Computer-assisted study on the reaction between pyruvate and ylide in the pathway leading to lactyl–ThDP Omar Alvarado • Gonzalo Jan ˜a • Eduardo J. Delgado Received: 9 May 2012 / Accepted: 5 July 2012 / Published online: 11 July 2012 Ó Springer Science+Business Media B.V. 2012 Abstract In this study the formation of the lactyl–thia- min diphosphate intermediate (L–ThDP) is addressed using density functional theory calculations at X3LYP/6- 31??G(d,p) level of theory. The study includes potential energy surface scans, transition state search, and intrinsic reaction coordinate calculations. Reactivity is analyzed in terms of Fukui functions. The results allow to conclude that the reaction leading to the formation of L–ThDP occurs via a concerted mechanism, and during the nucleophilic attack on the pyruvate molecule, the ylide is in its AP form. The calculated activation barrier for the reaction is 19.2 kcal/ mol, in agreement with the experimental reported value. Keywords L–ThDP  Mechanism  DFT  Fukui  Activation barrier Introduction Thiamin diphosphate (ThDP) is an important coenzyme in a variety of enzymes involved in the decarboxylation of a-keto acids in sugar metabolism. It is composed of two aromatic rings, a 4-aminopyrimidine ring and a thiazolium ring bridged by a methylene group [1, 2]. During the catalysis by ThDP enzymes, the 4 0 -aminopyrimidine moi- ety can interconvert among four ionization/tautomeric states: the 4 0 -aminopyrimidine (AP), the N1 0 -protonated 4 0 -amino pyrimidium (APH ? ), 1 0 ,4 0 -iminopyrimidine (IP), and the C2-ionizated ylide (Y1). In all ThDP-dependent enzymes, the catalytic cycle is initiated with the attack of the C2 atom of the ylide on the Ca of a pyruvate molecule to form the lactyl–ThDP (L–ThDP) intermediate, which then undergoes decarboxylation to form the hydroxy- ethylthiamin diphosphate (HEThDP) enamine/carbanion. Then, HEThDP reacts with a second molecule of an a-keto acid to form the intermediate AHA–ThDP, which finally leads to the product release and the ylide recover (Fig. 1). Despite the number of articles published on the catalytic cycle of ThDP-dependent enzymes, still there exist some aspects which remain unknown or controversial [3–6]. Specifically on the L–ThDP intermediate, there are some issues that stay unclear, namely, the protonation states of the N1 0 and N4 0 atoms, during the attack of C2 on the Ca of pyruvate, since not all of the ionization/tautomeric forms have been clearly characterized [2]. On the other hand, the manner the reaction occurs, i.e., via a stepwise or concerted mechanism, has not been clearly elucidated and remains as a controversial issue [7]. In this study the formation of the L–ThDP intermediate is addressed using high level density functional theory calculations, X3LYP/6-31??G(d,p). The study includes potential energy surface (PES) scans in order to identify and characterize critical points on it, transition state search, and intrinsic reaction coordinate calculations. Reactivity is analyzed in terms of Fukui functions. Electronic supplementary material The online version of this article (doi:10.1007/s10822-012-9589-3) contains supplementary material, which is available to authorized users. O. Alvarado  G. Jan ˜a  E. J. Delgado (&) Computational Biological Chemistry Group (QBC), Faculty of Chemical Sciences, Universidad de Concepcio ´n, Concepcio ´n, Chile e-mail: edelgado@udec.cl G. Jan ˜a Departamento de Ciencias Quı ´micas, Facultad de Ciencias Exactas, Sede Concepcio ´n, Universidad Andre ´s Bello, Concepcio ´n, Chile 123 J Comput Aided Mol Des (2012) 26:977–982 DOI 10.1007/s10822-012-9589-3