Rr. Aksamit et al., THE ROLE OF CYSTEINE-78 IN FLUOROSULFONYLBENZOYLADENOSINE INACTIVATION OF RAT-LIVER S-ADENOSYLHOMOCYSTEINE HYDROLASE, The Journal of biological chemistry, 269(6), 1994, pp. 4084-4091
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.