Bio 451 Examination I September 22, 1995 PLEASE PRINT YOUR NAME CLEARLY ON EVERY PAGE OF THIS EXAMINATION; IF THIS IS NOT DONE, WE WILL SET YOUR EXAM ASIDE WHEN THE EXAMS ARE TAKEN APART FOR GRADING. Answer questions I through X. Be as concise as possible. Please do not rewrite the question as part of your answer. You must write clearly; if we are unable to read your answer, we will not be able to give you credit. Please confine your answers to the page on which the question appears. The examination will begin at 8:20 a.m., and will end at 9:55 a.m. Score Summary: QUESTIONS MAX POINTS EARNED POINTS I... 12 II... 12 III.. 9 IV... 12 V... 7 VI.. 7 VII. 14 VIII. 5 IX.. 12 X... 10 1
I. (12 points) The following numbered statements seem to be contradictory. However, each is true, when taken in proper context; examples of experimental support for each statement were discussed in lecture and/or were clearly presented in specifically assigned sections of your text. You are asked to briefly summarize at the type of experimental support for each statement. For full credit, your answer must also include citation of at least one specific example for two of the three statements. A. The native conformation of a polypeptide is determined by its primary structure. B. The same primary segment can assume more than one stable conformation. C. Polypeptides having dramatically different primary structures can yield virtually identical tertiary conformations. 2
Amino Acid Three Letter One-letter Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Aspargine or Aspartic Acid Asx B Cysteine Cys C Glutamine Gln Q Glutamic Acid Glu E Glutamine or Glutamic Acid Glx Z Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V 3
II. (12 points) The composition of a peptide secreted by a culture of bacterial cells is: Amino Acid # Residues/peptide Cys 2 Arg 1 Glu 1 Ile 1 Leu 1 Considering ALL of the information listed below, deduce the most likely structure of the peptide. HINTS: Please use the single letter codes (see back of previous page) for the amino acids to write your final answer. ALSO: It helps (partial credit) to jot down what EACH observation tells you about the peptide. a. The titration curve for the peptide, over the range ph 2 thru 13, shows two, welldefined inflection points. b. The peptide: 1) does not react with iodoacetate 2) does not react with phenylisothiocyanate or dabsyl chloride 3) does not change in size when treated with trypsin c. The product of tryptic digestion reacts with fluorodinitrobenzene. d. After treatment with trypsin, three rounds of Edman degradation yielded the following partial sequence: Cys-Leu-Glu (Equipment malfunction prevented further analysis). e. Following incubation with excess 2-mercaptoethanol, the peptide binds two carboxymethyl groups when treated with excess iodoacetate. For full credit you must demonstrate that the structure you deduce is consistent with EACH of the stated experimental observations. 4
III. (9 points) ANSWER A OR B BUT NOT BOTH. IF YOU ANSWER BOTH QUESTIONS ONLY YOUR FIRST ANSWER WILL BE GRADED. A. Suppose you were given two frozen samples of purified collagen, one from a normal individual, and another from a person who is severely deficient in vitamin C. You are expected to compare certain of their structural characteristics. To your dismay you discover that, while thawing, the labels have fallen off in your water bath. A Bio 500 student suggests that you can readily identify the samples by determining their melting points. Explain the biochemical basis of this excellent suggestion. Be specific. HINT: Recall that vitamins act as cofactors for enzymes. B. Hemoglobin is a tetrameric protein consisting of two α and two β polypeptide subunits. The structure of the α and β subunits are remarkably similar to that of myoglobin. However, at a number of positions, hydrophilic residues in myoglobin have been replaced by hydrophobic groups in hemoglobin. How can this observation be reconciled with the generalization that hydrophobic residues fold into the interior of proteins? 5
IV. (12 points) The partial pressure of oxygen in the capillaries of active muscle is approximately 20 torrs, while that in the capillaries of the alveolar capillaries of the lungs is approximately 100 torrs. The P 50 of Mb and HbA for O 2 are 1 and 26 torrs, respectively. In Hb Ranier, Tyr 145 is replaced by His, thus modifying the salt bridges in deoxyhb Ranier ; P 50 for this mutant Hb is 12.9 torrs. Please address the following issues; in each instance, your rationale must be clear. A. Sketch the saturation curves for Mb, Hb, and Hb Ranier, and compare their relative affinities for O2. B. Would you expect the indicated salt bridge in deoxyhb Ranier to be stronger or weaker than that in deoxyhb A? C. Explain the fact that, as a means of compensating for the mutation, individuals with Hb Ranier exhibit polycythemia [an increase in numbers of erythrocytes (red blood cells) and in hemoglobin]. 6
V. (7 points) ANSWER A OR B BUT NOT BOTH. IF YOU ANSWER BOTH QUESTIONS ONLY YOUR FIRST ANSWER WILL BE GRADED. A. The graph below is a plot of v VS v/[s] for an enzyme which follows Michaelis-Menten kinetics. This type of plot is one of several linearized forms which are used to analyze such data. Which parameter(s) is (are) reflected by the slopes of the lines? What is the significance of the intercept on the v axis? One line represents data obtained in the absence of an inhibitor, while the other represents the results obtained with a noncompetitive inhibitor. Which is which? Explain your rationale. V A B v/[s] B. What is the significance of k 3 /K M or k cat /K M? Your rationale must be clear. 7
