Mechanism of inactivation of S-adenosylhomocysteine hydrolase by (Z)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine.

S-Adenosyl-L-homocysteine (AdoHcy) hydrolase, an enzyme important in the regulation of biological methylation reactions, was shown by McCarthy et al. (McCarthy, J.R., Jarvi, E.T., Matthews, D.P., Edwards, M.L., Prakash, N.J., Bowlin, T.L., Mehdi, S., and Bey, P. (1989) J. Am. Chem. Soc. 111, 1127-1128) to be inactivated by (Z)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (ZDDFA). In this study we have shown that the mechanism of this inactivation of AdoHcy hydrolase (NAD+ form) includes a rapid addition of water to the 5'-position of ZDDFA and elimination of fluoride ion, resulting in the formation of the 5'-carboxaldehydes 3 and 4. The 5'-carboxaldehydes 3 and 4 are then oxidized in a slower step to the 3'-keto-5'-carboxaldehydes 5 and 6 by reduction of the enzyme-bound NAD+ to NADH. Evidence in support of this mechanism includes the observation that the first step in this mechanism (i.e. elimination of fluoride ion and formation of the 5'-carboxaldehydes 3 and 4) can be catalyzed by apo-AdoHcy hydrolase and the NADH form of AdoHcy hydrolase. Incubation of ZDDFA with either the apo or NADH form of AdoHcy hydrolase resulted in rapid release of fluoride ion (determined by 19F NMR) and formation of the 5'-carboxyaldehydes 3 and 4 (determined by high performance liquid chromatography). The carboxaldehydes 3 and 4 were synthesized independently and were shown to be potent inhibitors of the NAD+ form of the enzyme. When the relative first-order rates of fluoride ion release (determined by 19F NMR) from ZDDFA, NAD+ reduction to NADH, and inactivation of the NAD+ form of the enzyme were compared, the release of fluoride ion was found to be approximately 20 times faster than NAD+ reduction or enzyme inactivation. Incubation of ZDDFA with the NAD+ form of AdoHcy hydrolase was shown to afford the 3'-keto-5'-carboxaldehydes 5 and 6, which were also formed upon incubation of the enzyme with the 5'-carboxaldehydes 3 and 4. The 3'-keto-5'-carboxaldehydes 5 and 6 were shown to be tightly (but not covalently) bound to the enzyme, since these products could be released by treatment of the ZDDFA-inactivated enzyme with HClO4 or other denaturing agents.