The thiol groups of rat liver cystathionase. Influence of pyridoxal phosphate, L-homoserine and L-alanine on the effect of p-chloromercuribenzoate and 5,5-dithiobis-(2-nitrobenzoate) on the enzyme.

Rat liver cystathionase (homoserine hydrolyase) catalyzes the deamination of homoserine and the desulfhydration of cysteine. A study has been made of the SH groups of the highly purified enzyme. The titration of the SH groups by p-chloromercuribenzoate and 5,5′-dithiobis-(2-nitrobenzoic acid) indicated that the enzyme contains approximatively 12 SH per mole (mol. wt. 170000) of native enzyme and 20 SH per mole of urea-denaturated enzyme. On the basis of their reactivity towards SH reagents the SH groups of cystathionase have been classified into 4 classes. Addition of pyridoxal phosphate, the coenzyme of cystathionase, prior to treatment with SH reagents did not modify the availability of SH groups for SH reagents; however, addition of homoserine, a substrate for cystathionase, or of l-alanine, a competitive inhibitor for both homoserine deamination and cysteine desulfhydration, led to a striking protection of several SH groups, 4 and 8, respectively. The over-all results are discussed in relation to the hypothesis of a “double” active center composed of one site necessary to the binding of homoserine and one other involved in the binding of cysteine. In addition the hypothesis that cystathionase may consist of subunits is considered, taking into account the symmetry of the SH groups in each class and the number of pyridoxal phosphate binding sites on the protein.