Interaction of Pyridoxal Analogues with Glutamate Apodecarboxylase and Aspartate Apoaminotransferase

The interaction of a variety of pyridoxal analogues with glutamate decarboxylase and aspartate aminotransferase has been studied. Pyridoxal phosphate N-oxide, N-methylpyridoxal phosphate, 3-O-methylpyridoxal phosphate, 4-de-(formyl)-4-vinyl pyridoxal phosphate, and pyridoxal analogues which contain modified substituents in position 5 (CH2CH2OPO3H2, CH(CH3)OPO3H2, CH2PO3H2, and CH2-CH2COOH) are bound at the pyridoxal phosphate binding site of the enzymes. The reaction of the analogues with the apoenzymes prevented activation with pyridoxal phosphate. Pyridoxal phosphate, pyridoxal phosphate N-oxide, O-methylpyridoxal phosphate, and the homologue of pyridoxal phosphate bind to apodecarboxylase to give absorption spectra characteristic of protonated Schiff bases. The rates of binding, the coenzyme constants, and the activation of the enzymes were determined for the analogues with aspartate apoaminotransferase and glutamate apodecarboxylase. The phosphate group of the coenzyme plays an important role in binding to the enzymes and in maintaining the enzymes and coenzymes in the proper conformation for catalysis. There are more stringent structural requirements for interaction of pyridoxal phosphate with the decarboxylase than with the transaminase. The free phenolic hydroxyl group and the free pyridine ring nitrogen are of importance in the interaction of pyridoxal phosphate with glutamate apodecarboxylase, but not as much as with aspartate apoaminotransferase. However, the phenolic hydroxyl group and the pyridine ring nitrogen are very important in the activation of the transminase. The addition of pyridoxal phosphate N-oxide to apodecarboxylase yields enzyme which is 29% as active as the native holoenzyme. The decarboxylase-N-oxide complex and the native glutamate decarboxylase have similar Km values for glutamate and Ki values for glutarate.