Name: Chem 351 Exam 1

Transcription

1 Use the pk a values below for all problems requiring pk a s: α-amino groups = 9.4 α-carboxyl groups = 2.3 Sidechain ionizable groups: ysteine = 8.5 Tyrosine = 10 Lysine = 10.5 Arginine = 12 istidine = 6.8 Glutamate/Aspartate = 4.0 Serine/Threonine = 13.5 Multiple hoice For Problems 1-15, pick the BEST answer. Write the letter corresponding to your choice in the box provided (2 points each). 1) By the generally accepted definition, uncompetitive inhibitors: A) can be detected by the changing slope of a Lineweaver-Burk plot. B) bind to the enzyme before or after a substrate is bound. ) reduce the turnover number of the enzyme. D) increase the Michaelis constant for a particular enzyme. E) None of the above statements is correct. F) More than one of the above statements is correct. 2) hymotrypsin provides a beautiful example of the complexity of enzyme-catalyzed reactions. Which of the following statements is true about chymotrypsin s structure and activity? A) The oxyanion hole refers to the catalytically active serine residue s nucleophilic character. B) The catalytic triad describes the inductive effect that is required for catalysis. ) One tetrahedral intermediate is predicted to form during the multi-step mechanism. D) The hydrophobic pocket empties with peptide bond cleavage and release of one fragment. E) None of the above statements is true. F) More than one of the above statements is true. 3) Which of the following is true about the ψ angle? A) It describes the relative rotational change that is observed for peptide bonds within a polypeptide. B) It is the bond angle defined by -N-α for a particular residue in a polypeptide. ) It is a rotational angle defining the relative movement of groups around a N-α bond within a polypeptide. D) It is the angle that can describe the relative positions of the -N bonds on either side of a rotating - bond within a polypeptide. E) It is the bond angle defined by α--o for a particular residue in a polypeptide. 4) entrifugation is used daily in a wet biochemistry lab. Which of the following statements about this technique is false? A) entrifugation is a likely next step when using ammonium sulfate to precipitate certain proteins. B) Isopycnic centrifugation uses an ion gradient to separate different organelles from each other. ) All centrifugation techniques make use of different spin times and speeds to separate components of different size and density. D) Supernatant is a term used to describe the soluble fraction of a differential centrifugation spin. E) More than one of the above statements is false. 5) ooperativity, as it applies to hemoglobin: A) explains how O 2 is better bound at the lungs than it is in the tissue. B) is demonstrated by a rightward shift of the oxygen binding curve as BPG decreases. ) is observed in the shape of protein s oxygen binding curve. D) allows hemoglobin to pick up more O 2 when the p of the tissue is lower. E) does all of above. 1

2 6) Mass spectrometry (MS) is becoming an increasingly important technique in protein analysis. Which of the following accurately describes this technique and its application? A) In a spectrum obtained from MS larger values represent a larger charged state for the analyte. B) In MS, the number of peaks in a spectrum corresponds to the number of polypeptide chains in the protein. ) The usual MS technique involves changing the charge carried by a purified sample. D) In MS, a mixture of proteins can be successfully separated and isolated from each other based on their differences in mass. E) None of the above. 7) Structural proteins are a distinct subclass of proteins. Which of the following statements about these proteins is false? A) Fibroin protein is enriched in small nonpolar residues for tighter β-sheet packing. B) ollagen α chains contain numerous disulfide cross-links for greater tensile strength. ) ydroxyproline is particularly important for the proper function of tendons and cartilage. D) Intermediate filaments are particularly strong because of their parallel two-chain coiled coils. E) None of the statements is false. F) More than one of the statements above is false. 8) Which of the following is a true statement about events/structures involved in muscle contraction? A) Tropomyosin heads have inherent ability to hydrolyze ATP. B) Troponins are polymers that bind across large lengths of actin fibers. ) Actin fibers are formed by the polymerization of many globular (G) actin monomers. D) ontraction requires the movement of the M lines within individual sarcomeres. E) None of the above is true. 9) onsidering the enzymatic reaction catalyzed by IV protease: A) hydrolysis is the net reaction observed. B) a peptide bond is cleaved on the carboxyl side of a proline residue. ) a serine residue serves as the nucleophile during the catalytic event. D) inhibitors have been designed to covalently link to the active site just like natural substrates do. E) None of the above is true. F) All of the above are true. 10) v 0 is a very important variable measured in Michaelis-Menten kinetic studies. It represents: A) the rate of a reaction only when all enzyme molecules are catalytically active. B) the rate of a reaction when k -1 is 0 based on the classic M-M scheme. ) the rate of a reaction when the [S] is at ½ saturation levels. D) the rate of the reaction obtained by the equation k 2 [ES] using the classic M-M scheme. E) None of the above is true. F) More than one of the above is true. 11) Which of the following statements accurately describes immunoglobulins? A) Monoclonal antibodies have a better chance at binding an antigen in a variety of techniques, denatured and native, compared to polyclonal antibodies. B) Antigen specificity is based on the variable domains of the F fragment. ) Ideally, in the immune response, each antibody is generated to detect several antigens. D) A monoclonal antibody would be detected as a single band on SDS-PAGE. E) The secondary antibody binds to the F c fragment of a primary antibody in ELISA. 2

