抄録
It was demonstrated that γ-cystathionase [EC 4. 4. 1. 1] incorporates the S-atom of L-cystine as a labile sulfane S-atom in the course of the cystine cleavage reaction.
The 35S-labeled native γ-cystathionase gradually released 35S. However, when the enzyme protein was denatured with 5 % trichloroacetic acid, the release of 35S from the enzyme protein was considerably suppressed. The release of 35S from the native enzyme protein was markedly enhanced by incubation with L-cystine. 35S was eliminated from the native enzyme with L-cystine as the sulfide ion, which was identified by trapping with lead acetate. The apparent Km of L-cystine for the 35S release (2.7×10-5 M) was comparable to that of [35S] cystine for 35S incorporation into the enzyme protein (2.5×10-5 M).
SH compounds such as dithiothreitol could also release the S-atom from the enzyme protein. The elimination reaction by these SH compounds seems to be nonenzymatic, since these SH compounds could eliminate 35S from the denatured 35S-labeled enzyme.
Iodoacetamide did not inhibit the homoserine deaminase, cystathionine lyase or cystine cleavage activities of γ-cystathionase. However, the incorporation of the S-atom from L-cystine into the enzyme protein and the release of the S-atom from the enzyme protein with L-cystine were markedly inhibited in the presence of 5mM iodoacetamide, indicating that iodoacetamide inhibits the S-atom incorporation by trapping the thiol formed from L-cystine by cystine cleavage. The thiol trapped with iodoacetamide was analyzed by using [14C] iodoacetamide, [14C] cystine or [35S] cystine. The compounds formed from the thiol and iodoacetamide were identified as cystine-thioglycolamide mixed disulfide and thioglycolamide disulfide, which was enzymatically derived from the mixed disulfide.
From these results, the mechanism of the cystine cleavage reaction catalyzed by γ-cystathionase is proposed to be as follows: (1) L-cystine is cleaved to pyruvate, NH3 and thiocysteine; (2) the thiocysteine interacts with a disulfide bond in the enzyme protein followed by incorporation of the S-atom of the thiocysteine to form the cystine trisulfide structure; (3) the cystine trisulfide structure is cleaved by consuming 2 mol of cysteine to eliminate the S-atom incorporated from the thiocysteine as a sulfide ion, accompanied by the reformation of a disulfide bond in the enzyme and the oxidation of cysteines to cystine.
