Binding of new PLP analogs to the catalytic domain of GABA transaminase.

The binding site of Pyridoxal-5-P in 4-aminobutyrate aminotransferase was studied by using analogs of the cofactor. A phosphorothioate analog (PLP(S] recognizes the catalytic site; it forms a stable complex with the apoenzyme (KD = 1nM) and serves as cofactor during catalysis. Replacement of a non-bridged oxygen by sulfur in the phosphate side chain renders a compound which preserves the negative charges needed for correct alignment of the cofactor at the catalytic site. This phosphorothioate analog of PLP can be used to investigate the catalytic site of vitamin B6 dependent enzymes by means of 31P NMR spectroscopy. A bulky P-pyridoxamine derivative, ie, N-4-azido-2-nitrophenyl pyridoxyl-5-P (NANP) competes with natural cofactor for its binding site. Upon illumination, the arylazide of P-pyridoxamine acts as an efficient photolabeling reagent of the protein. A characteristic feature of this photolabeling reagent, ie, its ability to recognize the cofactor binding site, can be exploited to ascertain the chemical nature of amino acid residues at the catalytic site.