in the pH gradient that is below the pI of the protein would (hw4)

migrate toward the cathode.

Which separation technique exploits the solubility differences of proteins? (hw4)

salting out by ammonium sulfate

The mass-to-charge ratios of denatured proteins are equivalent for different mass proteins. However, the cross-linked nature of the acrylamide media can limit migration through the polymer matrix. Gels with less cross-linked acrylamide (low % SDS gels) will do which of the following? (sw8)

separate larger proteins at the expense of smaller proteins, which will not resolve well

For reversible binding between a protein and a ligand (sw9)

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

Which of the following is not one of the three most common catalytic reaction mechanisms in an enzyme active site? (sw12)

using the histidine side chain to form covalent bonds with the substrate

Which of the following sequences would most likely be found in a transmembrane beta strand in the structure of a bacterial porin protein? Explain your choice. a. -Arg-Asn-Ser-Ile-Phe-Met-Lys-Glu-Gly- b. -His-Leu-Phe-Ala-Val-Asp-His-Lys-Asp- c. -Lys-Gln-Asp-His-Arg-Ser-Asn-Gly-Glu- d. -Asn-Val-Lys-Met-Glu-Ile-Arg-Leu-Gln- e. -Phe-Met-Leu-Val-Ala-Ile-Phe-Val-Leu- (ch6)

(d), which has alternating hydrophobic and hydrophilic residues. The beta strands must alternate in this way in order for the membrane side of the porin beta barrel to be hydrophobic and the channel side of the beta barrel to be hydrophilic.

Sodium dodecylsulfate (SDS) plays an important role in SDS PAGE. Select each correct description of what SDS does in denatured electrophoresis. (sw8)

-Because SDS is a detergent, it plays a role in denaturing the protein. -SDS is an amphipathic compound that binds to the hydrophobic portion of the protein, coating the mixture and giving the protein an overall negative charge proportional to the size of the protein

Which of the following statements are true about the poison ouabain? (sw10)

-Ouabain inhibits the Na+-K+ ATPase protein, and this particular inhibition stops muscles such as the heart and lungs from relaxing after contracting. -Ouabain is a poison found in the seeds of the climbing oleander plant, and is used by African tribesmen in poison arrows. However, in small doses, it can be used to treat patients with heart problems.

The Edman degradation is able to sequence up to 50 residues using a simple method. Place the description for each step in order of the Edman degradation technique. (sw8)

1. an unlabeled peptide is mixed with phenylisothiocynante to covalently bond to the amino terminal nitrogen. 2. peptide is treated with trifluroacetic acid to cleave the bond between the first and second residues. 3. the PITC-labeled amino acid is extracted using an organic solvent and further modified to a phenylthiohydantoin. 4. using standards, paper chromatography identifies the modified amino acid.

Calculate the purification of the target protein when there is a 30% decrease in activity and a 55% decrease in total protein after centrifugation. (hw4)

1.6-fold

Name three heterotropic effectors that alter the O2 binding affinity of hemoglobin. For each, state whether the effector is a positive or negative regulator of O2 binding affinity. Why is O2 considered a positive homotropic effector of O2 binding affinity for hemoglobin? (ch6)

2,3-bisphosphoglycerate (2,3-BPG), H+ (decreased pH), and CO2—all negative effectors of O2 binding. O2 is a positive homotropic effector because binding of one O2 molecule induces a conformational shift from the T to R state, which has a much higher affinity for O2.

Which of the following is not a critical aspect of enzyme structure and function? (The structural and functional aspects of enzymes vary widely, but for this question, be sure to focus on critical aspects that apply to virtually all enzymes, and not just specific enzymes.) (sw11)

A covalent bond forms between an enzyme and substrate following substrate binding.

The globular head domain of myosin has binding sites for actin and ATP that are far from each other in the tertiary structure of the protein, yet that protein cannot bind both ATP and actin at the same time. In terms of two distinct myosin protein conformational states that preferentially bind actin or ATP with high affinity, explain how ATP binding reduces the binding affinity of myosin for actin and vice versa. (ch6)

Actin and ATP must bind to different conformational states of the myosin head, so actin binding stabilizes a conformational state with low affinity for ATP, whereas ATP binding stabilizes a state with low affinity for actin.

Which of the following sample preparation steps is used for both native PAGE and SDS-PAGE? (sw8)

Addition of glycerol to the sample loading buffer

Which of the following statements are true about the role of calcium ions in muscle contraction? (sw10)

Although Ca2+ binding is required for muscle contraction, it does not actually bind to myosin. Calcium binding is an essential first step in the actin-myosin reaction cycle. When Ca2+ binds to the troponin complex, the resulting conformational change allows the myosin head to bind to actin.

