31P nuclear magnetic resonance of mitochondrial aspartate aminotransferase. The effects of solution pH and ligand binding.

~-Aspartate:2-oxoglutarate aminotransferase (EC 2.6.1.1) of higher organisms exists as cytoplasmic and mitochondrial isozymes with a high degree of sequence homology. This report describes an NMR study of the environment of the phosphate of the pyridoxal 5’-phosphate cofactor in the mitochondrial isozyme as revealed by the effect of pH on its 31P NMR chemical shift. Our previous study of the cytoplasmic isozyme revealed a phosphate resonance which was unaffected by pH (Martinez-Carrion, M. (1975) Eur. J. Biochem 54, 39-43). In contrast to this apparently static environment, the cofactor phosphate resonance of the mitochondrial isozyme is affected by pH, and this effect is different in the various enzyme forms previously identified as intermediates in the catalytic sequence of transamination. The effect of pH is described by a pK of 5.5 and by limiting chemical shifts of -2.4 and -3.5 ppm for the cofactor phosphate in the deionized enzyme. Chloride binds to the deionized enzyme, increasing the pK of the cofactor phosphate to 6.3. The substrate analogue a-methyl-L-aspartate broadens the resonance of the cofactor phosphate and results in a chemical shift constant at -3.8 ppm from pH 8.5 to about pH 6, decreasing slightly at lower pH. The substrate analogue erythro-/3-hydroxy-L-aspartate produces two overlapping 31P resonances. By contrast to all other forms of this isozyme, the phosphopyridoxamine form of the enzyme has a chemical shift which is unaffected by pH, remaining at -2.3 ppm. Succinate, a possible regulatory ligand which inhibits the enzyme, causes an increase in the cofactor phosphate pK to 7.6 and in the limiting chemical shifts to -3.1 and -3.9 ppm. The apotransaminase tightly binds inorganic phosphate, but it can be displaced by arsenate. The bound phosphate shows a chemical shift behavior somewhat similar to that of the phosphate ester of the bound cofactor. These observations are discussed in terms of the diversity between the isozymes of aspartate aminotransferase and the possible roles of the cofactor phosphate and its protein environment.