Mechanism of the irreversible inhibition of γ-aminobutyric acid-α-ketoglutaric acid transaminase by the neurotoxin gabaculine

Abstract

Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid), a naturally occurring amino acid isolated from Streptomyces toyocaenis, is an irreversible inhibitor of bacterial pyridoxal phosphate linked .gamma.-aminobutyric acid-.alpha.-ketoglutaric acid transaminase with t1/2 (25.degree. C) of 9 min at 3 .times. 10-7 M. Gabaculine is a substrate for .gamma.-aminobutyric acid transaminase. The measured KI [inhibition constant] is 2.86 .times. 10-6 M, and the Kcat [half-life] for its turnover is 1.15 .times. 10-2 s-1 at 25.degree. C. When gabaculine is transaminated by the enzyme, it is converted to a cyclohexatrienyl system with 1 exo double bond. Upon spontaneous aromatization, this high energy intermediate is transformed into a stable m-anthranilic acid derivative (m-carboxyphenylpyridoxamine phosphate), which results in the covalent and irreversible modification of the cofactor. This adduct is bound tightly to the active site of the enzyme and can be liberated under denaturing conditions.