Chemistry 27 Professor Matthew Shair. Harvard University Spring Hour Exam 2 Friday, March 24, :07 AM 12:00 PM

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1 ame: Chemistry 27 Spring 2006 Exam II Chemistry 27 Professor Matthew Shair arvard University Spring 2006 our Exam 2 Friday, March 24, :07 AM 12:00 PM iscussion Section (ay, Time): TF: irections: 1. o not write in red ink. othing in red ink will be graded. 2. Write your name on every page of the exam. 3. Provide your answers in the designated space. 4. The last page of the exam has a table of amino acids and cofactors. You may use the back of this page for scratch work but nothing written on this page will be graded. 5. You have 10 total pages. We have provided plenty of space for each answer. 6. All intermediates and proton transfers must be drawn as distinct steps unless explicitly stated in the question not to. 7. You do not need to draw out full structures of cofactors. Just draw out the reactive portions when required. 8. Molecular model kits and calculators are permitted. 9. A significant fraction of your exams are photocopied before they are returned. Question Score 1 /25 2 /20 3 /25 4 /30 Total /100 Page 1 of 10

2 ame: 1. [25 points] An artificial enzyme called Q synthase was engineered by creating multiple mutations in Q synthase. Q synthase was shown to efficiently convert 3deoxy arabinooctulosonic acid 7phosphate (AP) to methyldehydroquinate (Q) through intermediate 1. Both (7R) and (7S)AP were found to be substrates for the enzyme. C C (7R)AP (7S)AP 7 P P 2 3 Q synthase, A C 1 Q synthase Q (a) Provide plausible mechanisms by which (7R)AP and (7S)AP are converted to intermediate 1. You do not need to draw out mechanisms for steps involving A or A. You should show all other intermediates in your mechanisms. [15 points] (7R)AP A C B: or P syn elimination C P3 C (7S)AP A C P 3 2 C P 3 1 B: anti elimination 1 for showing A oxidations, 7 for each mechanism 4 for showing enolate formation, 3 for showing syn/anti elimination. 15 total Page 2 of 10

3 ame: (b) Why are (7R)AP and (7S)AP both converted to intermediate 1 with a single double bond geometry? What forces are responsible for controlling the double bond geometry of 1? Use structures you have drawn in part (a) to illustrate your answer. Explain your answer in, at most, two sentences. [6] (7R)AP minimize A 1,3 strain (7S)AP C C P3 P 3 minimize A 1,3 strain Syn elimination for (7R), anti elimination for (7S). A 1,3 strains dictates the stereochemistry of elimination 3 for showing each A1,3 strain. 6 total (c) raw the resulting structure of Q from intermediate 1, indicating the stereochemical configuration of the carbon containing the methyl group. Assume that the subsequent reactions catalyzed by Q synthase are the same as Q synthase. [4] C Q Stereochemistry at C containing = (R) 2 for structure, 2 for correct stereochemistry 4 total Page 3 of 10

4 ame: 2. [20 points] A mutation occurred in the E. coli Chorismate Mutase enzyme and the resulting mutant enzyme (CMmut) was found to convert deuteriated chorismate 2 to a mixture of products 3 and 4 as shown below. C C CMmut C C C C : 1 (a) raw the transition states by which 2 is converted to 3 and 4. [14 points] C C 3 boat TS C C 4 chair TS 7 for each structure 1 3, 4 correct structure/conformation, 2 stereochem total = 14 8 only, if correct but mixed up. Page 4 of 10

5 ame: (b) Below, the Xray crystal structure of CM bound to a transition state analog reveals the active site. Replacement (via mutation) of one amino acid in the CM active site caused CMmut to favor 3 over 4. Your answer need only mention one of two possible amino acid substitutions to get full credit. Explain your answer in, at most, two sentences. [6 points] Mutate Arg11 to Glu11 or Asp11 (R11 E or R11 ). This causes repulsive interactions between the carboxyl group of Glu/Asp11 and the carboxyl on the substrate, resulting in the favor of the BAT transition state. 2 for mutating Arg11', 2 for mutating to Glu/Asp, 2 for explanation 6 total 1 only, for mutating to hydrophobic residue and mentioning sterics Page 5 of 10

6 ame: 3. [25 points] An amazing new enzyme, epoxyreductase (ER) was discovered that uses two cofactors, PLP and A. This enzyme converts the epoxy aminoacid X to amino acid Y in an overall reductive reaction. C 2 2 X R PLP C 2 ER Y C 2 2 PLP R C 2 (a) Provide a plausible mechanism for the ERdependent conversion of X to Y. You can start with the PLPenzyme imine and you do not need to draw imine hydrolysis or transimination. o not worry about rationalizing the stereochemistry in this answer. [20 points] B: A LysEnz transimination C 2 C 2 C 2 C 2 2 C 2 R C 2 aminolysis or hydrolysis C 2 A C for indicating transimination from PLPenz imine, 4 for alpha deprotonation and pushing electrons into sink, 4 using electrons in sink to open epoxide, 6 for A attack on double bond and pushing electrons into sink, 2 for using electrons in sink for alpha protonation, 2 for indicating hydrolysis/aminolysis. 20 total Page 6 of 10

7 ame: (b) The ERdependent reduction of X stereospecifically affords Y as shown in the equation given. raw one image in 3 that rationalizes the stereochemical control of the formation of the deuteriated carbon in Y in this reaction. We suggest that you use one of your intermediates from part (a), but drawn in 3. [5 points] R C 2 C 2 or R C 2 C 2 Page 7 of 10

8 ame: 4. [30 points] A process known as trans splicing allows the ligation of extein fragments that exist on different polypeptides (see diagram). The trans splicing activity is derived from the polypeptide that contains the Cterminus extein (i.e. extein 2). 3 EXTEI 1 PEPTIE 1 C 2 3 C 2 SERIE PRTEASE/ITEI 2 EXTEI 2 C 2 TSplice Trans Splicing 3 EXTEI 1 EXTEI 2 C 2 A novel enzyme TSplice that performs trans splicing was found to possess features of a typical serine protease and some features of an intein as shown above. Page 8 of 10

9 ame: (a) Provide a plausible trans splicing mechanism using the initial template provided. ote that the Asp, is and Ser on TSplice still make up the catalytic triad of a typical serine protease. You T need to draw out all tetrahedral intermediates in the mechanism BUT you need to show all necessary interactions and proton transfers. [30 points] 3 EXTEI 1 PEPTIE 1 C 2 2 EXTEI 2 C 2 SERIE PRTEASE/ITEI 3 EXTEI 1 PEPTIE 1 C 2 2 EXTEI 2 C 2 2 PEPTIE 1 C EXTEI 1 SER PR A 3 EXTEI 2 C 2 EXTEI 1 B: A EXTEI 2 C 2 B: 3 EXTEI 1 EXTEI 2 C 2 Page 9 of 10

10 ame: 10 for serine protease mechanism 2 for showing Glu/is interaction and is deprotonation of of Ser, 2 for Ser attack on amide, 4 for abbreviated arrows on carbonyl, 2 for protonation of leaving R by is. (0 for protonation by general base) 20 for Splicing mechanism 8 for using Asp 2 to attack amide bond of AspSer, 2 for drawing resulting fivemembered ring pdt, 8 for using alpha 2 to attack acylser ester, 2 for drawing final spliced pdt. 30 total Page 10 of 10