BioChem 384 Exam 2

Rank the steps in the sequential order in which they occur as hemoglobin binds oxygen, with the first step/conformation at the top and the last step/conformation at the bottom. Iron moves into the same plane as the heme moving the F helix toward distal Histidine Asp94 Hydrogen bonds with Asn102, while Tyr42 breaks its hydrogen bond with Asp99 Fe2+ Is coordinated in the heme, and the heme is in a puckered conformation

1. Fe2+ Is coordinated in the heme, and the heme is in a puckered conformation 2. Iron moves into the same plane as the heme moving the F helix toward distal Histidine 3. Asp94 Hydrogen bonds with Asn102, while Tyr42 breaks its hydrogen bond with Asp99 if there is no oxygen bound, then the iron (+2 oxidation state) is not in the plane of the heme, and therefore pulls the heme into a puckered conformation. When oxygen binds to iron, it also hydrogen bonds to the distal histidine, thereby pulling the iron into the same plane as the heme group. This shift in the position of the iron within the heme causes helices to move, which leads to the forming and breaking of hydrogen bonds between Asp94 and Asn102 and Tyr42 and Asp99, respectively. This hydrogen-bond rearrangement changes the sub-unit from the T state (deoxygenated) to the R state (oxygenated).

Hemoglobin is composed of ___ individual polypeptide chain(s). Each of these individual polypeptide chains contains ___ heme co-factor(s) with ___ iron atom(s). Therefore, one hemoglobin molecule can potentially bind ___ O2 molecule(s).

1. Four (in order to function, four individual polypeptide chains must fold and associate with one another to form the tetramer structure of hemoglobin) 2. One (each individual polypeptide chain contains one heme group, and this non-amino acid group is essential to coordinating an iron atom) 3. One (each heme co-factor contains one iron atom) 4. Four (one O2 molecule binds to each iron-coordinated heme group, meaning a total of four O2 molecules can potentially bind to a single hemoglobin molecule)

Review the image and list in order each step of tandem mass spectroscopy analysis for a peptide. Sample ionization Peptide fragmentation to sub-fragment peptides by collision with helium Peptide separation based on m/z Separation of subfragments based on m/z Like an Edman degradation tryptic digest, each fragment can be sequenced using a proteomics search algorithm Each peptide is attracted to the detector

1. Sample ionization 2. Peptide separation based on m/z 3. Peptide fragmentation to sub-fragment peptides by collision with helium 4. Separation of subfragments based on m/z 5. Each peptide is attracted to the detector 6. Like an Edman degradation tryptic digest, each fragment can be sequenced using a proteomics search algorithm

Evaluate which of the following statements is/are true about enzymes and the transition from reactants to products. Choose one or more: A. An enzyme does not change the energy of the reactants or product, nor does it change the equilibrium constant of a reaction. B. The transition state theory states that in the transition state, the molecule can either become a product or remain a substrate, and this reaction intermediate can be physically isolated for short periods of time in both enzymatic and non-enzymatic reactions. C. One enzyme is needed for every substrate molecule in order to catalyze the reaction. D. 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.

A. An enzyme does not change the energy of the reactants or product, nor does it change the equilibrium constant of a reaction. D. 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. enzymes work by lowering the activation energy of a reaction, but they do not change the energy of the reactants, products, or the equilibrium constant of the reaction.

Which of the following is/are true regarding initial and maximal velocity of enzyme-mediated reactions? Choose one or more: A. At very low substrate concentrations, the initial velocity of an enzyme reaction will be lower than the maximal velocity of the enzyme reaction. B. 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. C. 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. D. Initial velocity is maintained until maximal velocity is reached.

A. At very low substrate concentrations, the initial velocity of an enzyme reaction will be lower than the maximal velocity of the enzyme reaction. B. 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. C. 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 of a reaction is determined by plotting the amount of product made on the y-axis and the time of a reaction on the x-axis. The slope of the line at the very beginning of this plot, when the reaction rate is at its fastest, is the initial velocity. This plot is made for an enzyme reaction using many different substrate concentrations. Then, these initial velocities are plotted against the substrate concentration on a Michaelis-Menten plot. The value on the y-axis, where additional substrate does not change the initial velocity very much, is the maximal velocity of the reaction.

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? Choose one: A. It is one of the residues connecting a β sheet to an α helix. B. It is one of the residues connecting one α helix to another α helix. C. It is part of a β sheet. D. It is one of the residues connecting one β sheet to another β sheet. E. It is part of an α helix.

A. It is one of the residues connecting a β sheet to an α helix. the lysine residue that is integral to the reaction mechanism of aldolase is positioned between a leucine residue and a proline residue. This lysine is one of the residues connecting a β sheet to an α helix.

Which of the following is false regarding the protein pepsin? Choose one: A. Pepsinogen is the active form of pepsin. B. Pepsinogen self-cleaves at the pH found in the stomach. C. Pepsin is a type of protease. D. Pepsin hydrolyzes peptide bonds.

