The mechanism of action of S-adenosylhomocysteinase.

S-Adenosylhomocysteinase catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine (AdoHcy) to adenosine and L-homocysteine without added cofactors. A mechanism is proposed which involves oxidation of the 3'-hydroxyl group of AdoHcy by enzyme-bound NAD+. Following oxidation, L-homocysteine is eliminated, alpha-beta, to give 3'-keto-4'-5'-dehydroadenosine. This compound reacts with water in a Michael type addition to form 3'-ketoadenosine which is then reduced to adenosine. This mechanism is supported by these facts. 1) The enzyme contains 1 tightly bound NAD+ per subunit. Upon addition of substrate, this NAD is converted to NADH. 2) The enzyme catalyzes the exchange of the 4'-proton of substrate with solvent. This exchange is an integral part of the catalytic mechanism. 3) The hydrolysis of [4'-2H]S-adenosyl-L-homocysteine has a Vmax isotope effect of 1.44. This provides additional evidence that cleavage of the C-4' C-H bond is a step on the reaction pathway. 4) 4',5'-Dehydroadenosine is oxidized by the enzyme, then converted into adenosine or into AdoHcy in the presence of L-homocysteine. 5) An adenosine analog, 5'-deoxyadenosine, is oxidized by the enzyme to yield 3'-keto-5'-deoxyadenosine, and an analog of the proposed intermediate, 3'-ketoadenosine. 6) The enzyme catalyzes the exchange of the C-4' proton of 5'-deoxyadenosine. Since the enzyme catalyzes proton abstraction without OH elimination, it was concluded that the elimination of H2O from adenosine proceeds by a carbanion mechanism and not by a concerted elimination. Substrate analogs in which the 5'-OH group of adenosine is replaced by -F, -Cl, or -SMe are not substrates for the enzyme.