Winter 2008 Final Key

Winter 2008 Final Key - lof3 BIS 102 Name k 29 Winter, 2008...

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Unformatted text preview: lof3 BIS 102 Name k 29 Winter, 2008 Last First K. Hilt Final Exam Score (200): Equations: pH = pK, + log {[b]/[a}} K, = 2/(y-x) Kb = x2/ (y-x) pH = (pKa‘ + pKa2)/2 (K,)(Kb) = 1 x 10'14 1==(q1 q2)/ar2 AG=AH-TAS v.,= {me[S]}/ {Km+[S]} Amino acid pKa’s: or-carboxyl group (2.1), (x-amino group (9.6) side chains: D (3.9) E (4.2) H (6.0) C (8.3) Y (10.1) K (10.5) R (12.5) Oligopeptide pKa’s: C-terminal carboxyl group (3.6), N—terminal amino group (7.4) This final is all about enzyme Z, the enzyme that you have purified and are characterizing. l. (20 pts.) To purify enzyme Z, you need to make up 20 liters of 50 mM phosphate buffer, pH 7.6. The stock solutions that you have are 3.0 M H3PO4 and 2.0 M NaOH. The pKa’s of phosphoric acid are 2.12, 7.21, and 12.32. Put your answers for the following items, on each line. To receive credit, you must show your calculations on the back of this page. 333 m13.0 M H3PO4 8’55 ml 2.0 M NaOH lg 81 2 ml H20 + 3 + H + 3 2. Amino acid analysis of your pure protein Z was done. (10 pts.) a) How would that be accomplished? Assume that you have pure enzyme Z in your pH 7.6 bufier mentioned in question #1. What are all of the reagents and steps that you will need to determine the amino acid analysis of your enzyme, in your lab? ‘ I +3 /. 6” 7161‘ 710°C, WM‘JM- ————-? £7“— 44-5 +3 2, (M «W column +— Ina/Hus (30 pts.) b) Let’s imagine that the amino acid analysis of your pure enzyme Z gives the following data: --2 {—w “~755ij Number of w.ch l (or Residues in line: ’ 1?. ‘7 8' . To receive credit, show all calculations on the back of this or another page. + 7. (10 pts.) c) Based on the data in part “b” above, which cutting tool sin, or chymOtrypsin, would be best to use to make best use of the protein sequenator, for sequencing? Explain. M M Fir-«9W ac. it.ng ‘40 4.64. “914431454? E‘nw-E- 11% +2 q ‘ ~ I 20 ream. H0 :g', M m 5— ”; ‘ 20f3 BIS 102 Name ég 3. (20 pts.) Your enzyme Z was completely digested with trypsin and an internal peptide (i.e. not derived from the N- or C-terminals) was purified. Determine its sequence from the following information: a) amino acid analysis of one mole of the peptide yields three moles D, one mole M, three moles S, one mole R, and one mole W; b) treatment of the intact peptide with asp-N gives a tetrapeptide containing S, M, D and W; free D; free S; and a tripeptide containing R, D, and S; 0) treatment of the original intact peptide with CNBr yields a tetrapeptide and a pentapeptide; d) treatment of the original intact peptide . . . . . . p v N ' p ‘ w1th chymotrypsm yields a tripeptrde and a hexapeptrde. 