S-adenosylhomocysteinase: mechanism of inactivation by 2'-deoxyadenosine and interaction with other nucleosides

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Abstract
S-Adenosylhomocysteinase (SAHase) [bovine liver], a tetrameric enzyme, is inactivated by 2''-deoxyadenosine (2''dAdo) in a time-dependent process. Inactivation may involve oxidation of 2''dADo at C-3'' by enzyme-bound NAD, subsequent proton abstraction at C-2'' and elimination of adenine. This results in irreversible formation of enzyme-bound NADH and of adenine (Ade) and inactivation. Upon inactivation of SAHase with deoxy[2''(R)-3H]adenosine, 3H2O is formed. This is consistent with the proposed mechanism and establishes that a trans elimination occurs. The stoichiometry of 3H2O release shows that maximally 2 of the 4 subunits participate in the reaction that results in 3H2O release. Reaction of SAHase with 2''dAdo results in reduction of 2 of the enzyme-bound NAD molecules. All 4 NAD molecules can be reduced by NaBH4, by only 2 are reduced to C-4 NADH. When the enzyme is inactivated with adenine-labeled 2''dAdo, radioactivity corresponding to 0.5-1.0 .mu.mol of 2''dAdo binds tightly per .mu.mol of subunit. This radioactive material is not removed from the enzyme by extensive dialysis but can be displaced by unlabeled 2''dAdo or Ade. After denaturation of the complex, radioactive material is released. Of this material 80-90% is adenine and < 1% 2''dAdo. 2''dAdo also binds tightly to the enzyme reduced with NaBH4. Upon denaturation mostly adenine (80-90%) is released. Reaction of [2''-3H]2''dAdo with enzyme reduced with NaBH4 does not result in 3H2O formation. The enzyme catalyzes the release of adenine from 2''dAdo by 2 mechanisms: one involves formation of 3''-keto-2''dAdo and subsequent elimination of adenine. The other does not involve oxidation of 2''dAdo and probably is a hydrolytic process. The ability of the enzyme to carry out the hydrolytic process may be a direct consequence of the manner in which 2''dAdo as well as the normal substrate binds to the enzyme, i.e., H-bond interaction of the protein with the adenine moiety and distortion of the ribose ring. When adenine-labeled adenosine is added to the enzyme, radioactivity corresponding to 0.5 .mu.mol/.mu.mol of subunit is associated with the protein after gel filtration. Of the radioactive material bound to the protein, 20% is adenine, 15% is adenosine and the remaining radioactivity is present in unidentified compounds. The adenine bound to the enzyme does not participate in the catalytic process, and it is probably bound to 2 of the subunits that do not participate in catalysis. Possibly, these 2 subunits have a regulatory function. SAHase probably consists of 2 nonequivalent pairs of subunits. Only 1 pair participates in catalysis, but all 4 subunits probably bind Ado and 2''dAdo. The carbocyclic analog of adenosine inactivates SAHase. Inactivation is accompanied by reduction of enzyme-bound NAD. After denaturation of the inactivated enzyme, the major compound isolated is adenine.