Glutamic-Aspartic Transaminase REACTION OF HOLOENZYME WITH SUBSTRATES AND OF APOENZYJIE WITH VITAMIN Be DERIVATIVES*

The general subject of enzymatic transamination has been extensively reviewed by several workers (Braunstein (I) ; Snell (2) ; Meister (3)). Glutamic-aspartic transaminase (L-aspartate : 2-oxoglutarate aminotransferase, EC 2.6.1.1) from pig heart. has been prepared by Jenkins, Yphantis, and Sizer (4) and shown to contain 2 molecules of pyridoxal phosphate per enzyme molecule of molecular weight of about 116,000. Under special conditions the enzyme can be resolved and reconstituted by the addition of pyridoxal phosphate (5-7). In the case of reactivation with pyridoxamine phosphate some workers obtained none, while others found a retarded reactivation (8-10). The addition of dicarboxylic keto acid substrates to the enzyme, which has a maximum absorption at 362 rnp, results in the formation of a complex with a maximum in the region of 430 rnp in which the carboxyl groups are reversibly bound to the enzyme (4, 11). Amino substrates also react with the enzyme as indicated by t.he shift of the maximum absorption peak to 333 rnp (11, 12). This change produced by amino substrates has been interpreted by Jenkins and Sizer (12) and Lis et al. (13) as indicating the formation of a pyridoxamine-P enzyme plus keto acid, and by Evangelopoulos and Sizer (11) as suggesting the formation of a pyridoxal-P enzyme-amino acid complex. The possibility that an enzyme-amino acid complex was an intermediate in the formation of pyridoxamine-P enzyme was suggested previously by Jenkins and Sizer (12). Since there is evidence that both pyridoxamine-P enzyme and a transaminase-amino acid complex absorb at about 330 rnp (11, 14), it is difficult to distinguish between them on the basis of spectral characteristics. In the present paper an attempt is made to obtain further information on the mechanism of transamination by a measurement of the reaction of transaminase with amino substrates to produce enzyme-substrate complex, pyridoxamine-P enzyme, and keto acids. We have also studied the interaction of several vitamin B6 derivatives with apotransaminase in order to correlate spectral changes with restoration of enzymatic activity produced by pyridoxal-P and pyridoxamine-P.