Amino acid residues that affect the basicity of the catalytic glutamate of the hydrolytic aldehyde dehydrogenases

In the catalytic mechanism of hydrolytic aldehyde dehydrogenases (ALDHs) the role of Glu268 (mature human ALDH2 numbering) as a general base is of major relevance. Since Glu268 basicity depends on its protein environment, here we explore its interactions with other amino acid residues in the three different conformations observed in ALDH crystal-structures: inside, intermediate and outside. In all of them Glu268 is in a hydrophobic environment. In the inside conformation, the theoretical pK(a) estimated by PROPKA3 is the result of the effects of hydrogen bonds with the protonated thiol of the catalytic Cys302 and/or the main-chain amide nitrogen of the highly conserved Gly270, and of charge-charge interactions with neighboring side-chains-Lys178, Glu/Asp476, His465 or Glu399 depending on the enzyme. In the intermediate conformation Glu268-carboxyl pK(a) is influenced by interactions with Glu/Asp476, Arg/Lys475, Lys/Arg178, His465 or Arg459, also depending on the enzyme. In the outside conformation, the effects on Glu268-carboxyl pK(a) arise from hydrogen bonds with the side chains of the strictly conserved Thr224 and/or of Lys/Arg178, and from charge-charge interactions with Lys/Arg/Asp178, Glu476, or Arg459. The estimated pK(a)s and interactions of Glu268-carboxyl in the intermediate and outside conformations are consistent with their previously proposed roles in activating the hydrolytic water and in a proton relay mechanism, respectively. Water channels connecting Glu268 with the bulk water were found in all hydrolytic ALDHs. In the inside conformation the theoretical pK(a)s of the Glu268-carboxyl and Cys302-thiol groups suggest that the carboxyl cannot receive the proton from the thiol. We propose that a protonated Cys302 might perform the nucleophilic attack on the aldehyde, which can be facilitated by Glu268 in the intermediate conformation. Finally, the conservation of the residues influencing Glu268 basicity between and within ALDH families suggests that these residues, not previously studied, are important for the catalytic mechanism of many ALDH enzymes. (C) 2015 Elsevier Ireland Ltd. All rights reserved.