Which of the following is true about sickle cell anemia? (sw9)

An effective method of treatment may include inducing the adult overexpression of the gene that codes for the γ subunit of fetal hemoglobin. A mutation of residue 6 of hemoglobin from glutamate to aspartic acid would likely not cause sickle cell symptoms. A mutation of residue 6 of hemoglobin from glutamate to isoleucine would likely cause sickle cell symptoms.

Evaluate which of the following statements is/are true about enzymes and the transition from reactants to products. (sw11)

An enzyme lowers the activation energy of a reaction, which means the transition state is not as energetically unfavorable as it would be without the presence of an enzyme. An enzyme does not change the energy of the reactants or product, nor does it change the equilibrium constant of a reaction.

Why can't an enzyme use an induced-fit mechanism to achieve catalytic perfection? (ch7)

An induced-fit mechanism requires some energy to drive conformational change in the protein upon substrate binding, which reduces the energy that could be used to bind the transition state and lower the activation energy.

Which of the following is/are true regarding initial and maximal velocity of enzyme-mediated reactions? (sw12)

At very low substrate concentrations, the initial velocity of an enzyme reaction will be lower than the maximal velocity of the enzyme reaction. When an enzyme behaves with Michaelis-Menten kinetics, the initial velocity of that enzyme reaction changes with substrate concentration, but the maximal velocity of that particular enzyme remains the same at a constant enzyme concentration and identical reaction conditions. The initial velocity is determined by the slope of the line at the beginning of a reaction when one plots the experimental results of reaction time versus product formed. Maximal velocity is then determined by when the initial velocity no longer significantly changes with increasing substrate concentration.

Which of the following statements about the concerted and sequential models of allostery is false? (sw9)

Both models are "symmetrical" in that all complexes have symmetry since they do not contain mixtures of subunits in the T and R states.

Which resin should you subject your mixture to get the best separation? Choose the resin with the correct attribute of the resin and protein that will separate the proteins. (sw7)

CMC cellulose, because the pH is above two of the contaminating proteins and less than YPOI and contaminant protein 3. These two proteins can then be separated by ionic conditions (salt gradient).

You have a collaborator who thinks she has created a compound to bind and inhibit an enzyme involved in lung cancer metastasis. Which of the following experiments using lysate from a tumor should be considered to test whether or not this drug would bind to the protein? (sw7)

Fix the drug compound to a chromatography bead and perform an affinity chromatography on the lysate to detect the protein that binds to the drug.

Statin drugs are used to reduce cholesterol in patients exhibiting high cholesterol levels. Statin drugs target _________________ , reducing the activity of the enzyme. This enzyme helps lower ______________ levels because it is in the cholesterol biosynthetic pathway. (sw11)

HMG-CoA reductase, cholesterol

Each human red blood cell has about 2x10^5 aquaporin monomers. If water flows through the membrane at 5x10^8 molecules per aquaporin tetramer per second, and the volume of a red blood cell (RBC) is 5x10^-11 mL, how rapidly would an RBC lose half its volume if put in a high-salt environment? The density of water in 55.5mol/L. (ch6)

Half volume = 2.5x10^-11mL it would flow out in 33.4ms

Describe the major structural differences between fetal hemoglobin (HbF) and maternal adult hemoglobin (HbA). Explain how these structural differences affect the ligand binding properties of the two hemoglobins and what impact this has on O2 delivery from the maternal red blood cells to the fetal red blood cells in the placenta. (ch6)

HbF contains two alpha and gamma chains (a2y2); HbA has two alpha chains and two beta chains (a2B2). The gamma chains in the HbF contain Ser143 residue in place of the His 143 residue found in the beta subunit. With two fewer positively charged groups on HbF (the gamma chains), 2,3-BPG binds more weakly to HbF than to HbA, so that HbF has a higher O2 affinity than does maternal HbA. At compatible 2,3-BPG concentrations in maternal and fetal blood, HbF will bind O2 more tightly than HbA, and O2 from maternal Hb can transfer to fetal Hb in the placenta, thus being "delivered" to the HbF through the placenta.

Which of these mutations is the most likely to eliminate the ability of chymotrypsin to perform its function? (Amino acids are represented by their three-letter code, with the residue number immediately following the name of the amino acid. The amino acid listed before the arrow is the original amino acid, and the amino acid listed after the arrow is the amino acid into which it has been mutated.) (sw12)

His57→Tyr

When using tandem mass spectrometry for peptide sequence determination, what is the input into the second mass spectrometer from the collision chamber? (sw8)

Isolated and fragmented peptides

Which of the following metabolite transformation reactions produces the same molecular formula for the product and the substrate? (sw11)

Isomerization reaction

A particular genomic caretaker protein, Protein X, has an affinity for both Ligand Y and Ligand Z. When you have 0.23 microM of Protein X in a solution and mix it with 0.11 microM of Ligand Y, the resulting solution contains 0.20 microM of free Protein X, 0.09 microM of free Ligand Y, and 0.02 microM of the protein-ligand complex, after equilibrium has been reached. However, when you have 0.23 microM of Protein X in a solution and mix it with 0.11 microM of Ligand Z, the resulting solution contains 0.14 microM of free Protein X, 0.02 microM of free Ligand Z, and 0.09 microM of the protein-ligand complex, after equilibrium has been reached. Which ligand has a greater affinity for Protein X? (sw9)

