4‐Aminobutyrate Aminotransferase

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Abstract
The analogs 5-phospho-pyridoxal-aminooxyacetate and 4-vinyl-pyridoxal 5-phosphate inhibit reduced [pig brain] 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 moleucle 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 .ANG. (2.7 nm) separates the 2 catalytic binding sites.

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