Enantiomers of 4-amino-3-fluorobutanoic acid as substrates for γ-aminobutyric acid aminotransferase. Conformational probes for GABA binding

Michael D. Clift, Haitao Ji, Gildas P. Deniau, David O'Hagan*, Richard B. Silverman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

γ-Aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5′-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5′-phosphate (PLP) to pyridoxamine 5′-phosphate (PMP). The enzyme then catalyzes the conversion of α-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches.

Original languageEnglish (US)
Pages (from-to)13819-13828
Number of pages10
JournalBiochemistry
Volume46
Issue number48
DOIs
StatePublished - Dec 4 2007

ASJC Scopus subject areas

  • Biochemistry

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