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Gluten Destruction: Future Directions

Kinetic Characterization

We intend to develop a mass spectroscopy assay to measure the kcat (the rate of turnover per molecule of enzyme) and kM (the substrate binding constant) values for our best mutant.

Specificity Studies

Crystal Structure

Protein structures can be obtained by crystallizing the protein, then bombarding it with X-rays and analyzing the diffraction pattern. Several crystal structures for wild-type Kumamolisin-As have already been published. To obtain further structural information, we hope to eventually obtain a crystal structure of our best mutated enzyme (G319S, D358G,D368H, N291D) bound to the PQLP model substrate. This will involve mutating one of the residues in the catalytic triad, so that the substrate will remain bound without being cleaved. The structural information that we may be able to glean from such a structure will allow us to better characterize, and perhaps further improve our mutant.

Biophysical Characterization

Once the ideal mutations are isolated, we intend to test the best mutants at gastric pH and in the presence of other gastric enzymes for a short period of time, mimicking the environment after enzyme ingestion and prior to enzyme uptake by the small intestine. We suspect that the thermostable properties of Kumamolisin-As will render our mutant enzyme reasonably resistant to degradation by gastric enzymes such as pepsin. However, if this proves not to be the case, we intend to reengineer the mutant for enhanced resistance to pepsin and other such gastric enzymes. Once ensured that the mutated Kumamolisin-As remains active under stomach conditions, this ideal mutated enzyme will be ready for in vivo experimentation.

Comparison of Theoretical Pills of ScPEP and Kumamolisin (G319S, D358G,D368H, N291D)

Therapeutic Promise

After successful completion of in vivo experimentation and clinical trials, treatment in capsule-form can be released for commercial use. Due to the much higher activity of our Kumamolisin mutant over SC-PEP, the amount of enzyme necessary for treatment can be greatly reduced.