3 12) Which of the following statements about protein sequences/sequencing is true? A) It is essential to use only one protease when digesting the protein into smaller pieces so that the primary sequence can be easily re-established. B) The primary sequence does not offer any important information about a protein s structure because intramolecular interactions can be between distant sites in a protein s sequence. ) SDS-PAGE performed under reducing conditions offers a great deal of information regarding tertiary structure. D) Reagents like dansyl chloride and dabsyl chloride selectively react only with the amino terminus of polypeptide chains. E) None of the above is true. 13) Which of the following statements about elution profiles is false? A) Elution profiles can allow for the calculation of specific activity of a protein of interest. B) Elution profiles generally show when protein loaded onto a column comes off the column. ) Elution profiles may be used to follow the activity of some protein specifically. D) Elution profiles always show larger proteins eluting before smaller proteins. E) None of the above. F) All of the above 14) The presence or absence of water is extremely important in biological systems, and, as a result, describing water is also essential. Which of the following terms cannot be used to describe aqueous environment(s)? A) ionic strength B) osmotic pressure ) p D) i E) all of the above can be used to describe aqueous environments. 15) Based on your understanding of the various levels of protein structure, which of the following statements is true? A) A domain is defined as a secondary structural motif having its own distinct function. B) Primary amino acid sequence predicts α-helical formation, since this motif is the result of regularly spaced hydrogen bonds that occur between the sidechains of the residues. ) ydrogen bonds that stabilize parallel β-sheets are parallel with respect to each other, making the sheets far more stable than antiparallel sheets. D) β-sheets can form as a result of interactions between distant residues, but β-turns are stabilized by hydrogen bonding that must occur between nearby residues. E) More than one of the above is true. 3

4 Short Answer. Show your work for all calculations! Protein Analysis: 1) β-neoendorphin is a natural opioid peptide that functions as a neurotransmitter. It has the sequence Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro, and its ability to bind to its receptor is affected by the charge that it carries. Using the pk a s listed on the front of your exam, sketch a complete theoretical titration curve for this nonapeptide, label axes and buffer zones, and indicate net formal charge on the molecules at all equivalence points. 2) onsidering the sequence of the nonapeptide above (and only considering potential interactions between adjacent residue sidechains): a) Which tyrosine residue would be expected to have the lower pk a? Tyr 1 Tyr 8 Both would have the same pk a b) A nonapeptide can be constructed with an Arg 6 Gly 6 mutation (all other residues are the same). ompare the pk a of the sidechain in the Lys 7 residue in the original nonapeptide with the Lys 7 of this altered form; which would have the higher pk a? Lys 7 (original) Lys 7 (mutant) Both would have the same pk a c) Another nonapeptide can be constructed with an Leu 5 Asp 5 mutation (all other residues are the same). ompare the pk a of the sidechain of the Arg 6 residue in the original nonapeptide with the Arg 6 of this altered form; which would have the higher pk a? Arg 6 (original) Arg 6 (mutant) Both would have the same pk a 3) Now consider the interaction between the sidechains of Lys 7 and Arg 6 at a p of 7.2. onsider all of the variables that would impact the strength of an interaction when you answer the following questions, and assume that the distance separating their sidechains stays about the same. ow would the strength and nature of their interaction change a) if the nonapeptide was placed in an environment of p 7.0? b) if the nonapeptide was placed in an environment of significantly increased ionic strength? 4

