Recently, we found (Qiu, J.; Pingsterhaus, J. M.; Silverman, R. B. J. Med. Chem. 1999, 42, 4725-4728) that conformationally rigid analogues of the GABA aminotransferase (GABA-AT) inactivator vigabatrin were not inactivators of GABA-AT. To determine if this is a general phenomenon of GABA-AT inactivators, several mono- and di-halogen-substituted conformationally rigid analogues (7-15) of other GABA-AT inactivators, 4-amino-5-halopentanoic acids, were synthesized as potential inactivators of GABA-AT. Four of them, (+)-7, (-)-9, (+)-10, and (+)-15, were inactivators, although not as potent as the corresponding open-chain analogues. The maximal inactivation rate constants, k(inact), for the fluoro- and bromo-substituted analogues were comparable, indicating that cleavage of the C-X bond is not rate determining. Consistent with that observation is the finding that [3-(2)H]-10 exhibits a deuterium isotope effect on inactivation of 3.3, suggesting that C-H bond cleavage is the rate-determining step. The rate of inactivation of GABA-AT by the fluorinated analogue 7 is 1/15 that of inactivation by the corresponding open-chain analogue, 4-amino-5-fluoropentanoic acid (3a). Whereas inactivation by 3a releases only one fluoride ion, inactivation by 7 releases 148 fluoride ions, accounting for the less efficient inactivation rate. Inactivation leads to covalent attachment of 2 equiv of inactivator after gel filtration; upon urea denaturation, 1 equiv of radioactivity remains bound to the enzyme. This suggests that, unlike the open-chain anlogue, the conformationally rigid analogue becomes, at least partially, attached to an active-site residue. It appears that the conformational constraint has a larger effect on inactivators that inactivate by a Michael addition mechanism than by an enamine mechanism.