K. J. Oh, J. Churchich
Aug 10, 1974
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Journal
The Journal of biological chemistry
Abstract
Abstract The enzyme cystathionase from rat liver is inactivated by incubation with 5,5'-dithiobis(2-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 (20), 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.