Ligand Z

Which component of a tandem mass spectrometer determines the mass of subfragments? (sw8)

Mass spectrometer 2

Cytochrome c protein (pI=12) and cytochrome c peroxidase protein (pI=5) can be isolated from yeast mitochondria. Both proteins are apparently "bound" to a DEAE ion-exchange column in a low-ionic-strength (10mM) buffer at pH7. When a buffer with increasing amounts of salt (a salt gradient) is applied to the column, cytochrome c elutes before cytochrome c peroxidase. From this information, can you determine whether cytochrome c is bound directly to the DEAE column or is more likely indirectly bound through association with cytochrome c peroxidase? Justify your answer using pI values, the pH, the net charge on the proteins, and the chrage on the ion-exchange column. (ch5)

On the basis of the pI values, at pH7 cytochrome c is positively charged and cytochrome c peroxidase is negatively charged. DEAE is an anion-exchange resin (positively charged), so cytochrome c cannot be directly bound to resin; it must be bound to cytochrome c peroxidase, which is in turn bound to resin. Increasing the salt concentration weakens the interaction of cytochrome c with cytochrome c peroxidase, causing it to elute first.

__________ is a positive allosteric effector of hemoglobin, increasing its affinity for oxygen, while _______ is a negative allosteric effector of hemoglobin, reducing its affinity for oxygen. (sw9)

Oxygen, 2,3-BPG

Part 1- How many of the four subunits of the potassium channel contain membrane-spanning helices? (Note that the initial view contains the ribbon structure of all four subunits visible.) Part 2- Within the selectivity channel, which component of the protein do these ions interact with? Note that in the molecular structure, two potassium ions and a water molecule are shown in the selectivity channel. Part 3- Why is a water molecule always observed between potassium ions in the selectivity filter of a channel like this? (sw10)

Part 1- Four Part 2- with loops between alpha helices Part 3- The water molecule provides a spacer between two nearby K+ ions, which would otherwise repel each other and prevent net transport of K+ ions through the channel.

One step in the reaction mechanism of aldolase is represented in this molecular structure. Which of the following best describes the stage of the aldolase mechanism that is captured here? Part 1- You may need to rotate the ball-and-stick or the space-filling model of the atomic representation so that you can observe the amino acid side chain. The ribbon structure will provide the least amount of help. Additionally, be sure to view the amino acid sequence and observe that the side chain of interest is flanked by a leucine residue and a proline residue. Part 2- The lysine residue that is integral to the reaction mechanism of aldolase is positioned between a leucine residue and a proline residue. Which of the following is true about this lysine? Part 3- A genetic mutation that causes which of the following substitutions would be the least likely to destroy the catalytic mechanism that relies upon the lysine residue discussed in Parts 1 and 2 regarding the aldolase enzyme? (sw11)

Part 1- The dihydroxyacetone phosphate is covalently bound to a lysine side chain. Part 2- It is one of the residues connecting a β sheet to an α helix. Part 3- K to R

Part 1- Protein secondary structure is important to the function of proteins and consists of three main types: alpha helix, beta strand, and beta turn. What type of protein secondary structure is highlighted? Part 2- Review the molecule again. Manipulate it in order to identify the specific lysine residue that serves as the attachment site for lipoamide in the enzyme glycine decarboxylase. You may want to view the hint if you need help. Part 3- There are nine lysine residues within PEA gylcine carboxylase. Why would a specific lysine attachment site for lipoamide be conserved among orthologous decarboxylase proteins? (sw11)

Part 1- alpha helix Part 2- Lysine 63 Part 3- The lipoamide needs to be precisely located near the enzyme active site.

Part 1- Experiments are performed to determine the initial reaction velocity of an enzyme-catalyzed reaction. What is NOT held constant so that the initial velocities can be used to plot the Michaelis-Menten graph? Part 2- Which of the following statements is false, considering Michaelis-Menten enzyme behavior and plots? (sw12)

Part 1- substrate concentration Part 2- Decreasing the concentration of enzyme will result in a decrease of Vmax and a decrease of Km.

Part 1- The enzyme ATCase is regulated by allosteric mechanisms. The binding of ATP to ATCase ______________ the activity of ATCase, while the binding of CTP _____________ the activity of ATCase. When ______ binds to ATCase, the R state conformation of ATCase forms, causing the catalytic site to become _____________ . Part 2- Which of the following is false regarding the protein pepsin? (sw12)

Part 1- upregulates, downregulates, ATP, activated PArt 2- Pepsinogen is the active form of pepsin.