5 4) AE (angiotensin converting enzyme) has been studied in various medical treatments and AE inhibitors are used to treat congestive heart failure and hypertension. One class of inhibitors being studied includes simple tripeptides; Leu-Gly-Pro is one of the most effective. Draw this tripeptide as it would exist at a p of 7.2; be careful to show stereochemistry. (5 points) 5) onsider the polypeptides listed below. A: MEREDIT B: DWIGT : PAM D: PYLLIS E: STANLEY Write the letter(s) corresponding to the polypeptide(s) that fulfill each of the characteristics described below. If none of the polypeptides fulfill the specific requirements, write NONE in the box (7 points). a) as/have the highest A 280, assuming each was measured in solution at the same concentration: b) as/have the highest net charge at a p below 2: c) as/have the lowest net charge at a p of 12: d) Would be cleaved into smaller fragments by chymotrypsin: e) as/have the fewest formally charged atoms at a p of 7: f) If all five of these fragments were loaded onto an anion exchange column run at a p of 6.8 (using an increasing salt gradient to elute everything), in what order would they elute? (if any are expected to co-elute, circle them): 6) DMT (dimethyltryptamine), shown in its protonated form at right (pk a 8.8), is a psychedelic compound found in many plants and observed to act as a neuromodulator in animals. a) This compound has been found to bind to dopamine (D1) and other receptors in vitro with fairly high affinity based on its charged state. What is the ratio of protonated to deprotonated forms of DMT that exist when the solution has a p of 9.2? DMT N N b) When isolating this compound from plants, a p adjustment is done. Starting with 50 ml of a 1.5 M concentration of DMT at a p of 9, how much 0.1M l is needed to bring the p of the solution to 8.5? c) Generally, DMT is stabilized in a salt with fumarate (shown in its fully protonated form at right). Fumaric acid has two acidic protons with pk a s of 3.01 and At what p will fumarate carry a formal charge of -1.8? O O O O fumaric acid 5

6 7) A fully developed erythrocyte containing 35 mm NaO 3, 20 mm K 2 PO 4, and 20 mm K 2 PO 4 (as well as a negligible amount of other solutes) is traveling through the capillaries of the muscle where the osmolarity of the plasma was measured to be about 180 mosm. A) alculate the osmolarity of the environment within the erythrocyte: B) If the osmolarity of the surrounding plasma is due primarily to the buildup of sodium lactate (see structure at right), what is the molar concentration of this metabolite in those capillaries? blue O ONa O sodium lactate red ) Several protein-ligand binding curves are shown in the figure. What position(s) might best represent the hemoglobin in this erythrocyte under the conditions described above? Draw in where the hemoglobin should be in this figure under these conditions and label both axes. D) T/F about this situation and hemoglobin in general: a) The hemoglobin found within these erythrocytes would be expected to be mostly in the relaxed state. b) The erythrocyte is traveling in a hypotonic environment. c) The hemoglobin is likely to contain a maximum number of salt bridges. d) Osmotic pressure is directed into the erythrocyte. e) The hemoglobin is likely to be binding O 2 with a higher affinity than it will be as the erythrocyte passes the lungs. f) An increasing [BPG] means hemoglobin is able to pick up more O 2 even in the mountains when O 2 is scarce. g) The effect of BPG is best shown by a slight leftward shift of hemoglobin s binding curve with increasing [BPG]. E) In addition to binding oxygen, globins also function as heme-binding proteins. Globins bind to heme cofactors with a behavior that can be described using classical ligand-binding graphs. alculate the K d and K a for a pool of globin proteins using the following observation: 39% of the proteins have bound hemes when [heme] is 0.36 mm. Show your work (all equations) clearly; remember your units. (6 points) 6

7 8) Assume that each of the O 2 binding proteins is being assessed under the same conditions and concentration. For each protein, write the letter(s) of the observations (A-K) that would apply. Some descriptions may be used more than once, and some may not be used at all. (3 points each box) emoglobin Myoglobin A) ontains primary structure. B) ontains quaternary structure. ) Enzyme D) ontains heme E) Intermolecular salt bridges F) yperbolic ligand binding curve G) BPG is a ligand ) Localized to tissue I) Single O 2 binding state J) Typically found within nucleated cells K) α-helical structure predominates 9) To fully study how enzymes impact the rate of a chemical reaction by influencing the energy changes that occur, the active site of enolase is shown below with the outline of an energy diagram. (12 points) i) omplete the drawing on the left by adding the necessary cofactors (circles) and drawing in the structure of the substrate as it would be bound in the active site. ii) omplete the energy diagram by adding labels next to the letters below. iii) Draw the structure of the product of this reaction. iv) In your energy diagram, indicate where the enolase picture you drew on the left and the product you drew on the right would be represented in that diagram (use an arrow and * on the diagram itself). Fill in circles and substrate Draw product of reaction A E D B A: B: : D: E: 7

