S-Adenosylhomocysteine (AdoHcy) hydrolase is irreversibly inactivated by (E)-5',6'-didehydro-6'-deoxy-6'-halohomoadenosines (EDDHHAs, halogen = I, Br, and Cl). The inactivation is concomitant with the reduction of the enzyme-bound NAD+ (E.NAD+) to NADH, the release of halide ion, and the formation of adenine (Ade) from the EDDHHAs. The mechanism of this inactivation involves two catalytic pathways. Pathway a involves a rapid addition of water to the 5',6'-bond of EDDHHAs and elimination of halide ion, resulting in the formation of 6'-carboxaldehyde 1 which then degrades chemically, resulting in the formation of Ade. Alternatively, 6'-carboxyaldehyde 1 can be oxidized by E.NAD+ to form 3'-keto-6'-carboxaldehyde 3 and the NADH form (inactive) of the enzyme. Like 6'-carboxaldehyde 1, the 3'-keto derivative 3 degrades chemically to form Ade. Pathway b involves the oxidation of EDDHHAs to 3'-keto-EDDHHAs 2 by E.NAD+, as the first step, and the subsequent release of halide ion to form 3'-keto-6'-carboxaldehyde 3. Evidence in support of these mechanisms includes the observations that incubation of EDDHHAs with AdoHcy hydrolase generated large molar excesses of halide ions and Ade, that Ade was shown to eliminate spontaneously from 6'-carboxaldehyde 1, and that the more rapid the halide ion release (Cl- > Br- > I-) from the EDDHHAs or the greater the partition ratios (nonlethal turnovers/lethal event), the lower the enzyme inactivation efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)