Kumamolisin is a serine protease discovered in extremophilic bacteria that live

Question

Kumamolisin is a serine protease discovered in extremophilic bacteria that live in hot, highly acidic geothermal pools. Since it has high activity at pH 3, it is being developed as a digestive aid to break down the protein gluten. People with celiac disease, who are allergic to gluten, could take a pill of modified kumamolisin with their meals to destroy the gluten protein in the acidic stomach.

The crystal structure of kumamolisin covalently complexed via serine-278 with a peptide intermediate has been solved. The image below shows the active site. The peptide substrate is dark grey, and kumamolisin is white. Oxygen atoms are black, and nitrogen is grey.

A. What are the likely roles of glutamate-78, aspartate-82 and serine-278?

B. Which catalytic triad residue of kumamolisin is different from the serine protease chymotrypsin? Why?

C. Draw the mechanism of peptide bond hydrolysis by kumamolisin. Label oxyanion intermediate/s.

Explanation / Answer

A) Glutamate-78, aspartate-82 and serine-278 forms the catalytic triad of kumamolisin which catalysis the hydrolysis of peptide bond. Glutamate and Aspartate are negatively charged acidic amino acids while serine is a polar amino acid which acts as a nucleophile at the enzyme's active site. And, glutamate and aspartate helps in nucleophile activation.

2) Glutamate-78 is the catalytic triad residue of kumamolisin which is different from the serine protease chymotrypsin. Histidine is part of cataytic triad of chymotrypsin which is replaced by glutamate in kumamolisin. Because, glutamate is acidic amino acid which is essential for the enzyme to be active in extremophilic bacteria while histidine is a positively charged or basic amino acid.

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