Ionization state of the coenzyme 5'-phosphate ester in cytosolic aspartate aminotransferase. A Fourier transform infrared spectroscopic study

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Abstract
In order to determine the ionization state of the 5''-phosphate of bound pyridoxal phosphate, a Fourier transform infrared spectroscopic study of cytosolic aspartate aminotransferase has been carried out. Dianionic and monoanionic phosphate monoesters give rise to two bands each in the infrared spectrum [Shimanouchi, T., Tsuboi, M., and Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498]. These bands can be identified in infrared spectra of the free coenzyme in solution. Due to interfering bands arising from the protein, only the band assigned to the symmetric stretching of the dianionic phosphate is observed in holoenzyme solutions. The integrated intensity of this band does not change with pH in the range 5.3-8.6, while for free pyridoxal phosphate, the integrated intensity of the same band changes with pH according to the pK value expected for the 5''-phosphate group in solution. Moreover, the value of the integrated intensity for the bound cofactor is close to the value given by free cofactor at pH 8-9. These results suggest that the 5''-phosphate of the bound cofactor remains mostly dianionic throughout the investigated pH range and disfavor other interpretations in terms of ionization of the phosphate group on the basis of the nuclear magnetic resonance 31P chemical shift-pH titration curve of holoenzyme [Schnackerz, K.D. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, E. A., Ed.) Part A, pp 195-208, Alan R. Liss, New York]. Instead, it seems likely that the 31P chemical shift may sense the ionization of the pyridoxal phosphate-Lys-258 Schiff''s base, as proposed for the mitochondrial isozyme [Mattingly, M. E., Mattingly, J. R., and Martinez-Carrion, M. (1982) J. Biol. Chem. 257, 8872].

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