Inactivation of γ-aminobutyric acid aminotransferase by l-3-chloroalanine hydroxamate Original Research Article

The mechanism of inactivation of γ-aminobutyric acid aminotransferase (GABA-AT) by l-3-chloroalanine hydroxamate (1) was investigated. Inactivation of [3H]PLP-reconstituted GABA-AT with 1 followed by denaturation gave no PMP or enamine adduct to the PLP; however, a new unknown metabolite was observed which was identical to the metabolite formed upon inactivation of GABA-AT by l-cycloserine. Time-dependent inactivation occurs, but the kinetics are second order; the rate of inactivation increases with time. After inactivation occurs the addition of fresh enzyme results in a faster rate of inactivation than prior to the initial inactivation. This indicates that the actual inactivator is generated from l-3-chloroalanine hydroxamate, and is not l-3-chloroalanine hydroxamate itself. Added gabaculine-inactivated enzyme to fresh enzyme does not increase the rate of inactivation, suggesting that the conversion of l-3-chloroalanine hydroxamate to the active form is not catalyzed by peripheral amino acid residues. l-3-Chloroalanine hydroxamate was shown to undergo buffer-catalyzed cyclization to l-cycloserine, which is the actual inactivator of GABA-AT.