A New Class of Conformationally Rigid Analogues of 4-Amino-5-halopentanoic Acids, Potent Inactivators of γ-Aminobutyric Acid Aminotransferase
journal contributionposted on 2000-02-04, 00:00 authored by Jian Qiu, Richard B. Silverman
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, kinact, 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 [3-2H]-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.
inactivation rate constantsk inactcovalent attachmenturea denaturationdeuterium isotope effect7 releases 148 fluoride ionsNew Classgel filtrationinactivation rateanalogue 72 equivenamine mechanismconformationallyfluoride ioninactivator vigabatrinMichael addition mechanismPotent InactivatorsGABA aminotransferase1 equiv