Biochemistry problem You have purified a protein secreted by a bacterium and ana

Question

Biochemistry problem
You have purified a protein secreted by a bacterium and analyzed its function. You have found that protein to be a protease which cleaves peptide bonds only after acidic R-groups (Asp and Glu). You suspect the enzyme to function in a manner similar to the mechanism of chymotrypsin. How might you chemically determine whether the enzyme contains a Ser and His in its active site similar to those in the active site of chymotrypsin? How would you expect the active site of this enzyme differ from that of chymotrypsin (be as specific as possible in your answer)?

Explanation / Answer

Serine proteases like Chymotrypsin consist of a catalytic triad of Serine, Histidine & Aspartate at its catalytic site.

Serine’s –OH group acts as nucleophile & mediates a nucleophilic attack on the carbonyl carbon of the peptide bond to be cleaved. Histidine helps in this attack by making the oxygen of the –OH group more nucleophilic.

One way that can determine whether Serine is present at the active site of the enzyme:

Use of Serine inhibitors:

Chymotrypsin is inactivated by DIPF (Di-Isopropyl Phospho Fluoride). DIPF reacts with Ser 195 of Chymotrypsin & modifies it, thereby inactivating chymotrypsin. If DIPF can also inactivate this protease, then we can understand that it consists of a Ser residue at its active site, which plays a central role in catalysis.

I think the catalytic triads at the active site in both chymotrypsin & this enzyme will be similar; but there will be a difference in the substrate binding pockets (S1 pocket) for these enzymes due to difference in substrate specificity.

Chymotrypsin cleaves the peptide bonds after aromatic amino acids. Chymotrypsin has a Ser 189 residue at its S1 pocket that allows aromatic amino acid residues to position itself such that the peptide bonds following them are cleaved.

But this enzyme cleaves the peptide bonds after acidic amino acids, so it must have a basic (positively charged at physiological pH) amino acid at its S1 to attract the acidic (negatively charged at physiological pH) residues & position them such that the peptide bond following them is positioned for cleavage.

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