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-fluorosulfonylbenzoyladenosine (FSBA) is associated with the formation of a disulfide bond between Cys-78 and Cys-112 (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 properties of four hydrolase proteins mutated at Cys-78 or Cys-112 were compared to those of the wild-type enzyme. When Cys-78 was mutated to either a serine or an alanine, proteins with greatly reduced enzymatic activity were obtained, large effects on kinetic constants were observed, and enzymatic activity was not affected by incubation with FSBA. When 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 kinetic constants were observed, and the enzyme was inactivated more rapidly upon incubation with FSBA. FSBA inactivation of the C112A mutant protein 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.
Aksamit
R R
RR
Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892.
Backlund
P S
PS
Moos
M
M
Caryk
T
T
Gomi
T
T
Ogawa
H
H
Fujioka
M
M
Cantoni
G L
GL
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D043383:Adenosylhomocysteinase; D000818:Animals; D001483:Base Sequence; D002918:Chymotrypsin / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D017931:DNA Primers; D011994:Recombinant Proteins; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D002918:Chymotrypsin; D000241:Adenosine; D003545:Cysteine