Theoretical Study on HF Elimination and Aromatization Mechanisms: A Case of Pyridoxal 5′ Phosphate-Dependent Enzyme

Abstract

Pyridoxal 5-phosphate (PLP), the phosphorylated and the oxidized form of vitamin B6 is an organic cofactor. PLP forms a Schiff base with the ϵ-amino group of a lysine residue of PLP-dependent enzymes. γ-Aminobutyric acid (GABA) aminotransferase is a PLP-dependent enzyme that degrades GABA to succinic semialdehyde, while reduction of GABA concentration in the brain causes convolution besides several neurological diseases. The fluorine-containing substrate analogues for the inactivation of the GABA-AT are synthesized extensively in cases where the inactivation mechanisms involve HF elimination. Although two proposed mechanisms are present for the HF elimination, the details of the base-induced HF elimination are not well identified. In this density functional theory (DFT) study, fluorine-containing substrate analogue, 5-amino-2-fluorocyclohex-3-enecarboxylic acid, is particularly chosen in order to explain the details of the HF elimination reactions. On the other hand, the experimental studies revealed that aromatization competes with Michael addition mechanism in the presence of 5-amino-2-fluorocyclohex-3-enecarboxylic acid. The results allowed us to draw a conclusion for the nature of HF elimination, besides the elucidation of the mechanism preference for the inactivation mechanism. Furthermore, the solvent phase calculations carried out in this study ensure that the proton transfer steps should be assisted either by a water molecule or a base for lower activation energy barriers.

Turnover and Inactivation Mechanisms for (S)-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase

“…It has been reported that the deprotonation of initial external aldimine M1 and stepwise fluoride elimination steps proposed in Scheme are typically considered as an E1cB elimination mechanism. , The electron-withdrawing effect of fluorine and the protonated nitrogen of the aldimine may stabilize the formed carbanion state during the elimination reaction. , Quinonoid transient state M2 is expected to form between the first and second external aldimines ( M1 and M3 ). However, the stopped-flow experimental results suggest that the antiperiplanar hydrogen and fluorine atoms in M1 undergo a Lys292-assisted E2 mechanism, i.e., concerted loss of a proton fluoride ion, with the formation of the alkene as a more favorable fluoride ion elimination pathway, affording the second external aldimine ( M3 ) directly as the single transient state species ( Pathway e ; Scheme ).…”

“…It has been reported that the deprotonation of initial external aldimine M1 and stepwise fluoride elimination steps proposed in Scheme 6 are typically considered as an E1cB elimination mechanism. 31,32 The electron-withdrawing effect of fluorine and the protonated nitrogen of the aldimine may stabilize the formed carbanion state during the elimination reaction. 32,33 favorable fluoride ion elimination pathway, affording the second external aldimine (M3) directly as the single transient state species (Pathway e; Scheme 6).…”

Metabolomic Profiling Identified Serum Metabolite Biomarkers and Related Metabolic Pathways of Colorectal Cancer

“…Pyridoxine is the metabolite of the vitamin B6 metabolic pathway, and there was a negative correlation between serum pyridoxine level and the risk of CRC [ 43 , 44 ]. Pyridoxine is converted to succinic semialdehyde in the vitamin B metabolic pathway [ 45 ]. The increased succinic acid semialdehyde level suggests that the vitamin B metabolic pathway is an activated state in colorectal cancer tissue, which decreases pyridoxine with protective effects.…”

Molecular dynamics simulations of apo, holo, and inactivator bound GABA‐at reveal the role of active site residues in PLP dependent enzymes

“…The residue Asp298 was reported as an active site residue that interacts with the pyridine ring of PLP via a hydrogen bond. In the literature, the nitrogen on the pyridine ring was depicted both as protonated or unprotonated . In case of a protonated nitrogen, the Asp298 residue should have a side chain with the charge of −1, while in the case of an unprotonated nitrogen on the pyridine ring a neutral Asp298 side chain should be considered.…”

“…In the cases of fluorine containing analogues, the reaction starts with a transimination followed by a HF elimination step for the inactivation. Both the experimental and theoretical studies have provided some clues regarding the nature of the catalytic mechanism . Vigabatrin, one of the natural substrate analogues, was reported as the most effective mechanism‐based inactivator of GABA‐AT with a high potency for treatment of epilepsy and it is used in >60 countries .…”