What is the biochemical difference between polyclonal and monoclonal antibodies? Why do you think that monoclonal antibodies, but not polyclonal antibodies, are suitable for human diagnostic and clinical applications? (ch5)

Polyclonal antibodies are a collections of immunoglobulin proteins that recognize different epitopes on the same antigen with various specificities and affinities. A monoclonal antibody is a single immunoglobulin that recognizes only one epitope. Monoclonal antibodies are suitable for human diagnostic and clinical applications for two mains reasons: (1) the antibody antigen interaction is highly specific and amenable to quality control, and (2) monoclonal antibodies are secreted by immortalized cell lines and can be produced indefinitely.

The first step of protein mass spectrometry is to get the protein (usually peptide fragments) into a gas phase as an ion. Which of the following describes matrix-assisted laser desorption/ionization (MALDI) ionization? (sw8)

Protein fragments are embedded in a solid mixture that absorbs light, and then a laser flashes on this mixture, leaving fragmented and ionized peptides in the gas phase.

Which of the following describes how thyroid diseases are diagnosed using radioactive iodine? (sw10)

Radioactive iodine is transported into thyroid gland cells by the Na+-I- symporter protein, a secondary active transporter. Some thyroid hormones, thyroxine and triodothyronine, will incorporate radioactive iodine, which leads to cell death as a useful diagnosis of and treatment for hyperthyroidism or thyroid diseases.

What is responsible for the "power stroke" of the actin-myosin cycle? (sw10)

Release of Pi from the myosin head

Which of the following reactions directly alters DNA and affects gene expression? (sw11)

Reversible covalent modification involving methylation

Your research project requires that you purify protein A away from proteins B and C using a combination of gel purification scheme using just these two columns that would accomplish your goal with the least amount of protein dilution. Include in your answer the types of running buffers you would use (ionic strength and pH) and the expected order of elution of the proteins from the two columns, given the physical properties of the proteins on the following table: (ch5)

Run the protein through a gel filtration column using a low-ionic-strength buffer (10mM NaCl) at pH6, which is a pH greater than the pI of protein A and protein B, but below the pI of protein C. Protein B will elute more rapidly from the column than A or C, which will likely co-elute. Because the pH is midwaybetween the pI of A and C, protein A will have a negative charge and C will have a positive charge. Running these proteins through a DEAE column (positively chrged) would cause protein A to bind tightly to the column, while C should elute. Protein A could then be eluted by increasing either ionic strength or pH of the buffer.

Antibodies can be useful to analyze specific target proteins both in vitro and in vivo; however, not all antibodies work equally well for both types of studies. Explain why an antibody might be able to detect its target proteins in rat liver call extracts by Western blotting of an SDS-PAGE gel but fail to detect the same protein using immunofluorescent staining of rat liver cell tissue sections. (ch5)

SDS-PAGE analysis separates denatured proteins and is more likely to expose the antigen to the antibody during Western blotting. In contrast, immunofluorescent staining traps proteins in the cell under conditions that could mask antigen sites as a intramolecular or intermolecular interactions.

Compare the order of migration of a small (10kDa) and large (80kDa) protein through a size-exclusion column and an 8% SDS-polyacrylamide gel. (ch5)

Size-exclusion column: The 80-kDa protein migrates faster (less interaction with the resin). SDS-PAGE: The 20-kDa protein migrates faster (smaller frictional coefficient).

Blood that has been stored for some time becomes depleted of 2,3-bisphosphoglycerate (2,3-BPG). What problems, if any, would this cause upon transfusion into a patient? Why? (ch6)

The 2,3-BPG-depleted red cells contain Hb that binds O2 too strongly and would not deliver O2 to the tissues.

Which of the following statements about oxygen binding to hemoglobin is NOT correct? (sw9)

The T state refers to oxyhemoglobin and the R state refers to deoxyhemoglobin

Would you expect the α and β subunits of hemoglobin to have more or fewer hydrophobic amino acids than myoglobin? Why? (ch6)

The alpha and beta subunits of hemoglobin are more hydrophobic because subunit interactions use hydrophobic amino acids.

Which of the following is FALSE when isolating proteins from whole cells? (sw7)

The amount of protein in each fraction isolated during centrifugation separation increases.

Which of the following is FALSE when isolating proteins from whole cells? (sw8)

The amount of protein in each fraction isolated during centrifugation separation increases.

In most enzymes, the active site consists of only a few residues. Why is the rest of the protein necessary? (ch7)

The bulk of the protein is necessary to produce the correct tertiary structure for the substrate binding site and for proper orientation of the catalytic residues.

Select the answer that is true regarding Gibbs free energy relationships in active and passive transport (sw10)

The delta G of the movement of a molecule into a cell in the absence of a transport protein is +14.4 kcal/mol. Transport of this molecule would require active transport.

