RESOLUTION OF PYRIDOXAL 5'-PHOSPHATE FROM O-ACETYLSERINE SULFHYDRYLASE FROM SALMONELLA-TYPHIMURIUM AND RECONSTITUTION OF APOENZYME WITH COFACTOR AND COFACTOR ANALOGS AS A PROBE OF THE COFACTOR BINDING-SITE

A procedure has been developed to prepare the apoenzyme of O-acetylser ine sulfhydrylase (apoOASS) by first converting the native enzyme to t he alpha-aminoacrylate intermediate and dialyzing against 5 M guanidin ium chloride, Aposulfhydrylase is stable for at least a month in buffe rs containing phosphate or phosphate analogues, Reconstitution of apos ulfhydrylase with pyridoxal 5'-phosphate (PLP), 2'-methyl PLP (2'-MePL P), and pyridoxal 5'-deoxymethylenephosphonate (PDMP) results in enzym atically competent proteins, Pyridoxal in the absence and presence of phosphate and pyridoxal 5'-phosphate monomethyl eater are unable to fo rm 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 enzyme s have been studied using P-31 NMR spectroscopy, The P-31 NMR of the a posulfhydrylase reconstituted with PLP exhibits a chemical shift of 5. 2 ppm, identical to that of native enzyme, The latter has been interpr eted 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. Schnacke rz, 1992, Biochemistry 31, 2298-2303). Reconstitution with 2'-MePLP gi ves a lower chemical shift of 4.95 ppm, suggesting a weaker ionic inte raction at the 5'-phosphate when compared to native enzyme, The PDMP-r econstituted enzyme gives a chemical shift of 23.7 ppm, consistent wit h the monoanionic form of the bound phosphonate, All of the chemical s hifts are pH independent. The apoenzyme has also been reconstituted wi th pyridoxal 5'-sulfate. Although the resulting enzyme is not active i n the overall reaction, it forms the external Schiff base. The PDMP- a nd 2'-MePLP-reconstituted enzymes have also been studied in the presen ce of amino acid reactants and analogues, and results are discussed in terms of the mechanism of OASS. (C) 1995 Academic Press, Inc.