The Mechanism of Inactivation of Human PlacentalS-Adenosylhomocysteine Hydrolase by (E)-4′,5′- Didehydro-5′-Methoxyadenosine and Adenosine 5′-Carboxaldehyde Oxime

The mechanisms by which (E)-4′,5′-didehydro-5′-methoxyadenosine (DMOA) and adenosine 5′-carboxaldehyde oxime (ACAO) inactivateS-adenosylhomocysteine (AdoHcy) hydrolase were elucidated in this study. Their inhibitory activities toward AdoHcy hydrolase were found to be time- and concentration-dependent, and DMOA and ACAO hadKiandk2values of 3.0 μmand 0.10 min−1and 0.67 μmand 0.16 min−1, respectively. The inactivation of AdoHcy hydrolase by DMOA (and ACAO) occurs concomitantly with the reduction of the enzyme-bound NAD+to NADH. The rates of enzyme inactivation correspond to the rates of NADH formation. Incubation of both DMOA and ACAO with the NAD+form of AdoHcy hydrolase resulted in formation of 3′-ketoadenosine (3′-keto-Ado) 5′-carboxaldehyde and its 4′-epimer. Incubation of DMOA and ACAO with the apo form of the enzyme afforded adenosine (Ado) 5′-carboxaldehyde and its 4′-epimer. These results show that DMOA and ACAO are “proinhibitors” of the enzyme. They are first converted to the inhibitors (Ado 5′-carboxaldehyde and its 4′-epimer) in the active site of the enzyme; these inhibitors then inactivate the enzyme by a type I mechanism. The results from this study demonstrated that this is a common mechanism by which 4′,5′-didehydroadenosine analogs, serving as substrates of both the 5′-hydrolytic activity and the 3′-oxidative activity of the enzyme, inactivate AdoHcy hydrolase. The results also provide further evidence supporting the hypothesis that AdoHcy hydrolase possesses a 5′-hydrolytic activity independent of the 3′-oxidation activity.