Precipitation of proteins can be accomplished by either heating a protein solution, followed by centrifugation, or by adding ammonium sulfate to the solution and then centrifuging the precipitate. With respect to an enzyme that was being purified by an activity assay, how would precipitation of this enzyme by these two methods affect the next purification step after resolubilization of the pelleted protein? (ch5)

The enzyme precipitated by heat denaturation will have little, if any, activity upon resolubilization, whereas the protein precipitated by ammonium sulfate should retain most of its activity upon resolubilization. The heat-denatured protein is nearly impossible to purify further because the activity assay will not work.

The four graphs here show the same reference curve for O2 binding by normal human adult hemoglobin at pH 7.4 in the presence of a physiologic concentration of 2,3-BPG. On each graph, sketch an additional O2 binding curve for the Hb as described in a-d, and briefly explain how this Hb differs from the Hb reference in terms of changes in the R-state-T-state equilibrium and P50 (pO2 at 50% saturation of O2 binding sites). a. For graph a: Hemoglobin with all of the 2,3-BPG removed from the O2 binding assay. b. For graph b: Hemoglobin at pH 7.2 rather than pH 7.4. c. For graph c: Hemoglobin mutant in which the predominant form of the protein is monomeric, with a very low binding affinity for O2. d. For graph d: Fetal hemoglobin in which His143 in the β subunit is replaced by Ser143 in the γ subunit. (ch6)

The four graphs here show the O2 binding curves of the various Hb proteins. a. In absence of 2,3-BPG, R-T equilibrium shifts towards R and the curve is slightly more hyperbolic (less sigmoidal) because of reduced cooperativity of O2 binding; the P50 will be lower (curve is shifted to the left). b. Higher [H+] (lower pH) shifts the R-T equilibrium toward T; the curve is more sigmoidal with a higher P50 (curve shifted to the right). c. Monomeric Hb has no cooperativity; the plot is purely hyperbolic with higher P50 (curve shifted to the right). d. Fetal hemoglobin has one less positive charge on each γ subunit in the central cavity, so 2,3-BPG binds less tightly and more hemoglobin is in the R state; the curve is mostly sigmoidal and shifted to the left (lower P50).

Explain the following observation: Enzyme A has a very broad pH optimum and exhibits the same catalytic activity at pH 6.5 as at pH 8.5. However, a competitive inhibitor, X, is effective at pH 6.5, but not at pH 8.5. (ch7)

The inhibitor loses activity as a result of deprotonation at pH 8.5 relative to pH 6.5, on a group found in the inhibitor molecule.

Using immunoprecipitation, you can isolate a protein (protein X) you think is involved in chronic myelogenous leukemia (CML), which is caused, in part, by a hyperactive tyrosine protein kinase called ABL. You think one of the targets of ABL is the protein X that you can purify. In comparing tissues with and without the disease, you subject the samples to isoelectric focusing. Understanding how IEF works, how would you expect the samples with CML to migrate compared to the wild-type, non-diseased sample protein? Use the figure to help you consider your response. (sw7)

The non-phosphorylated proteins from the non-diseased samples will move further to the anode without the bulky phosphate group attached to a tyrosine. The phosphorylated protein will have a lower isoelectric point and thus migrate further toward the anode.

Which of the following is/are true regarding observed effects of pH on enzyme activity and rate, and the underlying causes of these effects? (sw12)

The rate of an enzyme-catalyzed reaction will slow down when outside the optimal pH range of the enzyme. The active site of an enzyme can change shape and overall charge when pH is not optimal, possibly reducing the enzyme's activity.

Which of the following statements is true regarding the potassium channel protein discussed in the animation? (sw10)

The selectivity channel of potassium channel proteins functions due to the amino acids in the channel desolvating potassium ions, but not sodium ions.

Considering the fractional saturation binding curve of hemoglobin as shown below, as well as the definition of fractional saturation, which of the following statements are true about hemoglobin's oxygen binding profile? (sw9)

The sigmoidal curve is indicative of cooperative binding. At partial pressures of oxygen of more than 12 pKa, hemoglobin is as saturated with oxygen as it can be, indicating that in the vast majority of hemoglobin proteins, all four oxygen binding sites are occupied.

Trypsin, chymotrypsin, and elastase are all serine proteases that cleave after different amino acids. What is responsible for the substrate specificity? (sw12)

The substrate binding pockets accommodate different amino acids.

Given a mixture of proteins of different molecular sizes shown below and a gel filtration column inclusion range of 10,000 to 100,000 Daltons, select the correct statements that follow. Protein A 10,000 Daltons Protein B 35,000 Daltons Protein C 50,000 Daltons Protein D 51,000 Daltons Protein E 75,000 Daltons Protein F 125,000 Daltons Protein G 200,000 Daltons (sw7)

This gel filtration column is a good choice to isolate protein A from the rest of the proteins. This is a good gel filtration column to separate proteins D and F.

A mutation in the active site of an enzyme results in a large increase in stabilization of the ES complex, while there is no change in the stabilization of the transition state complex. What effect will this have on the rate of product formation? (ch7)

This would decrease rate of product formation because stabilization of ES increases the activation energy (even though the transition state energy is unchanged).