VI. (7 points) Several terms related to protein isolation, structure or characterization are listed below. Select the single statement which most accurately defines the term or describes its significance. A. Salting out --- a method of protein purification which is based primarily on difference among proteins in net charge. --- a separation method in which one or more proteins in a mixture are separated from others on the basis of differences in solubility. B. Immunoblotting --- a mode of protein detection based on the specific ligand:protein recognition. --- a method of protein detection based primarily on how tight the antibody binds to a nitrocellulose membrane. C. Iodoacetic acid --- reacts with disulfide bonded residues. --- forms a stable thioether with cysteine side chains. D. Molecular Chaperone --- may favor the On pathway, usually binds to hydrophobic patches; release requires energy. --- catalyzes the formation of the proper orientation of X-Pro bonds during protein folding. E. Off pathway --- a term used to describe non-productive pathways in protein folding. --- a metabolic pathway subject to strong feedback inhibition. G. pi --- the ph value at which a protein has no charges. --- the ph at which the net charge on a protein is zero. H. Circular Dichroism --- used to affirm the fact that the amino acid residues in proteins are of the L-configuration. --- provides information regarding changes in conformation of polypeptides. 8
VII. (14 points) The points assigned for this question reflect the number of correct answers which are possible. Choose the answer(s) which is (are) correct. A. With respect to enzymatic catalysis, the term steady state is best described as an experimental condition in which: --- -d[s]/dt = 0 --- the rate of change in the concentration of ES is approximately zero. --- initial velocity is zero order with respect to [S]. --- [E total ] remains unchanged. --- d[p]/dt = 0 B. For an enzyme which follows Michaelis-Menten kinetics, the substrate concentration required to reach a velocity which is only 1% lower than V max is: --- 10K M --- 30K M --- 80K M --- 99K M --- 300K M C. Those noncovalent interactions which affect k CAT --- are most important in the transition state. --- have little to do with recognition or affinity of substrate --- enhance K M by several orders of magnitude --- can include hydrophobic and ionic interactions as well as hydrogen bonds --- cannot be explored by site-directed mutagenesis D. Aspartate Trancarbamoylase (ATCase) --- a) Along with hemoglobin, the paradigm of allosteric proteins --- b) Decreases in activity on treatment with organomercurials --- c) Catalyzes the committed step in purine nucleotide synthesis --- d) Catalyzes the formation of carbamoyl aspartate from aspartic acid and carbamoyl phosphate --- e) Has interacting regulatory and catalytic sites on a single polypeptide chain F. Transition state analogs --- Fit better in the active site than the substrate --- Increase the rate of product formation --- Can be used to produce catabolic antibodies --- Are usually distorted or strained molecules --- Are potent inhibitors of enzymes (Continued) 9
G. Characteristics of the Bohr effect include --- Lowering the ph shifts the oxygen dissociation curve of hemoglobin to the right --- The acidic environment of an exercising muscle allows hemoglobin to bind O 2 more strongly --- The affinity of hemoglobin for O 2 is diminished by high concentrations of CO 2 --- In the lung, the presence of higher concentrations of H + and CO 2 allows hemoglobin to become oxygenated --- In the lung, the presence of higher concentrations of O 2 promotes the release of CO 2 and H + VIII. (5 points) The following five proteins are listed with their corresponding molecular weights and isoelectric points (pi s). If the five proteins were separated in an isoelectric-focusing experiment, what would be their distribution between the positive (+) and negative (-) ends of the gel? Molecular weight pi (daltons) --------------------- -- (a) α-antitrypsin 45,000 5.4 (b) Cytochrome c 13,400 10.6 (c) Myoglobin 17,000 7.0 (d) Serum albumin 69,000 4.8 (e) Transferrin 90,000 5.9 Indicate the high and low ph ends. Cathode (-) (+) Anode 10
IX. (12 points) Certain chloromethylketones, acting as affinity labeling reagents, irreversibly inactivate certain serine proteases by alkylating His residues. Since pancreatic proteases are very potent and similar in structure, it is often difficult to obtain completely pure samples devoid of contaminating enzymes having different specificities. Preparation of meaningful peptide maps requires the use of monospecific protease preparations. A frequent problem is requirement to preferentially inactivate chymotrypsin or trypsin, so as to obtain a preparation with a single enzymatic activity. This can be done by use of an appropriate chloromethyl ketone. A. Why would the reaction with the chloromethylketone inactivate a pancreatic protease? How many His residues per molecule of protease would you expect to react? Be as specific as possible. Your rationale must be clear. B. If you wished to obtain monospecific chymotrypsin [devoid of trypsin activity], which of the following chloromethylketones would you use to pretreat your protease preparation. Be as specific as possible. Your rationale must be clear. C. Such reagents do not react with denatured pancreatic proteases. Why? 11
X. (10 points) Shown below are three binding isotherms, each collected at a different temperature, for the association of ATP with a two-site transcriptional regulatory protein. From previous studies you that K 1 = K 2 (i.e., ATP binds to either site with equal affinity), and that these two association constants are temperature-invariant over the range 20 to 37 degrees Celsius. Using the binding expressions given below, explain how temperature affects the properties of this system in terms of the binding equilibria. What can be said about the cooperative binding of ATP [X] to this protein? Be specific. _ (K 1 + K 2 )[X ] + 2K 1 K 2 K c [X] 2 Y = ---------------------------------------- 1 + (K 1 + K 2 ) [X } + K 1 K 2 K c [X ]2 FOR EXTRA CREDIT: (5 points) A common method for representing the T-dependence of binding data is a van t Hoff plot of 1n K versus 1/T. Given the expression below, what would a van t Hoff plot of your data in part A reveal about the enthalpy ( H) of ATP binding in this case? Show your work. G = -RT1nK = H -T S 12