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

Abstract

S-Adenosylhomocysteinase (SAHase), a tetrameric enzyme, is inactivated by 2'-deoxyadenosine (2'dAdo) in a time-dependent process [Hirshfield, M. S. (1979) J. Biol. Chem. 254, 22-25]. It has been proposed that inactivation involves oxidation of 2'dAdo at C-3' by enzyme-bound nicotinamide adenine dinucleotide (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 [Abeles, R. H., TAshjian, A. H., Jr., & Fish, S (1980) Biochem. Biophys. Res. Commun. 95, 612-617]. It has now been established that upon inactivation of SAHase with deoxy[2'(R)-3H]adenosine, 3H2O is formed. This is consistent with the proposed mechanism and of 3H2O release shows that maximally two of the four subunits participate in the reaction that results in 3H2O release. Reaction of SAHase with 2'dAdo results in reduction of two of the enzyme-bound NAD molecules. However, all four NAD molecules can be reduced by NaBH4, but only two are reduced to C-4 NADH. When the enzyme is inactivated with adenine-labeled 2'dAdo, radioactivity corresponding to 0.5-1.0 mumol of 2'dAdo binds tightly per micromole 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 less than 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. We conclude that the enzyme catalyzes the release of adenine from 2'dAdo by two 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. It is proposed that the ability of the enzyme to carry out the hydrolytic process is a direct consequence of the manner in which 2'dAdo as well as the normal substrate binds to the enzyme, i.e., hydrogen-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 mumol/mumol 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 we conclude that it is bound to two of the subunits that do not participate in catalysis. Possible, these two subunits have a regulatory function. SAHase probably consists of two nonequivalent pairs of subunits. Only one pair participates in catalysis, but all four subunits probably bind Ado and 2'dAdo. We have confirmed the fact that the carbocyclic analogue of adenosine inactivates SAHase [Guranowski, A., Montgomery, J. A., 110-115]...