Mechanisms of Inactivation of Human S-Adenosylhomocysteine Hydrolase by 5′,5′,6′,6′-Tetradehydro-6′-deoxy-6′-halohomoadenosines † Xiaoda Yang, ‡ Dan Yin, ‡ Stanislaw F. Wnuk, § Morris J. Robins, | and Ronald T. Borchardt* ,‡ Department of Pharmaceutical Chemistry, The UniVersity of Kansas, Lawrence, Kansas 66047, Department of Chemistry, Florida International UniVersity, Miami, Florida, 33199, and Department of Chemistry and Biochemistry, Brigham Young UniVersity, ProVo, Utah 84602 ReceiVed June 30, 2000; ReVised Manuscript ReceiVed September 8, 2000 ABSTRACT: In an effort to design more specific and potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, we investigated the mechanisms by which 5′,5′,6′,6′-tetradehydro-6′-deoxy-6′-halohomoad- enosines (X ) Cl, Br, I) inactivated this enzyme. The 6′-chloro (a) and 6′-bromo (b) acetylenic nucleoside analogues produced partial (∼50%) loss of enzyme activity with a concomitant (∼50%) reduction of E-NAD + to E-NADH. In addition, Ade and halide ions were released from the inhibitors in amounts suggestive of a process involving enzyme catalysis. AdoHcy hydrolase, which was inactivated with compound a, was shown to contain 2 mol of the inhibitor covalently bound to Lys318 of two subunits of the homotetramer. These data suggest that the enzyme-mediated water addition at the 5′ position of compound a or b produces an R-halomethyl ketone intermediate, which is then attacked by a proximal nucleophile (i.e., Lys318) to form the enzyme-inhibitor covalent adduct (lethal event); in a parallel pathway (nonlethal event), addition of water at the 6′ position produces an acyl halide, which is released into solution and chemically degrades into Ade, halide ion, and sugar-derived products. In contrast, compound c completely inactivated AdoHcy hydrolase by converting 2 equiv of E-NAD + to E-NADH and causing the release of 2 equiv of E-NAD + into solution. Four moles of the inhibitor was shown to be tightly bound to the tetrameric enzyme. These data suggest that compound c inactivates AdoHcy hydrolase by a mechanism similar to the acetylenic analogue of Ado described previously by Parry et al. [(1991) Biochemistry 30, 9988-9997]. AdoHcy 1 hydrolase (EC 3.3.1.1) catalyzes the reversible hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy) (1, 2). This enzyme regulates all S-adenosylmethionine (AdoMet)-dependent transmethylations by controlling the intracellular levels of AdoHcy, which is a potent inhibitor of AdoMet-dependent methyltransferases (1, 2). Thus, AdoHcy hydrolase has become an attractive target for the design of antiviral, antiparasitic, antiarthritic, immunosuppressive, and antitumor agents (1, 2). Recently, X-ray crystal structures of the human (3) and rat (4) AdoHcy hydrolases have been solved. These structures provide for the first time insights into the nature of the amino acid residues used by AdoHcy hydrolase to catalyze the various chemical transformations proposed by Palmer and Abeles (5, 6). The first reaction catalyzed by AdoHcy hydrolase involves oxidation of the 3′-OH group of the substrate AdoHcy (or Ado) by E-NAD + to form E-NADH and 3′-keto-AdoHcy (or 3′-keto-Ado) (3′-oxidative step). The C4′-proton is then abstracted, presumably by a water molecule at the active site of the enzyme, which has been activated by hydrogen-bonding interactions with Asp131 and His55 (3) (4′-proton abstraction step), followed by -elim- ination of Hcy (or water) to form 3′-keto-4′,5′-didehydro- 5′-deoxy-Ado. His301 is then proposed (3) to participate in the activation of a water molecule (or Hcy) at the active site, which adds in a Michael-type addition to the 4′,5′ double bond (5′-hydrolytic step), generating 3′-keto-Ado (or 3′-keto- AdoHcy). Reduction of 3′-keto-Ado (or 3′-keto-AdoHcy) by E-NADH yields Ado (or AdoHcy), which is then released from the active site of the enzyme (3′-reductive step); thus, completing the catalytic cycle. The 5′-hydrolytic activity (also referred to as 5′/6′ hydrolytic activity) has been shown to function independently of the 3′-oxidative activity (7-9). In the past, significant effort has been made to design potent and selective inhibitors of AdoHcy hydrolase (1, 2). Most inhibitors are Ado analogues that are irreversibly oxidized to their 3′-keto derivatives with concomitant conversion of the NAD + -form of the enzyme (active) to the NADH-form (inactive). These inhibitors are referred to as type I mechanism-based inhibitors. However, significant effort has also been made to identify type II mechanism- based inhibitors that are designed to be transformed by the † This work was supported in part by a National Institutes of Health Grant (GM-29332) and American Cancer Society Grant (DHP-34). * To whom correspondence should be addressed. Phone: (785) 864- 3427. Fax: (785) 864-5736. E-mail: rborchardt@ukans.edu. ‡ The University of Kansas. § Florida International University. | Brigham Young University. 1 Abbreviations: Ade, adenine; Ado, adenosine; AdoHcy, S-adeno- sylhomocysteine; AdoMet, S-adenosylmethionine; BDDFHA, 6′-bromo- 5′,6′-didehydro-6′-deoxy-6′-fluorohomoadenosine; DHCeA, 2′,3′- dihydroxycyclopent-4-enyladenine; E-NAD + , enzyme bound NAD + ; E-NADH, enzyme bound NADH; FAB, fast-atom bombardment; Hcy, homocysteine; HPLC, high-performance liquid chromatography; IC, ion chromatography; MW, molecular weight. 15234 Biochemistry 2000, 39, 15234-15241 10.1021/bi0015055 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/14/2000