Resolution of pyridoxal 5'-phosphate from O-acetylserine sulfhydrylase from Salmonella typhimurium and reconstitution of apoenzyme with cofactor and cofactor analogues as a probe of the cofactor binding site

A procedure has been developed to prepare the apoenzyme of O-acetylserine sulfhydrylase (apoOASS) by first converting the native enzyme to the alpha-aminoacrylate intermediate and dialyzing against 5 M guanidinium chloride, Aposulfhydrylase is stable for at least a month in buffers containing phosphate or phosphate analogues, Reconstitution of aposulfhydrylase with pyridoxal 5'-phosphate (PLP), 2'-methyl PLP (2'-MePLP), and pyridoxal 5'-deoxymethylenephosphonate (PDMP) results in enzymatically competent proteins, Pyridoxal in the absence and presence of phosphate and pyridoxal 5'-phosphate monomethyl eater are unable to form a Schiff base with apoOASS, The reconstitution of apoOASS with PLP is highly cooperative judged by the initial rate of activity regained and shows no evidence of saturation with PLP, The reconstituted enzymes have been studied using P-31 NMR spectroscopy, The P-31 NMR of the aposulfhydrylase reconstituted with PLP exhibits a chemical shift of 5.2 ppm, identical to that of native enzyme, The latter has been interpreted in terms of a strong ionic interaction between enzyme and the 5'-phosphate of PLP (P. F. Cook, S. Hara, S. Nalabolu, and K. D. Schnackerz, 1992, Biochemistry 31, 2298-2303). Reconstitution with 2'-MePLP gives a lower chemical shift of 4.95 ppm, suggesting a weaker ionic interaction at the 5'-phosphate when compared to native enzyme, The PDMP-reconstituted enzyme gives a chemical shift of 23.7 ppm, consistent with the monoanionic form of the bound phosphonate, All of the chemical shifts are pH independent. The apoenzyme has also been reconstituted with pyridoxal 5'-sulfate. Although the resulting enzyme is not active in the overall reaction, it forms the external Schiff base. The PDMP- and 2'-MePLP-reconstituted enzymes have also been studied in the presence of amino acid reactants and analogues, and results are discussed in terms of the mechanism of OASS. (C) 1995 Academic Press, Inc.