8 10) The following short answer questions are about protein isolation methods. Two-dimensional gel electrophoresis was performed on a mixture of proteins that are described on the table at right. Isoelectric focusing was used first, then an SDS-PAGE further separated the proteins in the second step (electrodes are labeled for each dimension). Protein pi M r (kda) Quaternary composition A None B α None D α 2 a) Based on the properties of these proteins, map the relative position of each on the gel oriented for you below and be sure to label each band with the protein letter clearly; if more than one band is expected to migrate together, label the band with each letter: (6 points) 1-D 2-D b) If these same four proteins were run on a molecular sieve (gel filtration) column, in what order would they elute? (if any elute together, please circle them as a group). c) If these same four proteins were run on a cation exchange column, (all proteins were initially bound and a salt gradient was used to elute all of them) in what order would they elute? (if any elute together, please circle them as a group). d) 100 mg of total protein (A, B,, D) were loaded onto the gel filtration column, and the fraction containing B contained only 12 mg of total protein. Assuming all of the activity of this protein (B) was retained, calculate the enrichment in protein purification (by specific acitivity) that occurred in this step. e) The column fraction containing B (12 mg from part d) was loaded onto another column and protein B eluted in a fraction containing 10 mg of protein but only half of the original activity. A single band was observed in native-page. Where would you expect to see this band compared to molecular weight standards, and what can you conclude about this step of the purification? 8

9 Name: hem 351 Exam 1 11) As you know, cyanogen bromide (BrN) is a chemical reagent that cleaves very specific peptide bonds. Its mechanism of action is very similar to the mechanism observed using phenylisothiocyanate in the Edman degradation process. First, a nucleophilc substitution (S is the nucleophile) leads to the release of bromide and a high energy, charged intermediate. In the second step, an intramolecular substitution results in the formation of a 5-membered ring (containing an oxygen atom) within the polypeptide substrate, and 3SN is released. This ring contains a resonance-stabilized carbocation. Using the abbreviated polypeptide below, show the curved arrow mechanism for the two-step reaction that leads to this resonance-stabilized intermediate. Show the carbocation product and its BEST allclosed shell resonance contributor. (12 points total) R N O N O R R 2 2 S Br N 3 12) The drawing at the right is a zoomed in view of the proteins that play a [B] role in muscle contraction. What do the letters represent? [A] [A] the individual unit: [B] the individual unit: : D) Assuming that contraction has just been stimulated, and all necessary ingredients are present, place the following figures (D-G) in order (1-4) as the events would unfold during contraction. D E F G E) Which of the above figures represents the high energy release causing the conformational change known as the power stroke? 9

10 13) Aromatase is a member of the cytochrome P450 family, many of which are involved in steroidogenesis. Aromatase catalyzes the formation of estradiol from testosterone. Inhibitors of aromatase have been used in the treatment of breast cancer, especially when that cancer has been found to be estrogen receptor positive. ertain mushrooms produce inhibitors of this enzyme, so they are being studied for their medicinal properties. Pertinent Information oncentration of Aromatase used in each assay: 18 nm V max was attained using a [testosterone] of 400 µm, V max = 16 µm/s In one particular assay, [testosterone] = 24 µm, and the v 0 was determined to be 6.8 µm/s For each of the following, DEFINE each term that is being asked for, and then do the math. A) What is the turnover number for this enzyme? B) What is the K m for this enzyme? ) What is the catalytic efficiency (or specificity constant) of this enzyme? D) Sketch a Lineweaver Burk transformation plot using the data above. Label axes, define and calculate intercepts and slope (5 pts) E) What would happen to the enzyme activity in the presence of Arimidex, a compound known to be a competitive inhibitor of Aromatase? Explain, and draw the expected effect on the LB graph above - Label it. F) What would happen to the enzyme activity in the presence of Letrozole, if that compound was found to be a noncompetitive inhibitor of Aromatase? Explain, and draw the expected effect on the LB graph above - Label it N. Multiple hoice /30 Page 6 /18 Page 9 /20 Page 4 /17 Page 7 /18 Page 10 /17 Page 5 /20 Page 8 /15 Total /150 (155) 10