Reactions of phosphonate analogs of pyridoxal phosphate with apo-aspartate aminotransferase.

We have investigated reactions of the 5-phosphonoethyl and 5-phosphonoethenyl analogs of pyridoxal 5'-phosphate in the coenzyme site of cytosolic aspartate aminotransferase. Acid dissociation constants and equilibrium constants for hydration and for tautomerization have been evaluated for these compounds. In confirmation of previous results, both compounds are partially active. They bind to apoenzyme well and undergo conversion in the presence of glutamate to amine forms which show induced circular dichroism comparable to that of native enzyme. A normal "external" Schiff base is evidently formed with 2-methylaspartate, but the amounts of quinonoid intermediate formed with erythro-3-hydroxyaspartate are less than those formed with pyridoxal phosphate. The pKa of the imine group of the enzyme reconstituted with the phosphonoethyl analog is more than two units lower than that in the native enzyme. Binding of the dicarboxylates glutarate, 2-oxoglutarate, and succinate shifts the pKa upward. The absorption spectra of the resulting complexes indicate the existence of at least three low pH species. A shift of 2.3 to 2.9 ppm to a lower frequency was observed for the 31P NMR signal upon binding of these dicarboxylates or of 2-methylaspartate. Enzyme containing the analogs crystallizes. Polarized absorption spectra suggest that the coenzyme has an orientation similar to that of pyridoxal phosphate in the native enzyme.