5'-[p-(Fluorosulfonyl)benzoyl]adenosine-mediated inactivation of S-adenosylhomocysteinase.

Rat liver S-adenosylhomocysteinase (EC 3.3.1.1) is inactivated by 5'-[p-(fluorosulfonyl)benzoyl]adenosine following pseudo-first-order kinetics. A plot of the apparent first-order rate constant for inactivation vs. the 5'-[p-(fluorosulfonyl)benzoyl]adenosine concentration exhibits a hyperbolic curve indicative of the formation of a reversible enzyme-reagent complex prior to the inactivation. Values of 71.0 +/- 7.7 microM and 0.14 +/- 0.01 min-1 are estimated for Kd and k, respectively, at pH 8.25 and 25 degrees C. The substrate adenosine and a competitive inhibitor adenine completely protect the enzyme against inactivation. Values of dissociation constant for these ligands calculated from the protection experiments agree well with those obtained by other means, indicating that 5'-[p-(fluorosulfonyl)benzoyl]adenosine competes with these ligands for the same binding site. The inactivation is not reversed by dialysis against phosphate buffer or tris(hydroxymethyl)aminomethane hydrochloride buffer, but a full enzyme activity is regained by treatment with dithiothreitol. The inactivation is not accompanied by covalent attachment of the reagent but is correlated with the loss of two sulfhydryl groups per enzyme subunit. Thus, the inactivation appears to result from a reagent-mediated formation of a disulfide between two cysteine residues in close proximity. The 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified enzyme which is not capable of catalyzing the overall reaction can still catalyze the partial reactions such as the oxidation of the 3'-hydroxyl and the abstraction of the 4'-proton of adenosine.