493' 0;? (am “M: In”, w Give the one-letter code se ence of the original intact peptide 5" D ’ b N "' -- S ‘ K ‘i 9099*“ 5'D—wbD—b-S R ‘ M5 m ej-Haw curd-W ‘3 “6" WW"“ 4. (25 pts.) Enzyme Z catalyzes the reaction: S+ :1 P + H+ You are assaying enzyme Z from lefi to right, under Vmax conditions. Imagine that thirty I.U.’s of enzyme Z were catalyzing the reaction for two minutes, under Vmax conditions, in a 3.00 ml assay volume. The assay is buffered with your 50 mM phosphate buffer, pH 7.6 (from question #1 of this exam). What will the pH be at the end of two minutes? Put your answer on this line: pH = Q . Yb . To receive credit, show your work on the back of this or another page. 5. (10 pts.) Enzyme Z shows Michaelis-Menten type of kinetics. Vmax for the enzyme is 80 nmol P/min. When the enzyme is assayed at 1.0 uM substrate concentration, the initial velocity is 15 nmol P/min. What is the KIn for the enzyme? Show all calculations, in the space below. Km=_i?7zzt1_(give units) 80 “we P/M {t CH) d : UM L31 [5 nmeQ9/Ming- 0 KM + [5-] 6. Phosphatidylcholine turns out to be a noncompetitive inhibitor of enzyme Z. (10 pts.) a) Draw the complete structure of phosphatidylcholine, in the form that would predominate at pH (10 pts.) b) Sketch what the Lineweaver—Burk diagram would look like, with and without inhibitor. Label your plots, axes, and x-axis and y—axis intercepts. 3of3 BIS 102 Name % 7. Pure enzyme Z is analyzed using the three gel types that we discussed in class: IEF, native PAGE, and SDS-PAGE. The intact enzyme turns out to be a hexamer. It consists of two identical 20,000 MW subunits, one 10,000 MW subunit, and three identical 40,000 MW subunits. 41,59. Ho.) 1: \1 0, mm (20 pts.) a) Sketch what each of the following gels should show, assuming that each gel is capable of showing maximally what it could, i.e. no gel is “hiding” anything. Label each band(s) in each gel with its MW. Native PAGE SDS-PAGE IEF + l * 5 + 5 + The positive electrode (+) is indicated for each gel. Both the native and SDS—PAGE gels have stacking gels, but do not worry about them. (15 pts.) b) Describe how the above subunit composition of enzyme Z was determined. Which gel(s) had to be used? What standards, if any, bad to be used? Be very specific and clear in your discussion. Restrict your answer to the space below. + 3 _+2. Ida-49- a. wad-W 5W.+o «30" W'- MHw*"Mw. COW +22 M Mu) Adaan W a; 595’ pkéfi gé ‘ MM) 5b$ NM.) Em 2%. +3 +2 8. 20 ts. Puree eZ ave the followin ' etic Ho tima data: ( P ) nzym g ‘ }“ kafmA’p‘L p/ caswfl. in hm hm l {oi Mafia. “ *7" : 1 12 H ka \ W 3 ekfié’wg' e ¢?r( W Which amino acid residues do you think might be at the active site of enzyme Z? What are they doing? Defend your igmre'tation of the data. Magim"‘g.,‘xukme that u a macaw .ar Wu cm .W‘ w as“? am su— mumj (awnj) 5+. g 1 k my...“ mm.» B M be gut/ti 04219. M N 'nuclIco-(VM)__ (20 2,)(50 M4 FWWB :- ézoLXomso M) = / W42. P / n’wee P = (x £jC3M lag/ML» x : 4.333i = 333 m2 or L7] man-ea 0h“- 0.003 2. kW "-—‘- 5‘0 MM 9%! 9H» \0 Wm *1 java. (.44 0.7l b a.’31 “a (0.7!)(5‘0 Mn) : 3&5 MM “L” . n+5 MM "‘3' _ 09‘3— [W] «"44 ’ «‘9 ” “’3 m +ch I 3513-- 20 = 7.2! + (01} [454.20 I5.5‘ us 7~2l 4' [07' 34,)" -.-. I7‘2; +- (a; 0.44? = 7.2: + (~ 0.3%?) ‘2. (9.3% 7:49 H ll ...
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This test prep was uploaded on 04/07/2008 for the course CHE 8B taught by Professor Lievens during the Fall '07 term at UC Davis.

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Winter 2008 Final Key - lof3 BIS 102 Name k 29 Winter, 2008...

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