THE ROLE OF CYSTEINE-78 IN FLUOROSULFONYLBENZOYLADENOSINE INACTIVATION OF RAT-LIVER S-ADENOSYLHOMOCYSTEINE HYDROLASE

Inactivation of rat liver S-adenosylhomocysteine hydrolase by the site -directed reagent 5'-p-fluorosulfonyl-benzoyladenosine (FSBA) is assoc iated with the formation of a disulfide bond between Cys-78 and Cys-11 2 (Takata, Y., and Fujioka, M. (1984) Biochemistry 23, 4357-4362; Gomi , T., Ogawa,H., and Fujioka, M. (1986) J. Biol. Chem. 261, 13422-13425 ). To characterize the inactivation mechanism more precisely, the prop erties of four hydrolase proteins mutated at Cys-78 or Cys-112 were co mpared to those of the wild-type enzyme. When Cys-78 was mutated to ei ther a serine or an alanine, proteins with greatly reduced enzymatic a ctivity were obtained, large effects on kinetic constants were observe d, and enzymatic activity was not affected by incubation with FSBA. Wh en Cys-112 was mutated to either a serine or an alanine, the activity was similar to the wild-type protein, only small changes in the kineti c constants were observed, and the enzyme was inactivated more rapidly upon incubation with FSBA. FSBA inactivation of the C112A mutant prot ein was accompanied by the formation of a disulfide between Cys-78 and Cys-52. The data indicate that FSBA initially reacts with Cys-78 and that Cys-78 has an important role in the structure of the enzyme.