Active site of trypsin-like enzyme from Streptomyces erythreus: Specific inactivation by new chloromethyl ketones derived from [formula omitted] and [formula omitted

Abstract

In order to elucidate the active site of the trypsin-like enzyme from Streptomyces erythreus by the use of site-specific reagents without isotope labeling, l-i-chloro-3-(2,4-dinitroanilino)-7-aminophetan-2-one (DLCK), i.e. N a -2,4-dinitrophenyllysine chloromethyl ketone has been examined in comparison with l-i-chloro-3-tosylamido-7-aminoheptan-2-one (TLCK) as an active site-directed irreversible inhibitor. Although the apparent rate of inactivation of the enzyme by DLCK was about half of that by TLCK, it was shown that complete inactivation by DLCK involves a stoichiometric reaction which can be determined by spectral analysis of the 2,4-dinitrophenyl group, and amino acid analysis of the inhibited enzyme reveals loss of one histidine residue. It was also shown that, like TLCK, DLCK inactivates bovine trypsin irreversibly, though with a rate of inactivation about five times less than that by TLCK. In addition, a detailed study of the inactivation reaction of the chloromethyl ketone from N a- tosyl- l-arginine , l-i-chloro-3-tosylamido-6-guanidonhexane-2-one (TACK) with trypsin and the trypsin-like enzyme from S. erythreus showed that there is little difference between the reactivities of TACK and TLCK to the trypsinlike enzyme, though TACK inactivates trypsin more rapidly than TLCK does.