Huang H, Yuan C S, Wnuk S F, Robins M J, Borchardt R T
Department of Biochemistry, University of Kansas, Lawrence 66045, USA.
Arch Biochem Biophys. 1997 Jul 1;343(1):109-17. doi: 10.1006/abbi.1997.0159.
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.
本研究阐明了（E）-4'，5'-二脱氢-5'-甲氧基腺苷（DMOA）和腺苷5'-羧醛肟（ACAO）使S-腺苷同型半胱氨酸（AdoHcy）水解酶失活的机制。发现它们对AdoHcy水解酶的抑制活性具有时间和浓度依赖性，DMOA和ACAO的K（i）和k2值分别为3.0 microM和0.10 min（-1）以及0.67 microM和0.16 min（-1）。DMOA（和ACAO）使AdoHcy水解酶失活的过程与酶结合的NAD +还原为NADH的过程同时发生。酶失活的速率与NADH形成的速率相对应。DMOA和ACAO与AdoHcy水解酶的NAD +形式一起孵育导致形成3'-酮腺苷（3'-keto-Ado）5'-羧醛及其4'-差向异构体。DMOA和ACAO与酶的脱辅基形式一起孵育得到腺苷（Ado）5'-羧醛及其4'-差向异构体。这些结果表明DMOA和ACAO是该酶的“前抑制剂”。它们首先在酶的活性位点转化为抑制剂（Ado 5'-羧醛及其4'-差向异构体）；然后这些抑制剂通过I型机制使酶失活。本研究的结果表明，这是4'，5'-二脱氢腺苷类似物作为该酶5'-水解活性和3'-氧化活性的底物使AdoHcy水解酶失活的常见机制。这些结果还提供了进一步的证据支持AdoHcy水解酶具有独立于3'-氧化活性的5'-水解活性这一假设。
