The mechanism of inactivation of human placental S-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) inactivate S-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 had K(i) and k2 values of 3.0 microM and 0.10 min(-1) and 0.67 microM and 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.