E)-5',6'-DIDEHYDRO-6'-DEOXY-6'-FLUOROHOMOADENOSINE - A SUBSTRATE THATMEASURES 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-homocystei ne (AdoHcy) hydrolase and thus a poor mechanism-based inhibitor, was s hown 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'-carboxald ehyde (HACA), and 6'-deoxy-6'-fluoro-5'-hydroxyhomoadenosine (DFHHA)] accompanied by a slow irreversible inactivation of the enzyme. The enz yme inactivation was shown to be time-dependent, biphasic, and concomi tant with the reduction of the enzyme-bound NAD(+) (E.NAD(+)) to E NAD H. The reaction of EDDFHA with AdoHcy hydrolase was shown to proceed b y three pathways: pathway a, water attack at the 6'-position of EDDFHA and elimination of fluoride ion results in the formation of HACA, whi ch degrades chemically to form Ade; pathway b, water attack at the 5'- position of EDDFHA results in the formation of DFHHA; and pathway c, o xidation 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 pathwa ys were determined to be k(3'):k(6'):k(5') = 1:29:79 with one lethal e vent (enzyme inactivation) occurring every 108 nonlethal turnovers. Ev idence in support of these mechanisms includes the observations that i ncubation of EDDFHA with AdoHcy hydrolase (NAD(+) form) generates a la rger molar excess of fluoride ion [determined by F-19 nuclear magnetic resonance spectroscopy (NMR)], Ade [determined by high-performance li quid chromatography (HPLC) and chemical ionization mass spectrometry ( CI-MS)], and DFHHA [determined by F-19 NMR, H-1 NMR, and fast atom bom bardment mass spectrometry (FAB-MS)]. In an earlier study we have show n 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 wea k 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 EDD FHA has been identified as a ''fairly specific'' substrate for measuri ng the hydrolytic activity independent of the oxidative activity.