A. Pepsinogen is the active form of pepsin. Pepsinogen is the inactive precursor of pepsin. Pepsinogen is activated in the acidic environment of the stomach so that a part of pepsinogen is cleaved off to form the active pepsin. Pepsin is the active form of the protein, which hydrolyzes the peptide bonds of proteins.

Sodium dodecylsulfate (SDS) plays an important role in SDS PAGE. Select each correct description of what SDS does in denatured electrophoresis. Choose one or more: A. 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. B. Because SDS is a detergent, it plays a role in denaturing the protein. C. Because SDS is a detergent, it supports the native state by interacting with the nonpolar portions of a protein, stabilizing the three-dimensional structure of a protein. D. SDS is a charged detergent that neutralizes the protein, allowing the protein to migrate through the gel based on size.

A. 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. B. Because SDS is a detergent, it plays a role in denaturing the protein. SDS both binds to the protein at the nonpolar regions and helps to denature the protein prior to electrophoresis.

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 isoelectic 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. Choose one: A. The phosphorylated protein will have a lower isoelectric point and thus migrate further toward the anode. B. The non-phosphorylated proteins from the non-diseased samples will move further to the anode without the bulky phosphate group attached to a tyrosine. C. The phosphorylated protein will be more attracted to the negative charge on the anode. D. Non-diseased proteins will migrate with a lower pI than those from diseased tissues and thus the samples from the non-diseased samples will migrate more to the anode than diseased protein.

A. The phosphorylated protein will have a lower isoelectric point and thus migrate further toward the anode. adding a phosphate group will decrease the charge at the pH at which the protein will be isoelectrically neutral.

Which of the following is responsible for peptide fragmentation in a tandem mass spectrometer? Choose one: A. collision chamber B. vacuum chamber where proteins are solubilized C. mass spectrometer 1 D. mass spectrometer 2

A. collision chamber this is where a selected peptide is fragmented.

Which of the following is not one of the three most common catalytic reaction mechanisms in an enzyme active site? Choose one: A. using the histidine side chain to form covalent bonds with the substrate B. protonation or deprotonation of an amino acid, or water, on the enzyme C. temporarily sharing electrons between two atoms D. utilizing positively charged metal ions to correctly orient the substrate, or to mediate redox reactions

A. using the histidine side chain to form covalent bonds with the substrate Although histidine residues may form transient covalent bonds in specific instances of enzyme catalysis, this is not a "generalized strategy." Histidine residues are more commonly used in acid-base catalysis due to their pKa being so close to physiological pH, and to coordinate metal ions in metal-ion catalysis.

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 ___

ATP upregulates the activity of ATCase so that the reaction is faster at lower substrate concentrations. the binding of CTP to ATCase downregulates the activity of the enzyme. when ATP binds to ATCase, the R state conformation of the enzyme is favored. the R state of ATCase causes the active site to become activated so that the reaction can proceed quickly.

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. A. Using standards, paper chromatography identifies the modified amino acid B. An unlabeled peptide is mixed with phenylisothiocyanate to covalently bond to the amino terminal nitrogen C. The PITC- labeled amino acid is extracted using an organic solvent and further modified to a phenylthiohydantoin D. Peptide is treated with trifluoroacetic acid to cleave the bond between the first and second residues

B) An unlabeled peptide is mixed with phenylisothiocynante to covalently bond to the amino terminal nitrogen D) Peptide is treated with trifluroacetic acid to cleave the bond between the first and second residues C) The PITC-labeled amino acid is extracted using an organic solvent and further modified to a phenylthiohydantoin A) Using standards, paper chromatography identifies the modified amino acid PITC is covalently bonded to the N-terminal peptide, where acid treatment will remove the labeled amino acid with the label to be detected by chromatography.

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? Choose one: A. Beta strand B. Alpha helix C. Beta turn

B. Alpha helix Alpha helices are among the most common elements of secondary protein structure. In an alpha helix, the trace of the peptide backbone rotates around an imaginary central rod in a right-handed, helical manner.

There are nine lysine residues within pea gylcine carboxylase. Why would a specific lysine attachment site for lipoamide be conserved among orthologous decarboxylase proteins? Choose one: A. Glycine decarboxylase is not subject to selective pressure and is unlikely to undergo mutation. B. The lipoamide needs to be precisely located near the enzyme active site. C. Allosteric regulators prevent lipoamide from attaching at the other lysine residues. D. Lysine residues in alpha helices have side chains that are sequestered from being able to bind cofactors.

B. The lipoamide needs to be precisely located near the enzyme active site. Glycine decarboxylase is a key enzyme in the catabolism of glycine. The catalytic mechanism involves the transfer of a methylamine group by lipoamide between components of the enzyme complex. Therefore, it is crucial for this cofactor to be located in proximity to the active site of the enzyme, placing a high selective pressure to conserve lysine at this residue throughout evolutionary history.

Which of the following is correct about turnover number (kcat) and the specificity constant for an enzyme? Choose one: A. The kcat reveals how well an enzyme works. B. The specificity constant is defined as kcat/Km. C. kcat = vmax/[ES] D. The specificity constant is defined as (kcat)(Km).