Looking at the figure below, to which of the following does an enzyme bind the strongest? (sw11)

Transition State

A functional assay was designed to isolate a putative wound-healing growth factor present in plant extracts made from and exotic flower found high in the forest canopy of the amazon rain forest. Initial field studies of the purified factor identified a tetradecapeptide (14 amino acid residues) that consisted of 2mol of glycine and 1mol of 12 other amino acids-- none of which were alanine, histidine, leucine, serine, threonine, tryptophan, or valine on the basis of available amino acid standards. Although the field laboratory was set up to use Edman degradation to deduce partial amino acid sequences, the efficiency of the reaction limited its application to tetrapeptides and smaller. Therefore, the Edman degradation data were augmented by a combination or proteolytic cleavage assays with trypsin, chymotrypsin, and V-8 protease, as well as chemical cleavage with cyanogen bromide. Using the following information collected by the field biochemist, determine the most likely sequence of this tetradecapeptide, written left to right from the N-terminal residue using the single-letter amino acid code. Explain your reasoning. 1. Cleavage with trypsin yielded a hexapeptide, a septapeptide, and a single amino acid that was identified with amino acid standards as glutamine. 2. Cleavage with chymotrypsin yielded a hexapeptide, a pentapeptide, and a tripeptide with the amino acid sequence G-I-F. 3. Cleavage with V-8 protease yielded a heptapeptide, a tripeptide with the sequence P-R-Q, and a tetrapeptide with the sequence G-Y-N-D. 4. Cyanogen bromide chemical cleavage yielded a decapeptide and a tetrapeptide with the sequence G-I-F-M. (ch5)

Trypsin cleaves on the carboxyl side of lysine or arginine residues. Because the 1st or 14th amino acid must be Q (it cannot be the 7th or 8th amino acid based on the substrate specificity of trypsin), the 6th or 7th amino acid and the 13th or 14th amino acid must be K or R. Chymotrypsin cleaves on the carboxyl side of tyrosine, tryptophan, and phenylalanine residues and generated G-I-F; cyanogen bromide cleaves on the carboxyl side of methionine and generated G-I-F-M, thus the N-terminal sequence must be G1-F2-I3-M4, and the C-terminal amino acid must be Q14. The V-8 protease cleaves on the carboxyl side of glutamate and aspartate, so the C-terminal end must be P12-R13-Q14, and the sixth amino acid must be K6, as the only G is represented twice. The V-8 tetrapeptide must be adjacent to this C-terminal end and be G8-Y9-N10-D11. The V-8 heptapeptide is the first seven amino acids: G1-F2-I3-M4-X5-K6-E7, in which X5 must be cysteine (C5), the only amino acid remaining.

Which of the following statements are true regarding hemoglobin? (sw9)

When hemoglobin shifts from the T-state conformation to the R-state conformation, many noncovalent interactions are broken and reformed. The R state of hemoglobin has a higher affinity for oxygen than the T state.

You have discovered a small organic compound that you think will cause a significant shift in the peptide loop covering the active site of the enzyme, inhibiting the protein's function. Which approach(es) would be most appropriate to test for this hypothesis? (sw8)

X-ray crystallography NMR spectroscopy

If you wanted to improve the catalytic efficiency of an enzyme, would you mutate amino acid residues to increase binding affinity for the substrate or increase the binding of the transition state? Explain. (ch7)

You make the transition state bind more tightly in order to lower the activation energy of the reaction. Binding the substrate more tightly would make a higher activation energy.

In secondary active transporters, (sw10)

a proton gradient is used to transport molecules across the membrane.

A target protein was purified through several steps as shown in the table here. Answer the following questions about this purification scheme as they relate to the "?" values. a. What is the specific activity of the target protein after the first and fourth purification steps? b. Calculate the total protein yield (in milligrams) after the second and third purification steps. c. Calculate the overall percentage yield of the target protein (final activity/initial activity). d. Using gel filtration chromatography, the target protein activity was associated with a protein of 100,000 kDa. To get a more accurate molecular-mass estimation of the target protein, a small sample of the protein present in the most active fraction after step 4 was analyzed by SDS-PAGE, and as shown in the graph, the relative mobility of the most abundant protein (red circle) was plotted, along with that of known molecular-mass standards (black circles). What is the molecular mass of this purified protein based on SDS-PAGE, and how do you reconcile this with the molecular-mass estimate from gel filtration chromatography? (ch5)

a. First step: 2 × 104 units/mg protein; fourth step: 2 × 108 units/mg protein. b. Second step: 1 × 105 mg; third step: 1 × 103 mg. c. 4 × 109 units/1 × 1010 units = 40% yield. d. ∼25,000 kDa; the native protein is a homotetramer: four subunits of 25,000 kDa each.

