Question

In Enzyme kinetics can someone explain how color develops by explaining the chemical reaction that occurs with BAPNA.

also Draw the structure of benzamidine and comment on the type of inhibitor that benzamidine is and how this relates to its structure.

Answer 1

Chemicla rection that occurs with BAPNA :

Trypsin, an enzyme produced by the pancreas, hydrolyzes proteins to small fragments (proteoses, peptones, and peptides).

BAPNA is a synthetic (man-made) protein substrate consisting of a dye covalently bound to an amino acid.

Trypsin hydrolysis of BAPNA cleaves the dye molecule from the amino acid, causing the solution to change from colorless to bright yellow.

Since the covalent bond between the dye molecule and the amino acid is the same as the peptide bonds that link amino acids together, the appearance of a yellow color indicates the presence and activity of an enzyme that is capable of peptide bond hydrolysis.

Because the color change from clear to yellow is direct evidence of hydrolysis, additional tests are not required when determining trypsin activity using BAPNA.

Benzamidine is a reversible competitive inhibitor of trypsin, trypsin-like enzymes and serine proteases.

It is often used as a ligand in protein crystallography to prevent proteases from degrading a protein of interest, the triangular diamine group at the bottom gives it a very obvious 'stick-man' shape which shows up in difference density maps.

The benzamidine moiety is also found in some pharmaceuticals, like dabigatran.