(E)-5',6'-DIDEHYDRO-6'-DEOXY-6'-FLUOROHOMOADENOSINE - A SUBSTRATE THAT MEASURES THE HYDROLYTIC ACTIVITY OF S-ADENOSYLHOMOCYSTEINE HYDROLASE

(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'-keta-EDDFHA, which could react with water at either the C5' or C6' positions. The partition ratios among the three pathways were determined to be k(3'):k(6'):k(5') = 1:29:79 with one lethal event (enzyme inactivation) occurring every 108 nonlethal turnovers. Evidence in support of these mechanisms includes the observations that incubation of EDDFHA with AdoHcy hydrolase (NAD(+) form) generates a larger molar excess of fluoride ion [determined by F-19 nuclear magnetic resonance spectroscopy (NMR)], Ade [determined by high-performance liquid chromatography (HPLC) and chemical ionization mass spectrometry (CI-MS)], and DFHHA [determined by F-19 NMR, H-1 NMR, and fast atom bombardment mass spectrometry (FAB-MS)]. In an earlier study we have shown that the formation of Ade was a measure of HACA production [Yuan, C. S., Liu, S., Wnuk, S. F., Robins, M. J., and Borchardt, R. T. (1994) Biochemistry 33, 3758-3765]. DFHHA was shown in this study to be a weak reversible inhibitor of AdoHcy hydrolase. In addition, incubation of EDDFHA with the NADH form of the enzyme also generates fluoride ion, Ade, and DFHHA. The results from these studies have provided valuable new insights into the hydrolytic activity of AdoHcy hydrolase, and EDDFHA has been identified as a ''fairly specific'' substrate for measuring the hydrolytic activity independent of the oxidative activity.