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-BPG, but rather by a different compound that functions as a 2,3-BPG analog. Answer the following questions on the basis of the mechanism by which 2,3BPG regulates the O2 binding affinity of human hemoglobin. a. Considering the chemical and physical properties of the following compounds, which is the most likely candidate for the 2,3-BPG analog in bird red blood cells? b. The bird 2,3-BPG analog binds to hemoglobin in the same way 2,3-BPG binds to mammalian hemoglobin. Briefly describe where in the structure of the tetrameric bird hemoglobin you would expect the compound to bind and by what type of bonds and/or interactions. c. Would you expect the compound to increase or decrease the O2 binding affinity of bird hemoglobin? (ch6)

a. Inositol hexaphosphate b. It should bind in the central cavity between the four globin subunits, by ionic interactions with positively charged residues. c. The 2,3-BPG analog should decrease O2 affinity by shifting the equilibrium to the T-state conformation.

Consider the simple reaction below, in which kF(uncat) with no catalyst is 10−5 s−1, kF(cat) with an enzyme catalyst is 107 s−1, and kR(uncat) with no catalyst is 10−2 s−1: S<-->P a. what is the Keq for the reaction? b. what is the kR(cat) with enzyme catalyst? c. The enzyme that catalyzes this reaction has one active site per molecule. What is the kcat value for this enzyme if the vmax value is 5 × 103 M s−1 at an enzyme concentration of 2 × 10−6 M? (ch7)

a. Keq = (kF(uncat))/(kR(uncat)) = 10^-3 (unitless) b. kR(cat) = (kF(cat))/(Keq) = 10^10/s c. kcat = (vmax)/[Et] = 2.5x10^9/s

Consider five proteins with the properties shown in the following table. Answer the questions below about these proteins and justify your answers. a. Which protein would elute last from a gel filtration chromatography column under nondenaturing conditions? b. Which protein would migrate the slowest in an SDS-PAGE gel? c. Which protein would elute last from an anion-exchange column (DEAE gel matrix) using running buffer at pH 6.5? d. What is the likely quaternary structure for protein D on the basis of the data in the table? (ch5)

a. Protein C (smallest-mass native protein) b. Protein E (largest-mass denatured protein) c. Protein E (largest-mass denatured protein). d. Protein D is a homotrimer: three subunits of 25,000 Da each

Most X-ray crystallography analyses are now performed at centralized government labs that provide high-powered X-ray beams, which are able to resolve electron densities better than what can be done with smaller institutional instruments, and without the high costs of maintaining an in-house facility. However, even with this improved data collection methodology, the rate-limiting step in X-ray crystallography is often the ability to grow diffractable crystals. a. If a protein of interest is not amenable to crystallization, even after testing a large number of buffer conditions and temperatures, what is the most likely explanation? b. What are some of the advantages and disadvantages of obtaining dirractable protein crystals using orthologous protein from another species? c. If no suitable orthologous proteins were available, how might you modify the protein of interest to increase your chances of obtaining diffractable crystals? d. If none of these alternative approaches result in the isolation of diffractable crystals, what other method could possible be used to obtain the molecular structure of your protein? What is the primary advantage and disadvantage of this other method relative to X-ray crystallization? (ch5)

a. Structural heterogeneity of the protein can inhibit crystal growth. b. The main advantage of orthologous proteins is that sequence differences between closely related proteins can result in subtle chemical and physical changes that allow diffractable crystals to form. A disadvantage is that the two proteins may have significant structure-function differences that limit the data interpretation. c. Recombinant DNA techniques used to cause minimal changes in amino acid sequences, not likely to alter protein function, may change chemical and physical properties to permit crystallization. For example, a few amino acid changes in hydrophobic regions or in ionic interactions can sometimes be enough to facilitate crystallization. d. An alternative method is nuclear magnetic resonance (NMR) spectroscopy, which identifies the realtive locations of atoms in a concentrated solution of purified protein, providing spatial information used to decifer three-dimentional structures. The main advantage of NMR is that it uses soluble protein in its native form; the disadvantage in that it is limited to proteins <30kDa.

The activity of chymotrypsin changes as the pH changes in the range of pH 5-9, as shown on the following graph. From your understanding of the chemical mechanism of the chymotrypsin reaction, explain the pH effect on chymotrypsin activity by answering parts a, b, and c. a. What amino acid functional group on which residue is most likely responsible for the effect of pH? b. What is the apparent pKa of that functional group? Indicate on the graph how you deduced the pKa. c. Briefly explain in terms of the chymotrypsin mechanism why the activity increases as the pH increases in this pH range. (ch7)

a. The imidazole of the active site His residue. b. The Ph at which [base] = [acid], so protein would be 50% active at pH 6.8. c. In the first catalytic step of the mechanism, the active site His is unprotonated because it acts as a general base to accept a proton from the active site Ser.

