Enantiomers of 4-amino-3-fluorobutanoic acid as substrates for gamma-aminobutyric acid aminotransferase. Conformational probes for GABA binding.
Gamma-aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter gamma-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 alpha-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.
Clift
Michael D
MD
Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
Ji
Haitao
H
Deniau
Gildas P
GP
O'Hagan
David
D
Silverman
Richard B
RB
D000612:4-Aminobutyrate Transaminase / Q000378:metabolism*; D000613:Aminobutyrates / Q000378:metabolism; D003198:Computer Simulation; D008958:Models, Molecular; D011487:Protein Conformation; D013237:Stereoisomerism; D013379:Substrate Specificity; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
C526889:4-amino-3-fluorobutanoic acid; D000613:Aminobutyrates; D005680:gamma-Aminobutyric Acid; D000612:4-Aminobutyrate Transaminase