Abstract
Most inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase function as substrates for the "3'-oxidative activity" of the enzyme and convert the enzyme from its active form (NAD+) to its inactive form (NADH) (Liu, S., Wolfe, M. S., and Borchardt, R. T. (1992) Antivir. Res. 19, 247-265). In this study, we describe the effects of a mechanism-based inhibitor, 6'-bromo-5', 6'-didehydro-6'-deoxy-6'-fluorohomoadenosine (BDDFHA), which functions as a substrate for the "6'-hydrolytic activity" of the enzyme with subsequent formation of a covalent linkage with the enzyme. Incubation of human placental AdoHcy hydrolase with BDDFHA results in a maximum inactivation of 83% with the remaining enzyme activity exhibiting one-third of the kcat value of the native enzyme. This partial inactivation is concomitant with the release of both Br- and F- ions and the formation of adenine (Ade). The enzyme can be covalently labeled with [8-3H]BDDFHA, resulting in a stoichiometry of 2 mol of BDDFHA/mol of the tetrameric enzyme. The 3H-labeled enzyme retains its original NAD+/NADH content. Tryptic digestion and subsequent protein sequencing of the [8-3H]BDDFHA-labeled enzyme revealed that Arg196 is the residue that is associated with the radiolabeled inhibitor. The partition ratio of the Ade formation (nonlethal event) to covalent acylation (lethal event) is approximately 1:1. From these experimental results, a possible mechanism by which BDDFHA inactivates AdoHcy hdyrolase is proposed: enzyme-mediated water addition at the C-6' position of BDDFHA followed by elimination of Br- ion results in the formation of homoAdo 6'-carboxyl fluoride (HACF). HACF then partitions in two ways: (a) attack by a proximal nucleophile (Arg196) to form an amide bond after expulsion of F- ion (lethal event) or (b) depurination to form Ade and hexose-derived 6-carboxyl fluoride (HDCF), which is further hydrolyzed to hexose-derived 6-carboxylic acid (HDCA) and F- ion (nonlethal event).
Highlights
Palmer and Abeles [11] elucidated the mechanism by which AdoHcy hydrolase catalyzes the conversion of AdoHcy to Ado and Hcy
Enzyme inactivation by ZDDFA does not involve covalent modification; instead, ZDDFA is converted into Ado-5Ј-carboxaldehyde by the enzyme’s hydrolytic activity, which inactivates the enzyme by the type I mechanism
We report the first type II mechanism-based inhibitor of AdoHcy hydrolase, 6Ј-bromo-5Ј,6Ј-didehydro-6Ј-deoxy-6Ј-fluorohomoadenosine (BDDFHA), which does not require activation by the 3Ј-oxidative activity, but instead is activated by the enzyme’s hydrolytic activity and subsequently inactivates the enzyme by forming a covalent linkage with an amino acid residue at or nearby the active site of the enzyme
Summary
Materials—AdoHcy, Ado, Hcy, and calf intestinal Ado deaminase (EC 3.5.4.4) were purchased from Sigma. Determination of Ade Formation—AdoHcy hydrolase and BDDFHA were incubated in buffer A under the same conditions as described for FϪ and BrϪ ion determination, except that at each time point the reaction was stopped by addition of 10 l of 5 N HClO4. The protein concentration, 3H radioactivity, and remaining enzyme activity in the filtrate were determined, and the ratio of bound BDDFHA (mole) to the inactivated enzyme (mole of subunit) was calculated as the stoichiometric amount of the total binding (covalent binding ϩ noncovalent binding). The enzyme-inhibitor complex was denatured with 8 M urea and passed through the spin column again to remove any noncovalently bound [8-3H]BDDFHA and to change the buffer to buffer A containing 1 M urea. The washings from the conversion flask and eluate from the HPLC column were collected and combined for determination of radioactivity
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