Question

Lysines and cysteines are often important active site residues, making them important residues for developing chemical uncaging strategies in enzymes. You would like to conduct an experiment in which you want to study the interplay between two enzymes: KINS1 is a kinase with a cysteine active site, and PROT1 is a protease with a lysine active site. You want to see how the activity changes between KINS1 and PROT1 when one is inactive. In your set of analytical tools are antibodies for known substrates of KINS1 and a fluorogenic substrates for PROT1.

A) Design an experiment in which you answer the question, "Does the activity of KINS1 increase or decrease when PROT1 is inactive?" Your answer must have an annotated scheme, appropriate controls, and your expected results.

Answer 1

Answer- For this the vectors will be prepared, one of them will be having both of the genes in a normal stage while in other vector the sequence will be inserted having mutation or variation in the gene KINS1 while the PROT1 will be normal, in third vector the sequence with hyperactivity will be inserted. The vector will express the gene accordingly and the antibodies will react with the substrate if it will be present. The observation of the reaction will reveal about the expression of these genes and their relatedness.