Question

Trypsin is a proteolytic enzyme that displays a similar mechanism to the studied for chymotrypsin
(serine protease). Tosyl-L-lysine chloromethyl ketone is a specific inhibitor of trypsin. Why is this
compound specific for trypsin?

Answer 1

Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride has been used:
• in chymotrypsin purification to prevent binding of trypsins to the affinity support by inhibiting them in the crude extract as a protease inhibitor in tissue and cell homogenization
as a trypsin inhibitor to determine the specific protease activity levels

Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) blocks the lipopolysaccharide (LPS)- or cytokine-induced activation of nuclear factor κB (NF-κB), which, in turn, blocks the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) transcription. Blocks activation of pp70s6k by all mitogens. Blocks apoptosis in cell lines by inhibiting the processing of caspases in some cell lines and to some stimuli.

Biological description

Irreversible inhibitor of trypsin and trypsin-like serine proteases. Blocks LPS- or cytokine-induced NF-κB activation. Inhibits caspase processing; blocks apoptosis. Active in vitro and in vivo.

TPCK is an irreversible inhibitor of chymotrypsin. Also inhibits some cysteine proteases such as caspase, papain, bromelain or ficin. It does not inhibit trypsin or zymogens.

TPCK is observed covalently bound in the active site of Caspase 3 in the crystal structure of the complex solved in 2010.The chloromethyl group reacts with the active site cysteine to form a covalent bond with the loss of the chlorine.

TPCK is chosen for the chemical labelling of active histidine in enzyme analysis. The phenylalanine moiety is bound to the enzyme because of specificity for aromatic amino acid residues at the active site (as in chymotrypsin, in which it binds to the Histidine-57 residue in the active site)