Question 4: The catalytic mechanism of anolein Studies have suggested that anole

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Question 4: The catalytic mechanism of anolein
Studies have suggested that anolein functions as an acetylcholinesterase using an active site serine, with a catalytic mechanism nearly identical to that seen in chymotrypsin.

CH CH 3 3 H20 CH3 ?.? CH ?? CH H2C ?? acetylcholine choline acetic acid a) Postulate a reasonable mechanism for anolein activity; include in your model the following features: nature of the catalytic triad, role of the oxyanion hole, nucleophile, tetrahedral intermediate, acid-base catalyst. You may use a diagram as long as all required elements are present and defined clearly Upon analysis of the x-ray crystal structure of anolein, you have detcrmined the location of the putative oxyanion holc. Compared with that obscrved in chymotrypsin whcre two o-carbon backbonc amide NH groups provide hydrogen bonds to the oxyanion, in anolein it appears that the side chain of Asn-155 provides one of these interactions. To test this hypothesis, you performed site-directed mutagenesis to substitute a leucine residue for Asn-155 b) Draw the structure of both leucine and asparagine as they would appear at pH 7.0, and explain why you chose this amino acid substitution a. b. Kinetic analysis of the Leu-155 mutant show a nearly 300-fold decrease in kcat with little to no effect on KM. Explain these observations

Explanation / Answer

a)

b)

c)

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