An enzyme has a single active site at which it can bind and hydrolyze either X or Y; however, the enzyme cannot bind X and Y at the same time. Answer the following questions regarding the Km and vmax of this enzyme. a. Will the Km for X be affected if Y is present in the reaction mixture? Explain. b. will vmax for X be affected if Y is present in the reaction mixture? Explain. c. Is it possible for vmax and vmax/Km to show a different dependence on pH? Explain. (ch7)

a. Yes, Y is a competitive inhibitor of X; the apparent Km for X will increase. b. No, the vamx for X will be unaffected. c. Yes, the pH dependence of vmax reflects the ionization of catalytic site residues, whereas pH dependence of vmax/Km will reflect the ionization state of substrate binding residues.

How does the addition of an enzyme to a chemical reaction affect each of the following parameters (no effect, increase, or decrease)? a. Standard free energy of the reaction b. Activation energy of the reaction c. Initial velocity of the reaction d. Equilibrium constant of the reaction e. Time to reach equilibrium (ch7)

a. no effect b. decrease c. increase d. no effect e. decrease

It was found for the reaction A<-->B, the forward rate constant (kf) in the absence of enzyme was 1x10^-2/s, whereas the kf in the presence of enzyme was 5x10^6/s. a. Calculate the rate enhancement provided by the enzyme for this chemical reaction. b. If the equilibrium constant (Keq) for the A<-->B reaction is 1x10^3 in the absence of enzyme, what is the Keq in the presence of enzyme? Explain. (ch7)

a. rate enhancement = (kf catalyzed)/(kf uncatalyzed) = 5x10^8 fold enhancement b. Catalysts do not change the Keq, so the Keq remains 1 × 103.

Answer the following questions about the bioenergetics of ion transport across the cell membrane by the Na+-K+ ATPase transporter protein at 37degreesC under conditions in which the membrane potential is 70mV (inside of the cell is negative relative to the outside) and the ion concentration are as follows: [K+]outside = 5mM [K+]inside = 140mM [Na+]outside = 150mM [Na+]inside = 10mM a. What is the energy requirment to transport 2 mol of K+ across the membrane? b. What is the energy requirement to transport 3 mol of Na+ across the membrane? c. On the basis of the proposed mechanism of ion transport by the Na+-K+ ATPase transporter protein, explain how the hydrolysis of a single ATP is sufficient to transport 2 mol of K+ into the cell and 3 mol of Na+ out of the cell, considering that the amount of energy available from ATP hydrolysis is only ~50kJ/mol under cellular conditions, and as much as ~72kJ could be required for both transport processes. (ch6)

a. to transport 2 mol of K+, ∆G= 1.83kJ/mol x 2mol = +3.66kJ b. to transport 3 mol of Na+, ∆G= 13.74kJ/mol x 3 mol= +41.22kJ c. The Na+-K+ ATPase exchanges 2 mol of K+ imported for every 3 mol of Na+ exported, and so hydrolysis of a single ATP is sufficient.

What is the method used to purify a protein by exploiting the specific binding of the protein to its ligand called? (sw7)

affinity chromatography

The binding of molecular oxygen to the iron within the heme of hemoglobin causes the puckered heme to become planar. This alteration in the heme geometry causes a conformational change in the structure of hemoglobin, as illustrated in the figure below. Which of the following situations would likely result in the F helix moving into its oxygen-bound conformation (the conformation shown in the bottom of this figure when heme is planar)? (sw9)

carbon monoxide binding to the iron in the heme

Which of the following is responsible for peptide fragmentation in a tandem mass spectrometer? (sw8)

collision chamber

The following are critical for enzyme structure and function, EXCEPT (sw11)

covalent modification affecting the bioavailability of an enzyme.

Within the aquaporin, each subunit contains a channel that allows H2O passage. Through which of the following views would you expect water to travel? Assume water is represented by the red dot. In what direction is water expected to move through this protein? (sw10)

from the cytoplasm to the extracellular space from the extracellular space to the cytoplasm

Often times, two X-ray defraction patterns are needed to make phase determinations that are required to determine a protein's structure. When the procedure for calculating phase determinations uses a second pattern from a crystal made with an electron-dense atom such as mercury or selenium, the procedure is called (sw8)

isomorphous replacement.

Generally, the chemistry of Fmoc blocking is straightforward for most amino acids during solid state peptide synthesis. There is one amino acid, however, that presents a problem for Fmoc blocking during solid state peptide synthesis. That amino acid is... (sw8)

lysine

Glucose is transported across the cell membrane by Glut proteins, which function as primary energy-independent passive transporters. Under normal physiologic conditions when the concentration of serum glucose outside the cell, [glucose]outside, is 5mM, what is the free energy change for transport of serum glucose inth the cell at 37degreesC, considering that the glucose concentration inside the cell, [glucose]inside, is 0.1mM? (ch6)

∆G=RTln(C2/C1)+ZF∆V= -10.1 kJ/mol