4-Aminobutyrate aminotransferase. Different susceptibility to inhibitors, microenvironment of the cofactor binding site and distance of the catalytic sites.
The analogues 5-phospho-pyridoxal-aminooxyacetate and 4-vinyl-pyridoxal 5-phosphate inhibit reduced 4-aminobutyrate aminotransferase reconstituted with pyridoxal 5-phosphate, but they do not affect the catalytic activity of the holoenzyme. The binding of the 5-phosphopyridoxal adducts to the holoenzyme was monitored by measuring the fluorescence properties of the protein and the ligands. The presence of nonequivalent binding sites in reduced samples of enzyme containing 1 mol and 1.8 mol P-pyridoxyl residues/mol dimer was detected by steady and nanosecond fluorescence spectroscopy. The spectroscopic properties (fluorescence lifetime tau = 1 ns, polarization = 0.36 and quantum yield = 0.01) of the first molecule of 5-phospho-pyridoxyl are different from the properties of the second molecule of 5-phospho-pyridoxyl covalently bound to the enzyme (tau = 2.1 ns, P = 0.16, QT = 0.1). The spectroscopic properties of pyridoxal 5-phosphate and 5-phospho-pyridoxal-aminooxyacetate bound to different sites on the holoenzyme can be used to deduce proximity relationships between the catalytic sites of 4-aminobutyrate aminotransferase. On the basis of resonance energy transfer measurements, it is proposed that a distance of 27 A (2.7 nm) separates the two catalytic binding sites.
D000612:4-Aminobutyrate Transaminase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D011730:Pyridoxal / Q000031:analogs & derivatives* / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000494:pharmacology* / Q000031:analogs & derivatives; D013050:Spectrometry, Fluorescence; D013552:Swine; D000637:Transaminases / Q000037:antagonists & inhibitors*
C036136:4-vinylpyridoxal-5-phosphate; D011730:Pyridoxal; D011732:Pyridoxal Phosphate; C036135:5-phosphopyridoxal-aminooxyacetate; D000637:Transaminases; D000612:4-Aminobutyrate Transaminase