(E)-5',6'-Didehydro-6'-deoxy-6'-fluorohomoadenosine (EDDFHA), which is a poor substrate for the oxidative activity of S-adenosyl-L-homocysteine (AdoHcy) hydrolase and thus a poor mechanism-based inhibitor was shown to be a good substrate for the hydrolytic activity of this enzyme. Incubation of EDDFHA with AdoHcy hydrolase (NAD+ form) produces a large molar excess of hydrolytic products [e.g., fluoride ion, adenine (Ade) derived from chemical degradation of homoadenosine 6'-carboxaldehyde (HACA), and 6'-deoxy-6'-fluoro-5'-hydroxyhomoadenosine (DFHHA)] accompanied by a slow irreversible inactivation of the enzyme. The enzyme inactivation was shown to be time-dependent, biphasic, and concomitant with the reduction of the enzyme-bound NAD+ (E.NAD+) to E-NADH. The reaction of EDDFHA with AdoHcy hydrolase was shown to proceed by three pathways: pathway a, water attack at the 6'-position of EDDFHA and elimination of fluoride ion results in the formation of HACA, which degrades chemically to form Ade; pathway b, water attack at the 5'-position of EDDFHA results in the formation of DFHHA; and pathway c, oxidation of EDDFHA results in formation of the NADH form of the enzyme (inactive) and 3'keto-EDDFHA, which could react with water at either the C5' or C6' positions. The partition ratios among the three pathways were determined to be k3':k6':k5' = 1:29:79 with one lethal event (enzyme inactivation) occurring every 108 nonlethal turnovers.(ABSTRACT TRUNCATED AT 250 WORDS)