B. The specificity constant is defined as kcat/Km. Which of the following is correct about turnover number (kcat) and the specificity constant for an enzyme?

What is the method used to purify a protein by exploiting the specific binding of the protein to its ligand called? A. Ni2+ chelating chromatography B. affinity chromatography C. ion exchange chromatography D. high binding interaction chromatography

B. affinity chromatography a ligand is used to capture the specific protein that binds the ligand.

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? Choose one: A. separate the smaller proteins based on the percentage gel at the expense of larger proteins B. separate larger proteins at the expense of smaller proteins, which will not resolve well C. separate proteins as in size-exclusion chromatography, with the smaller proteins migrating most slowly and the larger proteins migrating farther through the gel D. allow the negative charge contributed by the smaller SDS molecules to more easily migrate through a low-percent acrylamide because of the increase in negative charge

B. separate larger proteins at the expense of smaller proteins, which will not resolve well a low-percentage gel means that there is less overall cross-linked acrylamide and the matrix has larger "gaps" for the larger proteins to enter and begin to be separated.

On the 2-D gel shown in the figure below, where would a protein with a high pI and a high mass be found? Choose one: A B C D

C In 2-D gels, proteins are first separated based on their isoelectric points (pI) in an isoelectric focusing experiment. The pI-separated proteins are then separated again on the basis of mass in a standard SDS-PAGE gel. Thus, low pI proteins are on the left and high pI proteins are on the right, while high mass proteins are at the top and low mass proteins are at the bottom of the gel.

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? Choose one: A. Mix the compound with the lysate and then perform and NMR spectroscopy of the mixture. B. Perform an immunoprecipitation using antibodies against the protein of interest after adding the drug compound to the lysate. C. Fix the drug compound to a chromatography bead and perform an affinity chromatography on the lysate to detect the protein that binds the drug. D. Perform a size-exclusion chromatography with lysate both with and without the compound and analyze the elution fraction for changes in size.

C. Fix the drug compound to a chromatography bead and perform an affinity chromatography on the lysate to detect the protein that binds the drug. this is a technique to use an immobilized ligand to purify and identify specific binding proteins.

Lipoamide is a covalently attached coenzyme that plays a key role in several decarboxylase reactions, including glycine decarboxylase, and is attached to a lysine residue in the enzyme. 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. Choose one: A. Lysine 118 B. Lysine 8 C. Lysine 63 D. Lysine 17

C. Lysine 63 Dihydrolipoamide, the reduced form of lipoamide, is covalently attached to Lysine 63.

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?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. Choose one: A. The dihydroxyacetone phosphate is noncovalently associated to a lysine side chain. B. The glyceraldehyde-3-phosphate is noncovalently associated to a lysine side chain. C. The dihydroxyacetone phosphate is covalently bound to a lysine side chain. D. The glyceraldehyde-3-phosphate is covalently bound to a lysine side chain.

C. The dihydroxyacetone phosphate is covalently bound to a lysine side chain. the stage of the reaction mechanism that is seen in the molecular structure is dihydroxyacetone phosphate covalently bound to a lysine side chain. This lysine residue is positioned between a leucine residue and a proline residue.

Trypsin, chymotrypsin, and elastase are all serine proteases that cleave after different amino acids. What is responsible for the substrate specificity? Choose one: A. Each protease is made in a different cell type. B. Different catalytic mechanisms C. Different amino acids involved in the catalytic triad D. The substrate binding pockets accommodate different amino acids.

D. The substrate binding pockets accommodate different amino acids. Serine proteases share a common reaction mechanism and a common catalytic triad. The specificity for different amino acid substrates comes from the substrate binding pocket, which differs to accommodate different amino acids.

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? Choose one: K to E K to G K to R K to Y K to D

K to R the genetic mutation that causes the K to R substitution would be the least likely to destroy the catalytic mechanism that relies upon the lysine residue discussed in Parts 1 and 2. This is because the lysine side chain is positively charged and the arginine is as well. The positive charge is integral to the mechanism of catalysis.

Sort the following statements into the correct bins based on whether they most appropriately describe the binding pocket of chymotrypsin, trypsin, or elastase.

chymotrypsin, trypsin, and elastase are all serine proteases that bind different types of amino acids due to the different structures of their binding pockets. The serine and glycines of chymotrypsin, along with its relatively large size, allow larger aromatic amino acids to bind. The negatively charged binding pocket of trypsin (with aspartic acid and two glycines) allows positively charged amino acids such as arginine to enter it, while the small size of the elastase binding pocket (with threonine, valine, and serine) allows only small amino acids to enter it.

Sort the following statements into the appropriate bins based on the classes of enzymes they belong to or describe.

transferases transfer functional groups; ligases catalyze the formation of C—C, C—O, C—S, or C—N bonds; while lyases cleave such bonds without using oxidation or hydrolysis reactions. These are three types of enzymes (out of six possible), as classified by IUBMB. Decarboxylase is an example of a lyase; synthetase is an example of a ligase; and kinases and transaminases are examples of transferases.