Naja atra cardiotoxins enhance the protease activity of chymotrypsin

Abstract

Snake venom cardiotoxins (CTXs) present diverse pharmacological functions. Previous studies have reported that CTXs affect the activity of some serine proteases, namely, chymotrypsin, subtilisin, trypsin, and acetylcholinesterase. To elucidate the mode of action of CTXs, the interaction of CTXs with chymotrypsin was thus investigated. It was found that Naja atra CTX isotoxins concentration-dependently enhanced chymotrypsin activity. The capability of CTX1 and CTX5 in increasing chymotrypsin activity was higher than that of CTX2, CTX3, and CTX4. Removal of the molecular beacon-bound CTXs by chymotrypsin, circular dichroism measurement, and acrylamide quenching of Trp fluorescence indicated that CTXs bound to chymotrypsin. Chemical modification of Lys, Arg, or Met residues of CTX1 attenuated its capability to enhance chymotrypsin activity without impairing their bond with chymotrypsin. Catalytically inactive chymotrypsin retained the binding affinity for native and modified CTX1. CTX1 and chemically modified CTX1 differently altered the global conformation of chymotrypsin and inactivated chymotrypsin. Moreover, CTX1 did not reduce the interaction of 2-(p-toluidino)-naphthalene-6-sulfonate (TNS) with chymotrypsin and inactivated chymotrypsin. Together with previous results revealing that TNS can bind at the hydrophobic region of active site in chymotrypsin, our data suggest that CTXs can enhance chymotrypsin activity by binding to the region outside the enzyme's active site.

Keywords: Cardiotoxin; Chemical modification; Chymotrypsin; Modulation of enzymatic activity.