Reaction of cystathionase with the fluorescent probe bis(dansyl)cystine.

The enzyme cystathionase from rat liver is inactivated by incubation with 5,5’-dithiobis(Z-nitrobenzoic acid) at pH 7.4. The reaction of four -SH groups per mole of enzyme brings about 95% loss of the homoserine deaminase activity. Kinetically, the reactive --SH groups can be classified into two classes. The substrate L-homoserine or the competitive inhibitor L-alanine has no effect on the rate of inactivation. The thiol groups of the enzyme undergo an exchange reaction with the dye bis(5-dimethylaminonaphthalene sulfonyl)L-cystine (bis(dansyl)cystine). The reaction of approximately two -SH groups with the dansylated reagent causes 40% loss of the homoserine deaminase activity. The absorption and fluorescence properties of the dansylated enzyme were used to gain information about the microenvironment surrounding the reactive -SH groups. The fluorescence properties--emission maximum (515 nm), relative fluorescence yield (ZO), and fluorescence decay time (18 ns)-of the dye bound to the enzyme are identical to the fluorescence properties of bis(dansyl)cystine in a nonpolar mixture containing dioxane: water (95:5). On the basis of these results, it is proposed that the -SH groups which react with bis(dansyl)cystine are located in a nonpolar environment. The inactivation of the enzyme by bis(dansyl)cystine cannot be related to dissociation of the cofactor pyridoxal-5-P from the catalytic site.