Chemical inactivation of bacterial GABA aminotransferase.
The effects of three potential irreversible inhibitors of gamma-aminobutyrate aminotransferase from Pseudomonas fluorescens were studied in order to throw more light on the nature of the active site of the enzyme. The thiol group reagent mercuric chloride inactivated the enzyme in a concentration-dependent manner. Inhibition kinetics were consistent with a simple bimolecular reaction. The second-order rate constant was 4.2 x 10(3) +/- 0.61 M-1 sec-1. In contrast to either of the substrates, the cofactor pyridoxal 5'-phosphate could protect the enzyme from the inhibition, suggesting cysteinyl residues are important for cofactor binding at the active site. p-Chloromercuribenzoic acid produced a similar inactivation of the enzyme. 4-Amino-2-fluorobutanoic acid also inhibited enzymic activity but in this case the inhibition was reversible and competitive with respect to gamma-aminobutyric acid (GABA). The inhibitor constant (Ki) was 0.83 +/- 0.44 mM. We found no evidence that this fluorinated analogue of GABA could act as a substrate for the enzyme.
Tunnicliff
G
G
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Evansville 47712, USA.
Crites
G J
GJ
D000612:4-Aminobutyrate Transaminase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007656:Ketoglutaric Acids / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008627:Mercuric Chloride / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
C115800:4-amino-2-fluorobutanoic acid; D004791:Enzyme Inhibitors; D007656:Ketoglutaric Acids; D008627:Mercuric Chloride; D005680:gamma-Aminobutyric Acid; D020245:p-Chloromercuribenzoic Acid; D011732:Pyridoxal Phosphate; D000612:4-Aminobutyrate Transaminase