Suicide Inactivation of Dioldehydratase by Glycolaldehyde and Chloroacetaldehyde: an Examination of the Reaction Mechanism Gregory M. Sandala, ²,§ David M. Smith,* ,‡ Michelle L. Coote, § and Leo Radom* ,²,§ School of Chemistry, UniVersity of Sydney, Sydney, NSW 2006, Australia, Research School of Chemistry, Australian National UniVersity, Canberra, ACT 0200, Australia, and Rudjer BoskoVic Institute, 10002 Zagreb, Croatia Received May 5, 2004; E-mail: david.smith@irb.hr; radom@chem.usyd.edu.au Dioldehydratase (DDH) is a coenzyme B 12 -dependent enzyme that catalyzes the transformation of ethane-1,2-diol (1) and propane- 1,2-diol (2) into the corresponding aldehydes. 1 In early studies of DDH, it was found that the substrate analogues glycolaldehyde (3) 2a and 2-chloroacetaldehyde (4) 2b rapidly caused its deactivation. 5′- Deoxyadenosine (Ado-H) was found to be one of the products in the deactivation process, but until recently, the fate of the substrate analogues was not known. In elegant EPR experiments, Frey and Reed and co-workers identified the product derived from both 3 and 4 as cis-ethanesemidione (5). 3,4 They proposed mechanisms both for the formation of cis-ethanesemidione and for the inactiva- tion process on the basis of these experimental results. 3,4 The fact that glycolaldehyde (3) and 2-chloroacetaldehyde (4) inactivate DDH is very intriguing and makes a computational investigation of the detailed mechanism of the inactivation, and a determination of how and why it diverges from the functional catalytic mechanism, desirable. That is the purpose of the present study. The accepted mechanism for the DDH-catalyzed transformation of ethane-1,2-diol (1) to acetaldehyde (6) is displayed in Scheme 1. 1,5 After substrate-induced homolytic fission of the C-Co bond of adenosylcobalamin, the 5′-deoxyadenosyl radical (Ado•) is generated. This abstracts H from C1 of 1, to give the 1,2- dihydroxyethyl radical (7). An OH 1,2-shift yields the 2,2- dihydroxyethyl radical (8). Reabstraction of H from Ado-H, leads to ethane-1,1-diol (9), from which enzyme-catalyzed elimination of water yields the product acetaldehyde. The hydrates of glycolaldehyde and 2-chloroacetaldehyde, namely ethane-1,1,2-triol (10) and 1,1-dihydroxy-2-chloroethane (11), are structurally similar to the natural substrates for the DDH- catalyzed reactions. It is therefore not surprising that DDH is able to function (at least partially) on these substrate analogues. We have, therefore, examined computationally the process of suicide inactiva- tion by these two substrates in the context of the mechanism depicted in Scheme 1. Geometries and scaled vibrational frequencies were obtained with the MPW1K/6-31+G(d,p) density functional theory procedure, with improved relative energies (enthalpies) at 0 K calculated using the high-level G3(MP2)-RAD methodology. 6,7 We have chosen ethanol as a model for Ado-H as it has previously been shown to satisfactorily describe the H-abstraction steps for B 12 -dependent processes. 8 A mechanism for the DDH-catalyzed reaction of ethane-1,1,2- triol (10), closely related to that originally proposed, 3 is depicted in Scheme 2. 9 In a manner analogous to that pertaining to a catalytic substrate (Scheme 1), H abstraction by Ado• from C2 of 10 gives the 1,2,2-trihydroxyethyl radical (12), with a barrier of 34.0 kJ mol -1 and an exothermicity of 21.1 kJ mol -1 . These values are consistent with those calculated for ethane-1,2-diol (35.0 and 27.6 kJ mol -1 , Scheme 1). The presence of the additional hydroxyl group does not appear to have a significant effect on the initial H- abstraction step. In the case of a natural substrate, such as ethane-1,2-diol (1), the next step involves hydroxyl group migration (Scheme 1). 1,5 However, for 12 such a step would simply lead to an equivalent structure. Hence, it is proposed that dehydration of 12 occurs to give the glycolaldehyde radical (13), for which we calculate an exothermicity of 37.0 kJ mol -1 (Scheme 2). Interestingly, we find that the symmetric cis-ethanesemidione structure (5) deduced from EPR experiments 3 is a transition structure (TS) lying 38.0 kJ mol -1 above the glycolaldehyde radical (13) on the potential energy surface (PES). We will address this point further below. For the moment, we note that for a mechanism analogous to that of the catalytic pathway to continue, H abstraction by glycolaldehyde radical (13′) from Ado-H would be required. The calculated barrier for this process is an astounding 113.7 kJ mol -1 , with an endothermicity of 88.1 kJ mol -1 ! By comparison, the barrier ² University of Sydney. § Australian National University. ‡ Rudjer Boskovic Institute. Scheme 1. Proposed Mechanism for the DDH-Catalyzed Reaction of Ethane-1,2-diol (Relative Energies in Parentheses, kJ mol -1 ) Scheme 2. Proposed Mechanism of Suicide Inactivation of DDH by Ethane-1,1,2-triol (Relative Energies in Parentheses, kJ mol -1 ) Published on Web 09/10/2004 12206 9 J. AM. CHEM. SOC. 2004, 126, 12206-12207 10.1021/ja047377f CCC: $27.50 © 2004 American Chemical Society