Question

Any help would be appreciated!

N-tosyl L-AA-chloromethylketones (see structure to the right) have proven to be effective irreversible inhibitors of members of the family of serine proteases. Given what you have learned about the substrate specificity pocket in this class of enzymes, which of the compounds listed below would you expect to be the most effective inhibitor for elastase? CIRCLE the most correct answer. R = Met side chain R = Leu side chain R = Ala side chain R = Gly side chain R = None of the above To which of the following general classes of inhibitor do all the compounds in (1) above belong CIRCLE the most correct answer. You are working for a pharmaceutical company trying to come up with a clinical treatment for people with severe emphysema. To test the potency of your choice of elastase inhibitor, you first test out a fluorimetric assay for detecting elastase activity. The assay you choose measures the release of highly fluorescent 7-amino-4-methyl-coumarin (AMC) [ lambda max excitation = 380 nm; lambda max emission = 460 nm] from a non-fluorescent synthetic peptide substrate, Ac-AAX-AAY-AAZ-AAR-AMC [in which the amino group of AMC is in amide linkage to the C-terminal COOH of the tetrapeptide]. Your boss hands you a preparation of elastase that has been in the company's freezer for ten years. So, you first need to determine its specific activity. You take a 10 pi sample of the purified preparation [1 ml, 2 pg/ml] and add it to 90 pi of assay buffer containing 20 mu M Ac-AAX-AAY-AAZ-AAR-AMC and, as measured by the fluorometric assay, you find it was able to generate 100,000 nmoles of product when your assay was conducted for 10 min at 25 degree C. What is the specific activity of the purified enzyme (in units/mg, where one unit is 1 mu mole of product per min) in this decade-old preparation? SHOW your calculations and BOX your final answer clearly.

Answer 1

1. c. R= alanine side chain

2. a. Active-site affinity tag

3. I am not sure of a correct answer, please find it somewhere else.