Abstract The β-fluoroamines are commonly used as substrate analogs to determine the mechanistic details of enzymatic reactions. Presence of fluorine atom gives rise to the alterations in the electronic profile and the pKa of molecules which results in mechanistic deviations. The fluorine-substituted mechanism-based substrate analogs are widely used in the inactivation of pyridoxal 5′-phosphate (PLP)-dependent enzymes. The presence of fluorine atom also alters the sequence of reactions taking place in PLP-dependent enzymes where the HF elimination reaction appears in between the transimination and inactivation reactions. Despite the amount of the works on β-fluoroamines, the effect of stereoelectronic differences on the transimination and HF elimination reactions taking place in PLP-dependent enzymes has not been investigated yet. A density functional theory study is conducted to elucidate mechanistic details of the reactions occurring in PLP-dependent enzymes. In order to understand the mechanistic insights of different isomers and the effect of the fluorine atom, 4-amino-3-fluorobutanoic acid (3-F-GABA) enantiomers are chosen to be investigated besides 4-aminobutanoic acid (GABA), which is the natural substrate for γ-aminobutyric acid aminotransferase (GABA-AT). The investigated β-fluoroamines are the experimentally proposed potential inhibitors of PLP-dependent enzyme GABA-AT.