24] Two-protonic-state electrophiles as probes of enzyme mechanism

Abstract

A two-protonic-state electrophile is a compound that exhibits different electrophilic reactivities in each of two protonic or ionization states and can be used as reactivity probes with special characteristics that permit the detection and characterization of interaction in molecules in general and in enzyme active centers. In particular, it is used as reporter-group delivery vehicles and in the isolation of thiol-enzymes by covalent chromatography. This chapter presents the theoretical basis for the use as reactivity probes of two-protonic-state electrophiles in general and reviews the applications of pyridyl disulfides in the isolation and study of enzymes. Disulfides containing the 2-pyridyl moiety have been shown to be exceedingly versatile and valuable reagents for the isolation and study by both structural and kinetic methods of thiol-containing molecules. Covalent chromatography is a rapid isolation technique for thiol-enzymes and peptides, and provides considerable economies in both time and materials. It is reported that to draw mechanistic conclusions from kinetic data obtained by using reactivity probes, it is important to take account of the possibility that reactions of enzyme nucleophiles with site-specific reagents that are not formally substrate analogs might well proceed through the intermediacy of adsorptive complexes, albeit characterized by rather large dissociation constants.